Sample records for embryo research
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Haimes, E.; Taylor, K.
2009-01-01
BACKGROUND This article reports on an investigation of the views of IVF couples asked to donate fresh embryos for research and contributes to the debates on: the acceptability of human embryonic stem cell (hESC) research, the moral status of the human embryo and embryo donation for research. METHODS A hypothesis-generating design was followed. All IVF couples in one UK clinic who were asked to donate embryos in 1 year were contacted 6 weeks after their pregnancy result. Forty four in-depth interviews were conducted. RESULTS Interviewees were preoccupied with IVF treatment and the request to donate was a secondary consideration. They used a complex and dynamic system of embryo classification. Initially, all embryos were important but then their focus shifted to those that had most potential to produce a baby. At that point, ‘other’ embryos were less important though they later realise that they did not know what happened to them. Guessing that these embryos went to research, interviewees preferred not to contemplate what that might entail. The embryos that caused interviewees most concern were good quality embryos that might have produced a baby but went to research instead. ‘The’ embryo, the morally laden, but abstract, entity, did not play a central role in their decision-making. CONCLUSIONS This study, despite missing those who refuse to donate embryos, suggests that debates on embryo donation for hESC research should include the views of embryo donors and should consider the social, as well as the moral, status of the human embryo. PMID:19502616
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Embryo deaths in reproduction and embryo research: a reply to Murphy's double effect argument.
Devolder, Katrien
2013-08-01
The majority of embryos created in natural reproduction die spontaneously within a few weeks of conception. Some have argued that, therefore, if one believes the embryo is a person (in the normative sense) one should find 'natural' reproduction morally problematic. An extension of this argument holds that, if one accepts embryo deaths in natural reproduction, consistency requires that one also accepts embryo deaths that occur in (i) assisted reproduction via in vitro fertilisation (IVF) and (ii) embryo research. In a recent paper in this journal, Timothy Murphy criticises both the initial argument and its extension. Murphy argues that double-effect reasoning can justify embryo deaths both in natural reproduction and IVF, but not in embryo research. Thus, according to Murphy, one can, without being inconsistent, (1) believe the embryo is a person and accept natural reproduction and IVF, and (2) accept natural reproduction and IVF, while rejecting embryo research on the ground that it involves embryo deaths. I show that Murphy's argument is problematic because double effect cannot justify embryo deaths in standard IVF practices. The problem is that the proportionality criterion of double effect is not met by such practices. Thus, Murphy's argument fails to support (1) and (2). An implication of his argument failing to support (2) is that it does not defeat the position I have defended in the past-that if one accepts standard IVF practices one should also accept embryo research, including research with embryos created solely for that purpose.
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Bürgin, M T; Bürkli, P
2002-11-01
At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.
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Donation of surplus frozen pre-embryos to research in Israel: underlying motivations.
Raz, Aviad; Amer-Alshiek, Jonia; Goren-Margalit, Mor; Jacobi, Gal; Hochberg, Alyssa; Amit, Ami; Azem, Foad; Amir, Hadar
2016-01-01
The high number of IVF procedures performed in Israel has had an unforeseen consequence: accumulation of large amounts of surplus frozen embryos. After five years that the frozen embryos are kept for free, patients need to make an embryo disposition decision. One option is donation for research. The donation rate in Israel is very low. Our aim was to understand the attitudes, values and perceptions of female IVF patients that decided to donate their surplus frozen embryos to research. The study setting was a tertiary IVF unit which during the 2000-2009 period treated 241 patients who had their frozen pre-embryos stored for more than five years. The study population consists of the 12 patients (from among the 241) who had decided to donate their excess frozen pre-embryos to research. In-depth interviews were carried out with 8 of those 12 patients. IVF patients who donated their surplus frozen pre-embryos to research viewed the frozen embryo as a valuable resource that does not have human identity yet. The majority expressed a gradualist approach to the human status of the embryo as requiring successful implantation and development in the uterus. All the respondents chose donation to research not because it was their first choice but because they did not want or were unable to use the pre-embryos in the future, in addition to not willing to thaw them. For many of the respondents, donation to research was accompanied by a sense of uncertainty. All would have preferred to donate their pre-embryos to infertile women or couples, an option which is currently prohibited in Israel. The moral reasoning behind decisions that patients make regarding excess pre-embryos is important for health care practitioners to consider when offering decision-making alternatives and counseling. For our respondents, the scarcity of donating excess frozen pre-embryos to research may reflect patients' preference for embryo donation to infertile couples. Recommended ways to increase donation to research may include public education and awareness, as well as targeted communication with IVF patients by multi-professional IVF unit teams comprised of a medical doctor and a professional trained in bioethics.
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Bowens, Krietta Kai
2006-01-01
The legal status of embryos in American law is changing. At present, most states do not afford embryos the same protections as a born person, but some states are attempting to change this standard. Granting embryos the same legal status as born human beings poses a significant problem for industries that work with embryos, especially fertility treatment facilities and scientists researching stem cell and gene therapy technologies. This paper describes the methods of defining embryos in American law, and discusses the implications of granting embryos the same rights as born persons for the reproductive technology and scientific research industries.
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Davies, L
1994-03-01
Arguments in favour of embryo research Prevention of congenital defects. Reduction in number of miscarriages. Better understanding of infertility. Arguments against embryo research Morally unacceptable to take potential human life. The 14-day limit on research is arbitrary. Fears about the creation of 'superspecies' and 'designer babies'. Fear of embryo 'trade' in which embryos are bought and sold.
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'New embryos' - new challenges for the ethics of stem cell research.
Holm, Søren
2008-01-01
Among the many ethical issues raised by human embryonic stem cell research (in the following all references to 'stem cells' should be read as references to human embryonic stem cells), two have gained specific prominence: (1) whether stem cell research is ethically problematic because it entails the destruction of human embryos and (2) what kind of control embryo donors should have over the stem cell lines derived from their embryos. In the present paper, I will analyse how these two issues are engaged by various attempts to derive stem cells from anomalous embryos (e.g. embryos in cleavage arrest, embryos not implanted following pre-implantation genetic diagnosis or embryos created by altered nuclear transfer) or in ways that are claimed to be non-destructive for the embryo (e.g. blastocyst or blastomere biopsy). Copyright 2008 S. Karger AG, Basel.
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Use of "excess" human embryos for stem cell research: protecting women's rights and health.
Cohen, C B
2000-01-01
Proposed National Institutes of Health guidelines for stem cell research are too narrowly drawn and do not adequately protect the freedom of choice and health of women who donate embryos. They need to be expanded to cover not only the point of embryo donation, but also that of embryo creation. Guidelines are provided to ensure that donors undergoing hyperstimulation and egg retrieval gave voluntary informed consent to the production of embryos that might later prove in excess. A standard for determining when embryos have been overproduced is presented to address the possibility that additional embryos will be created for stem cell research in violation of the guidelines and at risk to women's health.
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Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.
Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko
2017-03-01
Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes. The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Kayssan, Marjaneh; Dolatian, Mahrokh; Omani Samani, Reza; Maroufizadeh, Saman
2017-01-01
Objective After the introduction of assisted reproductive techniques, human embryos were officially introduced into laboratories and now thousands of them are cryopreserved in such settings. Embryonic stem cells and the future application of such cells in the treatment of disease opened the door to further research on human embryos. These developments raise many ethical issues, some of which have religious aspects. The main question is: what is the embryo? Should we consider it a human being? Thus, the purpose of this study was to investigate attitudes towards the personhood of the embryo. Materials and Methods In this cross sectional study, 203 infertile patients (n=406), 54 clinic staff and 49 embryo researchers, selected using convenience sampling at the Royan Institute, completed a questionnaire on personhood of human embryo. The questionnaire had been developed following qualitative research and had satisfied face and content validity tests. Results At the pre-implantation stage the majority of participants in all three groups considered the human embryo as "not a human being". Also, at the post-implantation stage of development, the majority of infertile couples and clinic staff considered the embryo as "not a human being" but, half the researchers (51%) considered the embryo in this stage as a "potential human". Half of the infertile couples considered the human fetus before ensoulment time (19th week of pregnancy according to the Shiite Islamic scholars) as "not-human being", while more than half of researchers (55.1%) considered it as a "potential human". Conclusion Ensoulment time is a major and important border for personhood. Most infertile couples and clinic staff consider the human embryo as "not a human being" but majority of all study participants considered the human fetus to be a complete human after ensoulment time. PMID:28670524
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Where does New Zealand stand on permitting research on human embryos?
Jones, D Gareth
2014-08-01
In many respects New Zealand has responded to the assisted reproductive technologies (ARTs) as positively as many comparable societies, such as Australia and the UK. Consequently, in vitro fertilisation (IVF) and pre-implantation genetic diagnosis (PGD) are widely available, as is non-commercial surrogacy utilising IVF. These developments have been made possible by the Human Assisted Reproductive Technology (HART) Act 2004, overseen by its two committees, the Advisory Committee on Assisted Reproductive Technology (ACART) and the Ethics Committee (ECART). However, New Zealand stands apart from many of these other societies by the lack of permission for scientists to conduct research using human embryos. There is no doubt this reflects strongly held viewpoints on the part of some that embryos should be protected and not exploited. Legitimate as this stance is, the resulting situation is problematic when IVF is already designated as an established procedure. This is because the development of IVF involved embryo research, and continuing improvements in procedures depend upon ongoing embryo research. While prohibition of research on human embryos gives the impression of protecting embryos, it fails to do this and also fails to enhance the health and wellbeing of children born using IVF. This situation will not be rectified until research is allowed on human embryos.
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Human research cloning, embryos, and embryo-like artifacts.
Hyun, Insoo; Jung, Kyu Won
2006-01-01
Research suggests that cloning is incapable of producing a viable embryo when it is used on primate eggs. In fact, the entity created may not qualify as an embryo at all. If the results stand, cloning avoids the moral objections typically lodged against it, and cloning is itself an "alternative source" of stem cells.
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Scott, Rosamund; Williams, Clare; Ehrich, Kathryn; Farsides, Bobbie
2012-01-01
This paper analyses elements of the legal process of consent to the donation of ‘spare’ embryos to research, including stem-cell research, and makes a recommendation intended to enhance the quality of that process, including on occasion by guarding against the invalidity of such consent. This is important in its own right and also so as to maximise the reproductive treatment options of couples engaged in in vitro fertilisation (IVF) treatment and to avoid possible harms to them. In Part 1, with reference to qualitative data from three UK IVF clinics, we explore the often delicate and contingent nature of what comes to be, for legal purposes, a ‘spare’ embryo. The way in which an embryo becomes ‘spare’, with its implications for the process of consent to donation to research, is not addressed in the relevant reports relating to or codes of practice governing the donation of embryos to research, which assume an unproblematic notion of the ‘spare’ embryo. Significantly, our analysis demonstrates that there is an important and previously unrecognised first stage in the donation of a ‘spare’ embryo to research, namely: consent to an embryo being ‘spare’ and so, at the same time, to its disuse in treatment. This is not explicitly covered by the Human Fertilisation and Embryology (HFE) Act 1990, as amended by the HFE Act 2008. Having identified this important initial stage in the process of consent to the donation of a ‘spare’ embryo to research in conclusion to Part 1, in Part 2 we analyse the idea of consent to an embryo's disuse in treatment on the basis that it is ‘spare’ with reference to the legal elements of consent, namely information as to nature and purpose, capacity, and voluntariness. We argue that there are in fact three related consent processes in play, of which the principal one concerns consent to an embryo's disuse in treatment. If the quality of this first consent is compromised, in turn this will impact on the quality of the consent to the donation of that ‘spare’ embryo to research, followed by the quality of consent to future cycles of assisted reproduction treatment in the event that these are needed as a result of a donation decision. The analysis overall is of central relevance to the debate as to whether, and if so when, it should be permissible to request the donation of fresh embryos for research, as opposed to those that have been frozen and, for instance, have reached the end of their statutory storage term. This has a particular bearing on the donation of embryos to stem-cell research since there is a debate as to whether fresh embryos are most useful for this. PMID:22647978
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Patients' Attitudes towards the Surplus Frozen Embryos in China
Jin, Xuan; Wang, GongXian; Liu, SiSun; Liu, Ming; Zhang, Jing; Shi, YuFa
2013-01-01
Background. Assisted reproductive techniques have been used in China for more than 20 years. This study investigates the attitudes of surplus embryo holders towards embryos storage and donation for medical research. Methods. A total of 363 couples who had completed in vitro fertilization (IVF) treatment and had already had biological children but who still had frozen embryos in storage were invited to participate. Interviews were conducted by clinics in a narrative style. Results. Family size was the major reason for participants' (dis)continuation of embryo storage; moreover, the moral status of embryos was an important factor for couples choosing embryo storage, while the storage fee was an important factor for couples choosing embryo disposal. Most couples discontinued the storage of their embryos once their children were older than 3 years. In our study, 58.8% of the couples preferred to dispose of surplus embryos rather than donate them to research, citing a lack of information and distrust in science as significant reasons for their decision. Conclusions. Interviews regarding frozen embryos, including patients' expectations for embryo storage and information to assist them with decisions regarding embryo disposal, are beneficial for policies addressing embryo disposition and embryo donation in China. PMID:23509811
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The evolution of porcine embryo in vitro production.
Grupen, Christopher G
2014-01-01
The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.
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Factors associated with the donation and non-donation of embryos for research: a systematic review.
Samorinha, Catarina; Pereira, Margarida; Machado, Helena; Figueiredo, Bárbara; Silva, Susana
2014-01-01
Systematic knowledge on the factors that influence the decisions of IVF users regarding embryo donation for research is a core need for patient-centred policies and ethics in clinical practice. However, no systematic review has been provided on the motivations of patients who must decide embryo disposition. This paper fills this gap, presenting a systematic review of quantitative and qualitative studies, which synthesizes the current body of knowledge on the factors and reasons associated with IVF patients' decisions to donate or not to donate embryos for research. A systematic search of studies indexed in PubMed, ISI WoK and PsycINFO, published before November 2013, was conducted. Only empirical, peer-reviewed, full-length, original studies reporting data on factors and reasons associated with the decision concerning donation or non-donation of embryos for research were included. Eligibility and data extraction were performed by two independent researchers and disagreements were resolved by discussion or a third reviewer, if required. The main quantitative findings were extracted and synthesized and qualitative data were assessed by thematic content analysis. A total of 39 studies met the inclusion criteria and were included in the review. More than half of the studies (n = 21) used a quantitative methodology, and the remaining were qualitative (n = 15) or mixed-methods (n = 3) studies. The studies were derived mainly from European countries (n = 18) and the USA (n = 11). The proportion of IVF users who donated embryos for research varied from 7% in a study in France to 73% in a Swiss study. Those who donate embryos for research reported feelings of reciprocity towards science and medicine, positive views of research and high levels of trust in the medical system. They described their decision as better than the destruction of embryos and as an opportunity to help others or to improve health and IVF treatments. The perception of risks, the lack of information concerning research projects and the medical system and the conceptualization of embryos in terms of personhood were the most relevant motives for not donating embryos for research. Results relating to the influence of sociodemographic characteristics and reproductive and gynaecological history were mostly inconclusive. Three iterative and dynamic dimensions of the IVF patients' decision to donate or not to donate embryos for research emerged from this review: the hierarquization of the possible options regarding embryo disposition, according to the moral, social and instrumental status attributed to embryos; patients' understanding of expectations and risks of the research on human embryos; and patients' experiences of information exchange and levels of trust in the medical-scientific institutions. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Couples' willingness to donate embryos for research: a longitudinal study.
Samorinha, Catarina; Severo, Milton; Machado, Helena; Figueiredo, Bárbara; de Freitas, Cláudia; Silva, Susana
2016-08-01
Decision-making on embryo disposition is a source of distress and is subject to change over time. This paper analyzes the willingness of couples undergoing in vitro fertilization to donate cryopreserved embryos for research from 15 days after embryo transfer to 12 months later, taking into account the influence of psychosocial, demographic, and reproductive factors. Prospective longitudinal study, with 74 heterosexual couples undergoing in vitro fertilization in a public fertility centre in Portugal, recruited between 2011 and 2012. Participants were evaluated twice: 15 days after embryo transfer and 12 months later. A significant decrease in patients' willingness to donate embryos for research over time was observed [86.5% to 73.6%; relative risk (RR) = 0.85; 95% CI 0.76-0.95]. A higher education level (>12 years) [adjusted RR (RRadj ) = 0.79; 95% CI 0.64-0.96], considering research on human embryos to be important (vs. very important) (RRadj = 0.59; 95% CI 0.39-0.85) and practicing a religion less than once a month (vs. at least once a month) (RRadj = 0.73; 95% CI 0.53-1.00) seemed associated with unwillingness to donate embryos for research over time. Change towards non-donation happened mainly among couples who first considered that it was better to donate than wasting the embryos. Change towards donation occurred mostly among those stating that their priority at time 1 was to have a baby and who became pregnant in the meantime. Quality of care guided by patients' characteristics, values, preferences, and needs calls for considering the factors and reasons underlying couples' willingness to donate embryos for research over time as a topic in psychosocial guidelines for infertility and medically assisted reproductive care. © 2016 Nordic Federation of Societies of Obstetrics and Gynecology.
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Moral qualms, future persons, and embryo research.
Shaw, David Martin
2008-05-01
Many people have moral qualms about embryo research, feeling that embryos must deserve some kind of protection, if not so much as is afforded to persons. This paper will show that these qualms serve to camouflage motives that are really prudential, at the cost of also obscuring the real ethical issues at play in the debate concerning embryo research and therapeutic cloning. This in turn leads to fallacious use of the Actions/Omissions Distinction and ultimately neglects the duties that we have towards future persons.
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Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks
ERIC Educational Resources Information Center
Wellner, Karen L.
2014-01-01
In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…
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Bruno, C; Dudkiewicz-Sibony, C; Berthaut, I; Weil, E; Brunet, L; Fortier, C; Pfeffer, J; Ravel, C; Fauque, P; Mathieu, E; Antoine, J M; Kotti, S; Mandelbaum, J
2016-07-01
In couples who have chosen and confirmed the fate of surplus frozen embryos, which factors influence their decision, with a special emphasis on their symbolic representation of the embryo(s)? Embryo representation and gamete donation use significantly influence the fate of surplus cryopreserved embryos. Previous studies report difficulties for couples to decide whether or not to continue storing their frozen embryo(s) and different factors have been already highlighted which influence their decision, including embryo conceptualization, information and support provided by the medical institution, quality of embryo(s) and life events. Little is known, however, about couples who definitely decided to stop their parental project and finalized the process of decision-making about the fate of their cryopreserved embryo(s). This prospective study was conducted over a period of 3 years (2007-2010) and included IVF/ICSI patients with surplus frozen embryos, who made a final embryo disposition decision. Among the 280 eligible IVF/ICSI patients, 247 agreed to participate in the study. According to the available options, 91 persons chose to 'stop cryopreservation', 77 chose donation to 'research' and 48 'embryo donation' to infertile couples. Furthermore, 31 participants who chose embryo donation for a parental project were refused by the center as not compatible with their mandatory medical conditions. Among them, 27 participants then selected donation to research as a new option and were included in a fourth group: 'donation to research after Refusal of Embryo Donation for parental project' or 'research-RED' (n = 27). Four participants chose 'stop cryopreservation', however, given the small number of subjects this latter group was not included in the analysis. In all, 243 participants who made a final choice concerning the fate of their cryopreserved embryos were included in this study. Participants were sent a letter of invitation to a semi-structured interview of 30 min with a psychologist. Interviews were conducted separately for each partner, including a questionnaire with a common part and a specific part, according to the chosen option, and allowing a quantitative evaluation. A multivariate logistic regression model was used to assess the link between their embryo representation and their decision about their embryos' fate. After adjustment for age, gender, gamete donation, number of children and the different embryo representations, a choice to 'stop cryopreservation' is more frequent if the embryo is represented as a child [odds ratio (OR) adjusted = 3.29, 95% confidence interval (CI) = 1.62-6.66], P = 0.0009. Representing the embryo as a project prompts patients to choose 'donation to research' [OR adjusted = 3.76, 95% CI = 1.56-9.06], P = 0.0032. Respondents are more likely to choose 'embryo donation' if they represent the embryo as a potential person [OR adjusted = 3.77, 95% CI = 1.45-9.80], P = 0.0064. Furthermore, patients who benefited from gamete donation are ∼10 times more likely to donate their embryos to another couple [OR adjusted = 10.62, 95% CI = 3.99-28.30], P < 0.0001. For more than half the participants (57%) the decision-making was easy, however, deciding to stop cryopreservation was significantly more difficult than choosing research or embryo donation (P < 0.0001). Socio-economic status, moral and religious affiliations are known to influence the choice of couples but analyzing these factors was not an aim of the present study. When couples definitely decide to stop their parental project, the embryo symbolic representation remains the main factor that influences the fate of their frozen embryo(s). Moreover, this representation can evolve when influenced by external events and information provided. In order to support patients who are making this difficult decision, it could be helpful to explore this symbolic representation early in the IVF/ICSI procedure, before surplus embryo freezing, as a new tool enhancing the accuracy of counseling. this study was supported by a grant from the 'Agence de la biomedicine (ABM)', the national regulatory ART agency, under the authority of the French Ministry of Health. The authors have no conflict of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Sawai, Tsutomu; Hatta, Taichi; Fujita, Misao
2017-04-01
To understand the steps and objectives for which Japanese people are willing to accept human-animal chimeric embryo research using human induced pluripotent stem cells. An internet-based survey was conducted for the general public and researchers in Japan in 2016. Over 60% of the public and 83.8% of researchers supported the creation of human-swine chimeras and 81.0% of the public and 92.4% of researchers supported the creation of human-swine chimeric embryos. When presented with a graded view of human-swine chimeric embryo research with concomitant, specific objectives, a large majority of the general public as well as researchers are willing to accept this research with the aims of disease study, novel drug and treatment development, and transplantation.
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Ethics and policy in embryonic stem cell research.
Robertson, John A
1999-06-01
Embryonic stem cells, which have the potential to save many lives, must be recovered from aborted fetuses or live embyros. Although tissue from aborted fetuses can be used without moral complicity in the underlying abortion, obtaining stem cells from embryos necessarily kills them, thus raising difficult questions about the use of embryonic human material to save others. This article draws on previous controversies over embryo research and distinctions between intrinsic and symbolic moral status to analyze these issues. It argues that stem cell research with spare embryos produced during infertility treatment, or even embryos created specifically for research or therapeutic purposes, is ethically acceptable and should receive federal funding.
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Abortion, embryonic stem cell research, and waste.
Jensen, David A
2008-01-01
Can one consistently deny the permissibility of abortion while endorsing the killing of human embryos for the sake of stem cell research? The question is not trivial; for even if one accepts that abortion is prima facie wrong in all cases, there are significant differences with many of the embryos used for stem cell research from those involved in abortion--most prominently, many have been abandoned in vitro, and appear to have no reasonably likely meaningful future. On these grounds one might think to maintain a strong position against abortion but endorse killing human embryos for the sake of stem cell research and its promising benefits. I will argue, however, that these differences are not decisive. Thus, one who accepts a strong view against abortion is committed to the moral impermissibility of killing human embryos for the sake of stem cell research. I do not argue for the moral standing of either abortion or the killing of embryos for stem cell research; I only argue for the relation between the two. Thus the conclusion is relevant to those with a strong view in favor of the permissibility of killing embryos for the sake of research as much as for those who may strongly oppose abortion; neither can consider their position in isolation from the other.
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Lyerly, Anne Drapkin; Steinhauser, Karen; Voils, Corrine; Namey, Emily; Alexander, Carolyn; Bankowski, Brandon; Cook-Deegan, Robert; Dodson, William C.; Gates, Elena; Jungheim, Emily S.; McGovern, Peter G.; Myers, Evan R.; Osborn, Barbara; Schlaff, William; Sugarman, Jeremy; Tulsky, James A.; Walmer, David; Faden, Ruth R.; Wallach, Edward
2010-01-01
Objective To describe fertility patients' preferences for disposition of cryopreserved embryos and determine factors important to these preferences Design Cross-sectional survey conducted between June 2006 and July 2007 Setting Nine geographically diverse U.S. fertility clinics Participants 1020 fertility patients with cryopreserved embryos Interventions Self-administered questionnaire Main Outcome Measures Likelihood of selecting each of five conventional embryo disposition options: store for reproduction, thaw and discard, donate to another couple, freeze indefinitely, and donate for research; likelihood of selecting each of two alternative options identified in previous research: placement of embryos in the woman's body at an infertile time, and a disposal ceremony; importance of each of 26 considerations to disposition decisions; and views on the embryo's moral status. Results 54% of respondents with cryopreserved embryos were very likely to use them for reproduction, 21% were very likely to donate for research, 7% or fewer were very likely to choose any other option. Respondents who ascribed high importance to concerns about the health or well-being of the embryo, fetus, or future child were more likely to thaw and discard embryos or freeze them indefinitely. Conclusions Fertility patients frequently prefer disposition options not available to them or find available options unacceptable. Restructuring and standardizing the informed consent process and ensuring availability of all disposition options may benefit patients, facilitate disposition decisions and address problems of long term storage. PMID:19061998
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Mohler-Kuo, Meichun; Zellweger, Ueli; Duran, Aysun; Hohl, Michael K; Gutzwiller, Felix; Mutsch, Margot
2009-08-01
The purpose of this study was to investigate attitudes towards the donation of surplus embryos among couples with cryopreserved embryos/zygotes, and to identify correlates associated with attitudes toward the destinations of surplus embryos/zygotes. Eleven of 19 Swiss in vitro fertilization (IVF) centers in existence in 2004 participated in the survey. Questionnaires were sent to 888 eligible couples; 458 men (52%) and 468 women (53%) returned them. Fifty-two percent of the participants supported the donation of surplus embryos to other couples, but divided opinions on the disclosure of biological parents' identities were identified. About 70% of participants indicated that donations of surplus embryos for medical research or therapy should be allowed, following strict regulations. Multiple logistic regression analyses revealed couples' position on the moral status of an embryo as the strongest predictor of attitudes toward all destinations of surplus embryos. Having children due to IVF/Intra-Cytoplasmic Sperm Injection (ICSI) treatment was negatively associated with attitudes towards donations to other couples. Perceived importance of religion, age >40, being a resident of the French-speaking region and unsuccessful IVF/ICSI treatment experiences were predictive of supporting donations for medical research. Swiss couples with cryopreserved embryos/zygotes are open to different options related to donating, rather than discarding, surplus embryos.
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Vidal Martínez, Jaime
2006-01-01
This study deals with issues of research with human embryos obtained through in vitro fertilization in the context of the Spanish Law. The paper focuses on Act 14/2006 on techniques of human assisted reproduction, which replaces the previous Act from 1988. The author claims that the main goals of Act 14/2006 are, on the one hand, to eliminate the restrictions affecting research with human embryos put in place by Act 45/2003 and, on the other, to pave the way for a future legislation on biomedical research. This paper argues for the need of an effective and adequate juridical protection of human embryos obtained in vitro according to responsibility and precautionary principles.
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Developing the Digital Kyoto Collection in Education and Research.
Hill, Mark Anthony
2018-04-16
The Kyoto embryo collection was begun in 1961 by Dr. Hideo Nishimura. The collection has been continuously developed and currently contains over 44,000 human normal and abnormal specimens. Beginning online in 1997, the internet provided an opportunity to make embryos from the collection widely available for research and educational purposes (http://tiny.cc/Embryo). These embryonic development resources have been continuously published and available from that time until today. Published in Japanese as an Atlas of Embryonic Development. Published online as the Kyoto Human Embryo Visualization Project (http://atlas.cac.med.kyoto-u.ac.jp) and also as the Human Embryo Atlas (http://tiny.cc/Human_Embryo_Atlas). Published now electronically as a digital eBook (http://tiny.cc/Kyoto_Collection_eBook). This new digital format allows incorporation of whole embryo and histology manipulable images, labels, and a linked glossary. New imaging modalities of magnetic resonance imaging (MRI) and episcopic fluorescence image capture (EFIC) can also be easily displayed as animations. For research, the collection specimens and histological sections have been extensively studied and published in several hundred papers, discussed here and elsewhere in this special edition. I will also describe how the Kyoto collection will now form a major partner of a new international embryology research group, the Digital Embryology Consortium (https://human-embryology.org). The digital Kyoto collection will be made available for remote researcher access, analysis, and comparison with other collections allowing new research and educational applications. This work was presented at the 40th Anniversary Commemoration Symposium of the Congenital Anomaly Research Center, Graduate School of Medicine, Kyoto University, Japan, November, 2015. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.
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Stem cell research in Germany: ethics of healing vs. human dignity.
Oduncu, Fuat S
2003-01-01
On 25 April 2002, the German Parliament has passed a strict new law referring to stem cell research. This law took effect on July 1, 2002. The so-called embryonic Stem Cell Act ("Stammzellgesetz-StZG") permits the import of embryonic stem (ES) cells isolated from surplus lvF-embryos for research reasons. The production itself of ES cells from human blastocysts has been prohibited by the German Embryo Protection Act of 1990, with the exception of the use of ES cells which exist already. The debate on the legitimate use of ES cells escalated, after the main German research funding agency, the Deutsche Forschungsgemeinschaft (DFG), unexpectedly published new guidelines recommending a restricted use of human ES cells for research. Meanwhile, the debate has ethically divided society, political parties, government and church members into a group supporting and a group rejecting ES cell research. The arguments in favour of such a research can be summarized as arguments derived from a new "ethics of healing" calling for a therapeutic imperative, whereas the arguments against can be summarized as arguments violating the fundamental principle of human dignity as they imply the destruction of human embryos. This article will try to present and evaluate various ethical arguments founded on the latest biological and medical data on the potential use of stem cell technologies. It will finally come to the conclusion that ES cell research is opposed to human dignity, since the procedures of isolating ES cells require the destruction and instrumentalization of human embryos. Human embryos are human beings at a very early stage of their development, fully possessing the ability of completing their development. At this very early stage, human embryos are extremely dependent and fragile, and thus vulnerable corporealities. Vulnerability and human dignity demand the protection of the embryo's corporeal integrity. Hence, this essay will try to propagate research with adult stem (AS) cells, a procedure which does not require the destruction of human embryos; with regard to the necessary plasticity, it should be emphasized that AS cells very much resemble ES cells.
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Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos
Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene
2015-01-01
Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970
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Feminists on the inalienability of human embryos.
McLeod, Carolyn; Baylis, Francoise
2006-01-01
The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos.
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Current status of in vitro embryo production in sheep and goats.
Paramio, M-T; Izquierdo, D
2014-10-01
Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi-step methodology comprising the following procedures: (i) In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co-incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-free genetic movement and easier and cheaper germplasm commercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology. © 2014 Blackwell Verlag GmbH.
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Reporting of embryo transfer methods in IVF research: a cross-sectional study.
Gambadauro, Pietro; Navaratnarajah, Ramesan
2015-02-01
The reporting of embryo transfer methods in IVF research was assessed through a cross-sectional analysis of randomized controlled trials (RCTs) published between 2010 and 2011. A systematic search identified 325 abstracts; 122 RCTs were included in the study. Embryo transfer methods were described in 42 out of 122 articles (34%). Catheters (32/42 [76%]) or ultrasound guidance (31/42 [74%]) were most frequently mentioned. Performer 'blinding' (12%) or technique standardization (7%) were seldom reported. The description of embryo transfer methods was significantly more common in trials published by journals with lower impact factor (less than 3, 39.6%; 3 or greater, 21.5%; P = 0.037). Embryo transfer methods were reported more often in trials with pregnancy as the main end-point (33% versus 16%) or with positive outcomes (37.8% versus 25.0%), albeit not significantly. Multivariate logistic regression confirmed that RCTs published in higher impact factor journals are less likely to describe embryo transfer methods (OR 0.371; 95% CI 0.143 to 0.964). Registered trials, trials conducted in an academic setting, multi-centric studies or full-length articles were not positively associated with embryo transfer methods reporting rate. Recent reports of randomized IVF trials rarely describe embryo transfer methods. The under-reporting of research methods might compromise reproducibility and suitability for meta-analysis. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Byk, J C
1997-02-01
The objective of this paper is to stimulate academic debate on embryo and fetal research from the perspective of the drafting of a protocol to the European Convention on Biomedicine. The Steering Committee on Bioethics of the Council of Europe was mandated to draw up such a protocol and for this purpose organised an important symposium on reproductive technologies and embryo research, in Strasbourg from the 16th to the 18th of December 1996.
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Mizuno, Hiroshi; Akutsu, Hidenori; Kato, Kazuto
2015-01-01
Human-animal chimeric embryos are embryos obtained by introducing human cells into a non-human animal embryo. It is envisaged that the application of human-animal chimeric embryos may make possible many useful research projects including producing three-dimensional human organs in animals and verification of the pluripotency of human ES cells or iPS cells in vivo. The use of human-animal chimeric embryos, however, raises several ethical and moral concerns. The most fundamental one is that human-animal chimeric embryos possess the potential to develop into organisms containing human-derived tissue, which may lead to infringing upon the identity of the human species, and thus impairing human dignity. The Japanese Expert Panel on Bioethics in the Cabinet Office carefully considered the scientific significance and ethical acceptability of the issue and released its "Opinions regarding the handling of research using human-animal chimeric embryos". The Panel proposed a framework of case-by-case review, and suggested that the following points must be carefully reviewed from the perspective of ethical acceptability: (a) Types of animal embryos and types of animals receiving embryo transfers, particularly in dealing with non-human primates; (b) Types of human cells and organs intended for production, particularly in dealing with human nerve or germ cells; and (c) Extent of the period required for post-transfer studies. The scientific knowledge that can be gained from transfer into an animal uterus and from the production of an individual must be clarified to avoid unnecessary generation of chimeric animals. The time is ripe for the scientific community and governments to start discussing the ethical issues for establishing a global consensus.
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Avian models in teratology and developmental toxicology.
Smith, Susan M; Flentke, George R; Garic, Ana
2012-01-01
The avian embryo is a long-standing model for developmental biology research. It also has proven utility for toxicology research both in ovo and in explant culture. Like mammals, avian embryos have an allantois and their developmental pathways are highly conserved with those of mammals, thus avian models have biomedical relevance. Fertile eggs are inexpensive and the embryo develops rapidly, allowing for high-throughput. The chick genome is sequenced and significant molecular resources are available for study, including the ability for genetic manipulation. The absence of a placenta permits the direct study of an agent's embryotoxic effects. Here, we present protocols for using avian embryos in toxicology research, including egg husbandry and hatch, toxicant delivery, and assessment of proliferation, apoptosis, and cardiac structure and function.
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[Research with human embryo stem cells. Foundations and judicial limits].
Eser, Albin; Koch, Hans-Georg
2004-01-01
Research with human embryos, and particularly, the use for scientific purposes of human embryonic stem cells has given raise to different sort of problems at the international level. One of the most strict regulation in this field, is this lecture Professors Albin Eser and Hans-Georg Koch analyse the german legal framework in relation with the use of embryos and human embryonic stem cells for scientific purposes.
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The zebrafish embryo model in toxicology and teratology, September 2–3, 2010, Karlsruhe, Germany.
Busch, Wibke; Duis, Karen; Fenske, Martina; Maack, Gerd; Legler, Juliette; Padilla, Stephanie; Strähle, Uwe; Witters, Hilda; Scholz, Stefan
2011-05-01
The use of fish embryos is gaining popularity for research in the area of toxicology and teratology. Particularly embryos of the zebrafish offer an array of different applications ranging from regulatory testing to mechanistic research. For this reason a consortium of two research centres and a company with the support of the COST Action EuFishBiomed has organised the Workshop “The zebrafish embryo model in toxicology and teratology”, in Karlsruhe, Germany, 2nd–3rd September 2010. The workshop aimed at bringing together experts from different areas of toxicology using the (zebra)fish embryo and stimulating networking between scientists and representatives from regulatory bodies, research institutions and industry. Recent findings, presented in various platform presentations in the area of regulatory toxicity, high throughput screening, toxicogenomics, as well as environmental and human risk assessment are highlighted in this meeting report. Furthermore, the constraints and possibilities of the model as discussed at the workshop are described. A follow up-meeting was appreciated by the about 120 participants and is planned for 2012.
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Research on reproduction is essential for captive breeding of endangered carnivore species.
Jewgenow, K; Braun, B C; Dehnhard, M; Zahmel, J; Goeritz, F
2017-04-01
Assisted reproductive technology (ART) has great potential for conservation, but its successful application in captive breeding programmes of endangered species is often compromised by limited background on species' biology. Although carnivore species benefit from knowledge obtained in domesticated species (dogs, cats and ferrets), the focus of research is different. In pet animals, research in reproduction has mainly been focused on ovarian function and contraception, although substantial progress has also been made in the field of in vitro embryo production, transgenic embryos and cloning to aid relevant medical models. In endangered species, however, research should focus on characterizing reproductive traits (cyclicity and seasonality) to unravel species-specific endocrine principles of reproduction physiology. Based on this knowledge, it is crucial to enhance the ability to manipulate female reproductive cycles, especially those of embryo recipients. Furthermore, research conducted on molecular and cellular mechanisms of gamete and embryo development, as well as on cryopreservation protocols of gametes and embryos, is required for successful implementation of advanced ART to wild carnivores. This review will provide a summary on the state of the art with focus on ART contributing to conservation breeding of endangered carnivores. © 2016 Blackwell Verlag GmbH.
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Byk, J C
1997-01-01
The objective of this paper is to stimulate academic debate on embryo and fetal research from the perspective of the drafting of a protocol to the European Convention on Biomedicine. The Steering Committee on Bioethics of the Council of Europe was mandated to draw up such a protocol and for this purpose organised an important symposium on reproductive technologies and embryo research, in Strasbourg from the 16th to the 18th of December 1996. PMID:9055160
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Time-lapse microscopy and image analysis in basic and clinical embryo development research.
Wong, C; Chen, A A; Behr, B; Shen, S
2013-02-01
Mammalian preimplantation embryo development is a complex process in which the exact timing and sequence of events are as essential as the accurate execution of the events themselves. Time-lapse microscopy (TLM) is an ideal tool to study this process since the ability to capture images over time provides a combination of morphological, dynamic and quantitative information about developmental events. Here, we systematically review the application of TLM in basic and clinical embryo research. We identified all relevant preimplantation embryo TLM studies published in English up to May 2012 using PubMed and Google Scholar. We then analysed the technical challenges involved in embryo TLM studies and how these challenges may be overcome with technological innovations. Finally, we reviewed the different types of TLM embryo studies, with a special focus on how TLM can benefit clinical assisted reproduction. Although new parameters predictive of embryo development potential may be discovered and used clinically to potentially increase the success rate of IVF, adopting TLM to routine clinical practice will require innovations in both optics and image analysis. Combined with such innovations, TLM may provide embryologists and clinicians with an important tool for making critical decisions in assisted reproduction. In this review, we perform a literature search of all published early embryo development studies that used time-lapse microscopy (TLM). From the literature, we discuss the benefits of TLM over traditional time-point analysis, as well as the technical difficulties and solutions involved in implementing TLM for embryo studies. We further discuss research that has successfully derived non-invasive markers that may increase the success rate of assisted reproductive technologies, primarily IVF. Most notably, we extend our discussion to highlight important considerations for the practical use of TLM in research and clinical settings. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Optimized ex-ovo culturing of chick embryos to advanced stages of development.
Cloney, Kellie; Franz-Odendaal, Tamara Anne
2015-01-24
Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.
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Should we clone human beings? Cloning as a source of tissue for transplantation.
Savulescu, J
1999-01-01
The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910
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Atomic force microscopy as a tool to study Xenopus laevis embryo
NASA Astrophysics Data System (ADS)
Pukhlyakova, E. A.; Efremov, Yu M.; Bagrov, D. V.; Luchinskaya, N. N.; Kiryukhin, D. O.; Belousov, L. V.; Shaitan, K. V.
2012-02-01
Atomic force microscopy (AFM) has become a powerful tool for imaging biological structures (from single molecules to living cells) and carrying out measurements of their mechanical properties. AFM provides three-dimensional high-resolution images of the studied biological objects in physiological environment. However there are only few AFM investigations of fresh tissue explants and virtually no such research on a whole organism, since most researchers work with cell cultures. In the current work AFM was used to observe the surface of living and fixed embryos and to measure mechanical properties of naive embryos and embryos with overexpression of guanine nucleotide-binding protein G-alpha-13.
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Concerns about eroding the ethical barrier to in vitro eugenics: lessons from the hESC debate.
Pugh, Jonathan
2014-11-01
In his discussion of in vitrogametogenesis, Rob Sparrow claims that an ethical barrier to development of this technology is that many jurisdictions currently prohibit the practice of creating embryos solely for the purpose of research. However, he suggests that this ethical barrier will soon be eroded, in view of the fact that in vitro gametogenesis could serve as a powerful new technology to overcome infertility. In this commentary, I argue that Sparrow is being overly optimistic in his analysis here. I claim that the debate over so-called compromise positions in the human embryonic stem cell debate suggests that the purpose of the research for which a research embryo is created is unlikely to be considered as having any significant bearing on the moral permissibility of the practice for those who oppose it. Even though in vitro gametogenesis could serve as a powerful new technology to overcome infertility, I argue that opponents of the practice of creating embryos solely for research purposes would still view the creation of research embryos that the development of in vitro gametogenesis would require, as being incompatible with affording the embryo proper moral respect. I conclude by suggesting that Sparrow's analysis of the potential benefits of in vitro gametogenesis provides us with further reasons to scrutinise the unconvincing arguments that are often cited in favour of prohibiting the practice of creating embryos solely for research purposes. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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Automation of Technology for Cancer Research.
van der Ent, Wietske; Veneman, Wouter J; Groenewoud, Arwin; Chen, Lanpeng; Tulotta, Claudia; Hogendoorn, Pancras C W; Spaink, Herman P; Snaar-Jagalska, B Ewa
2016-01-01
Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines.To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research.
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[An exploratory study regarding the hypothetical human embryo donation in Chile].
Alvarez Díaz, J A
2007-12-01
To explore opinions of patients who undergone to complex ART towards gamete and embryo donation, as well as the reasons to do it or not. The seat was the Hospital Clínico de la Universidad de Chile. There were interviewed ten participants (seven women, three men), who had undergone at least to one ART, without comprising of donation programs. It was a cross-sectional study of descriptive bioethics, done with ethnographic qualitative methodology with a semistructured interview applying speech analysis to the resulting text. Regarding embryo donation, six participants would accept to donate them, five to fertility therapy and one to research. Regarding the cryopreservation, three participants would always accept it, and three with some restrictions, just one on them would rather to discard instead of donating a cryopreserved embryo. It could be suggested: gamete donation is more commented and generally accepted; embryo donation is a more conflicting and less discussed subject, as much to donate as to accept; cryopreservation is a complex subject, commented but also conflicting, whose acceptance or not, as well as the destiny of the probably cryopreserved embryos, depends on the believes that participants have about the origin of the life, personal ethics, and the religion. It could be possible to say that a hypothesis constructed in this study (to be verified in future quantitative researches) is that embryo donation could take place, for therapy of fertility, and exceptionally to research.
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The Virtual Embryo Project (v-Embryo™)
The v-Embryo is a far reaching new research program at the US EPA to develop a working computer model of a mammalian embryo that can be used to better understand the prenatal risks posed by environmental chemicals and to eventually predict a chemical's potential developmental tox...
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Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.
Beck, Aaron P; Watt, Roland M; Bonner, Jennifer
2014-02-28
The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.
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Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development
Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer
2014-01-01
The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734
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Challenges of primate embryonic stem cell research.
Bavister, Barry D; Wolf, Don P; Brenner, Carol A
2005-01-01
Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.
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Moreno-Moya, Juan Manuel; Ramírez, Leslie; Vilella, Felipe; Martínez, Sebastián; Quiñonero, Alicia; Noguera, Inmaculada; Pellicer, Antonio; Simón, Carlos
2014-03-01
To illustrate an efficient, complete, step-by-step protocol for studying implantation in mice. Video presentation of an animal model for research in reproductive biology. Mouse (Mus musculus). A nonsurgical embryo transfer system very similar to that used for human embryo transfer. The protocols with recipient and donor mice are performed in parallel in the same week. For the donor mice: the first step is ovarian stimulation, followed by ovulation induction and mating; finally, the mice are sacrificed, and the embryos are collected and cultured. For recipient mice: first estrous synchrony is induced, followed by mating with a vasectomized male, visualization of the vaginal plug, and nonsurgical transfer of the embryos. Finally (optionally), the implantation sites can be visualized on day 7.5 of development. (All animal experiments were performed with the approval of the institutional review board.) Implantation is an essential step in human reproduction although, because of technical and ethics considerations, still relatively little is known about human implantation and early development. Conversely, mouse models are well established and can be used for preliminary experiments. However, there are various bottlenecks in the procedure for obtaining and transferring murine embryos, which makes experimentation with this model more difficult. These difficulties include pseudopregnancy, ovarian hyperstimulation, and embryo collection, culture, and transfer. We have proposed a complete, efficient method for obtaining, culturing, and transferring mouse blastocysts that can be easily applied in research. Potential applications include testing new media components that do not affect preimplantation but do affect implantation and early development. The embryo transfer method proposed here has been demonstrated to achieve embryo implantation easier and faster than, and in approximately similar rates as other traditional surgery methods. This workflow is the first set of complete step-by-step instructions available that incorporate advances such as nonsurgical mouse embryo transfer. This will facilitate research into different reproduction events such as embryo development, embryo implantation, or contraception. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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Can artificial techniques supply morally neutral human embryos for research?
Cheshire, William P; Jones, Nancy L
2005-01-01
Amidst controversy surrounding research on human embryos, biotechnology has conceived a substitute in the artificial human embryo. We examine the claim that novel embryos constructed artificially should be exempt from ethical restraints appropriate for research on embryos that come into being through natural processes. Morally relevant differences in intrinsic value depend on the sense in which the entity may be artificial, whether in regard to constituent matter, genetic or cellular form, generative means, or intended purpose. Considering each of these Aristotelian categories from a physicalist viewpoint, technology can achieve only limited degrees of artificiality because redesigned embryos still retain most of their natural features and relationships. From an essentialist viewpoint, the very limits of technology preclude the capability of manipulating the fundamental nature or essence of the individual who, even at the embryonic stage of life, cannot be made to be artificial through and through. A human may possess artificially contributed attributes but cannot be an artificial being. Classification of novel human organisms as artificial, therefore, is insufficient grounds by which to relinquish the principle that human moral status should be recognized for all living beings of human origin. In uncertain cases, at least the possibility of special human moral status should be considered present in organisms that are derived asexually, are developmentally defective, or are otherwise technologically altered.
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Conception and development of the Second Life® Embryo Physics Course.
Gordon, Richard
2013-06-01
The study of embryos with the tools and mindset of physics, started by Wilhelm His in the 1880s, has resumed after a hiatus of a century. The Embryo Physics Course convenes online allowing interested researchers and students, who are scattered around the world, to gather weekly in one place, the virtual world of Second Life®. It attracts people from a wide variety of disciplines and walks of life: applied mathematics, artificial life, bioengineering, biophysics, cancer biology, cellular automata, civil engineering, computer science, embryology, electrical engineering, evolution, finite element methods, history of biology, human genetics, mathematics, molecular developmental biology, molecular biology, nanotechnology, philosophy of biology, phycology, physics, self-reproducing systems, stem cells, tensegrity structures, theoretical biology, and tissue engineering. Now in its fifth year, the Embryo Physics Course provides a focus for research on the central question of how an embryo builds itself.
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Human embryonic stem cell research debates: a Confucian argument
Tsai, D
2005-01-01
The author proposes that the "gradualist" position is more plausible than the other two positions. Confucius's moral principle of jen, which proposes a unique theory of "love of gradation", and the principle of yi, which advocates "due treatment for persons", are then explored. The author then argues that our moral obligations to do good to other living organisms, persons, and our families are different. Putting together the "gradualist" position on the human embryo, and Confucius's theories of "love of gradation" and "due treatment for persons", the author concludes that the early embryo has less ethical significance than the later fetus and adult human. The moral obligation we have toward persons is clearer and stronger than that which we have toward human embryos. Embryo research is justifiable if it brings enormous welfare to human persons that cannot be otherwise achieved. The "love of gradation" requires us, however, to extend love and respect towards other entities according to their different status. We should therefore be very cautious in using human embryos for research, acknowledging the gradualist nature of their moral status. PMID:16269558
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Role of the phagocytes on embryos: some morphological aspects.
Da Silva, José Roberto Machado Cunha
2002-06-15
Phagocytosis in embryos was studied by Elie Metchnikoff more than a century ago and is a pillar of the Phagocytic Theory. Throughout the last three decades phagocytosis in embryos has been studied from different perspectives, which this review describes and analyzes. The following branches were identified: 1) the search for the origin and first identification of well-known adult phagocytes in embryos, including their role after induced injuries; 2) the search for the occurrence of phagocytosis in embryos and its role during their physiological development; and 3) the search for phagocytosis in embryos, as a tool to study identity and self-recognition. It is possible to verify that different cell types are able to undertake phagocytosis, under a variety of different stimuli, and that the nature of what is phagocytosed also varies widely. Although the overwhelming majority of species described among metazoarians are invertebrates, most published articles in this field relate to mammals (particularly mice and humans) and birds (particularly chicks). In order to enrich this field of knowledge, research using a wider variety of vertebrate and invertebrate species should be undertaken. Furthermore, the present knowledge of phagocytosis in embryos needs a revised paradigm capable of embracing all the above-mentioned research trends under a single, more general, biological theory. In this sense, Metchnikoff's Phagocytic Theory, which is based on a broad biological paradigm and is thus capable of dealing with all research trends mentioned herein, should be revisited in order to contribute to this edification. Copyright 2002 Wiley-Liss, Inc.
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Efficient harvesting methods for early-stage snake and turtle embryos.
Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki
2016-04-01
Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.
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Integration of microfluidics in animal in vitro embryo production.
Wheeler, M B; Rubessa, M
2017-04-01
The in vitro production of livestock embryos is central to several areas of animal biotechnology. Further, the use of in vitro embryo manipulation is expanding as new applications emerge. ARTs find direct applications in increasing genetic quality of livestock, producing transgenic animals, cloning, artificial insemination, reducing disease transmission, preserving endangered germplasm, producing chimeric animals for disease research, and treating infertility. Whereas new techniques such as nuclear transfer and intracytoplasmic sperm injection are now commonly used, basic embryo culture procedures remain the limiting step to the development of these techniques. Research over the past 2 decades focusing on improving the culture medium has greatly improved in vitro development of embryos. However, cleavage rates and viability of these embryos is reduced compared with in vivo indicating that present in vitro systems are still not optimal. Furthermore, the methods of handling mammalian oocytes and embryos have changed little in recent decades. While pipetting techniques have served embryology well in the past, advanced handling and manipulation technologies will be required to efficiently implement and commercialize the basic biological advances made in recent years. Microfluidic systems can be used to handle gametes, mature oocytes, culture embryos, and perform other basic procedures in a microenvironment that more closely mimic in vivo conditions. The use of microfluidic technologies to fabricate microscale devices has being investigated to overcome this obstacle. In this review, we summarize the development and testing of microfabricated fluidic systems with feature sizes similar to the diameter of an embryo for in vitro production of pre-implantation mammalian embryos. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Developments in stem cell research and therapeutic cloning: Islamic ethical positions, a review.
Fadel, Hossam E
2012-03-01
Stem cell research is very promising. The use of human embryos has been confronted with objections based on ethical and religious positions. The recent production of reprogrammed adult (induced pluripotent) cells does not - in the opinion of scientists - reduce the need to continue human embryonic stem cell research. So the debate continues. Islam always encouraged scientific research, particularly research directed toward finding cures for human disease. Based on the expectation of potential benefits, Islamic teachings permit and support human embryonic stem cell research. The majority of Muslim scholars also support therapeutic cloning. This permissibility is conditional on the use of supernumerary early pre-embryos which are obtained during infertility treatment in vitro fertilization (IVF) clinics. The early pre-embryos are considered in Islamic jurisprudence as worthy of respect but do not have the full sanctity offered to the embryo after implantation in the uterus and especially after ensoulment. In this paper the Islamic positions regarding human embryonic stem cell research and therapeutic cloning are reviewed in some detail, whereas positions in other religious traditions are mentioned only briefly. The status of human embryonic stem cell research and therapeutic cloning in different countries, including the USA and especially in Muslim countries, is discussed. © 2010 Blackwell Publishing Ltd.
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GLUCOCORTICOID RECEPTOR REGULATION IN THE RAT EMBRYO: A POTENTIAL SITE FOR DEVELOPMENTAL TOXICITY?
Glucocorticoid receptor regulation in the rat embryo: a potential site for developmental toxicity?
Ghosh B, Wood CR, Held GA, Abbott BD, Lau C.
National Research Council, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. -
USDA-ARS?s Scientific Manuscript database
Previous research indicates cows ovulating a small dominant follicle (<_ 12 mm) had lower pregnancy rates than cows ovulating a large follicle (> 12 mm). We hypothesized cows ovulating a small follicle would have delayed embryo development and decreased embryo quality. Objectives of this study wer...
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[The destiny of cryopreserved embryos].
Karpel, L; Achour-Frydman, N; Frydman, R; Flis-Trèves, M
2007-12-01
To know the psychological motivations of couples who keep their embryos so long (five years and more) and do not make a decision about them. We studied 84 couples refrained from making a decision on their cryopreserved embryos for at least five years. They were invited to fill out a questionnaire focusing on three points: the reasons of the indecision, their own representation of the cryopreserved embryos and their choice for the future: donation to another couple, to research, pregnancy or no solution for the moment. Mean (S.D.) women's and men's age were respectively, 38.8 (2.5)- and 41.3 (2.5)-years old. On average, three (1-9) embryos are preserved since 7.5 (5-12) years. Most of couples are parents. Four major reasons explain their attitudes: feeling of being too aged (25%), fear of a multiple pregnancy (45%), disagreement between members of couple (20%) and fear of failure (42.5%). Multiple choices were given to the future of the embryos: 25% wanted a pregnancy, 8% wanted to give them to infertile couples, 20% to research and 27.5% did not find any solution. Twenty percent were hesitating. The representation of those embryos is more symbolic than material. Most of the time, they see them like a potential child, a hope for the future or a brother or sister of their alive children. Those embryos are symbolized. They are a proof of fertility, a hope for another child. So, whatever the legal statement, couples will be in a dilemma because it is never easy for an infertile person to renounce to embryos, and the hope for children.
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Microfluidics for mammalian embryo culture and selection: where do we stand now?
Le Gac, Séverine; Nordhoff, Verena
2017-04-01
The optimization of in-vitro culture conditions and the selection of the embryo(s) with the highest developmental competence are essential components in an ART program. Culture conditions are manifold and they underlie not always evidence-based research but also trends entering the IVF laboratory. At the moment, the idea of using sequential media according to the embryo requirements has been given up in favor of the use of single step media in an uninterrupted manner due to practical issues such as time-lapse incubators. The selection of the best embryo is performed using morphological and, recently, also morphokinetic criteria. In this review, we aim to demonstrate how the ART field may benefit from the use of microfluidic technology, with a particular focus on specific steps, namely the embryo in-vitro culture, embryo scoring and selection, and embryo cryopreservation. We first provide an overview of microfluidic and microfabricated devices, which have been developed for embryo culture, characterization of pre-implantation embryos (or in some instances a combination of both steps) and embryo cryopreservation. Building upon these existing platforms and the various capabilities offered by microfluidics, we discuss how this technology could provide integrated and automated systems, not only for real-time and multi-parametric monitoring of embryo development, but also for performing the entire ART procedure. Although microfluidic technology has been around for a couple of decades already, it has still not made its way into the clinics and IVF laboratories, which we discuss in terms of: (i) a lack of user-friendliness and automation of the microfluidic platforms, (ii) a lack of robust and convincing validation using human embryos and (iii) some psychological threshold for embryologists and practitioners to test and use microfluidic technology. In spite of these limitations, we envision that microfluidics is likely to have a significant impact in the field of ART, for fundamental research in the near future and, in the longer term, for providing a novel generation of clinical tools. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oup.com.
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Whole embryo culture: a "New" technique that enabled decades of mechanistic discoveries.
Ellis-Hutchings, Robert G; Carney, Edward W
2010-08-01
Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come. Copyright 2010 Wiley-Liss, Inc.
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Courts, legislators and human embryo research: lessons from Ireland.
Binchy, William
2011-01-01
When it comes to the matter of human embryo research law plays a crucial role in its development by helping to set the boundaries of what may be done, the sanctions for acting outside those boundaries and the rights and responsibilities of key parties. Nevertheless, the philosophical challenges raised by human embryo research, even with the best will of all concerned, may prove too great for satisfactory resolution through the legal process. Taking as its focus the position of Ireland, this paper explores the distinctive constitutional approach taken on this issue and addresses the difficulty of translating sound philosophy into judicial decrees and the difficulty of establishing expert commissions to make law reform proposals on matters of profound normative controversy. It concludes that the Irish experience does have useful lessons for those in other countries who are concerned with the legal approach to research on human embryos and points to the desirability of a diversity of normative positions in order to enrich the quality of the analysis so as to encourage more informed debate in society.
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Hens, Kristien; Dondorp, Wybo J; Geraedts, Joep P M; de Wert, Guido M
2013-05-01
What do scientists in the field of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) consider to be the future direction of comprehensive embryo testing? Although there are many biological and technical limitations, as well as uncertainties regarding the meaning of genetic variation, comprehensive embryo testing will impact the IVF/PGD practice and a timely ethical reflection is needed. Comprehensive testing using microarrays is currently being introduced in the context of PGD and PGS, and it is to be expected that whole-genome sequencing will also follow. Current ethical and empirical sociological research on embryo testing focuses on PGD as it is practiced now. However, empirical research and systematic reflection regarding the impact of comprehensive techniques for embryo testing is missing. In order to understand the potential of this technology and to be able to adequately foresee its implications, we held an expert panel with seven pioneers in PGD. We conducted an expert panel in October 2011 with seven PGD pioneers from Belgium, The Netherlands, Germany and the UK. Participants expected the use of comprehensive techniques in the context of PGD. However, the introduction of these techniques in embryo testing requires timely ethical reflection as it involves a shift from choosing an embryo without a particular genetic disease (i.e. PGD) or most likely to result in a successful pregnancy (i.e. PGS) to choosing the best embryo based on a much wider set of criteria. Such ethical reflection should take account of current technical and biological limitations and also of current uncertainties with regard to the meaning of genetic variance. However, ethicists should also not be afraid to look into the future. There was a general agreement that embryo testing will be increasingly preceded by comprehensive preconception screening, thus enabling smart combinations of genetic testing. The group was composed of seven participants from four Western Europe countries. As willingness to participate in this study may be connected with expectations regarding the pace and direction of future developments, selection bias cannot be excluded. The introduction of comprehensive screening techniques in embryo testing calls for further ethical reflection that is grounded in empirical work. Specifically, there is a need for studies querying the opinions of infertile couples undergoing IVF/PGS regarding the desirability of embryo screening beyond aneuploidy. This research was supported by the CSG, Centre for Society and Life Sciences (project number: 70.1.074). The authors declare no conflict of interest. N/A.
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Żuradzki, Tomasz
2014-12-01
In this article, I present a new interpretation of the pro-life view on the status of early human embryos. In my understanding, this position is based not on presumptions about the ontological status of embryos and their developmental capabilities but on the specific criteria of rational decisions under uncertainty and on a cautious response to the ambiguous status of embryos. This view, which uses the decision theory model of moral reasoning, promises to reconcile the uncertainty about the ontological status of embryos with the certainty about normative obligations. I will demonstrate that my interpretation of the pro-life view, although seeming to be stronger than the standard one, has limited scope and cannot be used to limit destructive research on human embryos.
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ERIC Educational Resources Information Center
Kass, Leon R.
1979-01-01
This article discusses the ethical issues involved in research on human "in vitro" fertilization, laboratory experimentation with human embryos, and the intrauterine transfer of such embryos for the purpose of assisting human reproduction. (Author/MC)
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Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system
Men, Hongsheng; Zhao, Chongbei; Wei, Si; Murphy, Clifton N.; Spate, Lee; Liu, Yang; Walters, Eric M.; Samuel, Melissa S.; Prather, Randall S.; Critser, John K.
2011-01-01
As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease. PMID:21458047
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Human embryo research and the 14-day rule.
Pera, Martin F
2017-06-01
In many jurisdictions, restrictions prohibit the culture of human embryos beyond 14 days of development. However, recent reports describing the successful maintenance of embryos in vitro to this stage have prompted many in the field to question whether the rule is still appropriate. This Spotlight article looks at the original rationale behind the 14-day rule and its relevance today in light of advances in human embryo culture and in the derivation of embryonic-like structures from human pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.
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NASA Astrophysics Data System (ADS)
Austin, Carlye Anne
This research explores the tissue distribution of silver, as well as adverse effects in pregnant mice and embryos, following prenatal silver nanoparticle (AgNP) exposure. Chapter one of this dissertation is a survey of the published literature on the reproductive and/or developmental toxicity of AgNPs. The available data indicate that AgNPs adversely affect sperm count, viability, and/or motility both in vivo and in vitro, and cause apoptosis and necrosis in spermatogonial stem cells and testicular cells. Additionally, AgNP exposure results in mortality and morphological deformities in fish embryos, but produces no adverse effects in chicken embryos. The current published research on in vivo AgNP exposure to mammals during gestation consists of only three studies, one of which is described in chapter two of this dissertation. These studies report results that may suggest a potential for adverse effects on fetal development (e.g. , decreased viability and fetal and placental weights, increased incidence of developmentally young embryos), but additional research is needed. Chapter two of this dissertation investigates the distribution of silver in tissues of pregnant mice and gestation day (GD) 10 embryos following intravenous maternal exposure to 50 nm AgNPs during early organogenesis (GDs 7-9). Examinations of embryo morphology and histology were also performed. Results demonstrated the presence of silver in all organs and tissues examined. Silver concentrations were highest in liver, spleen, and visceral yolk sac, and lowest in embryos. Groups of mice were also treated with soluble silver nitrate, and the pattern of silver tissue distribution following silver nitrate exposure was similar to that which followed AgNP treatment. Transmission electron microscopy-energy dispersive x-ray spectroscopy (TEM-EDS) confirmed the presence of vesicle-bound nanoparticulate silver in visceral yolk sac endoderm, but not mesoderm. This finding, along with the high silver concentration in visceral yolk sac and low silver concentration in embryos, suggests that visceral yolk sac tissue mitigates AgNP transfer to embryos. No significant treatment-related effects on embryo morphology or tissue histology were detected. Chapter three constitutes an expanded study of silver distribution in pregnant mice and developing embryos, with the addition of 10 nm AgNP treatment groups and examination of fetuses at GD16. Very low concentrations of silver were measured in GD10 embryos and GD16 fetuses following 10 nm AgNP treatment or in GD16 fetuses following 50 nm AgNP treatment. Highest silver concentrations were measured in maternal liver, spleen, and visceral yolk sac. AgNP particle size (10 or 50 nm) did not consistently affect silver tissue distribution. At GD10, 50 nm AgNP treatment resulted in significantly higher silver concentrations than 10 nm AgNP treatment for liver, spleen, and visceral yolk sac only; at GD16, in visceral yolk sac only, 10 nm AgNP treatment resulted in a significantly higher silver concentration than 50 nm AgNP treatment. In liver, spleen, visceral yolk sac, and uterus, absolute silver concentrations following 10 nm AgNP treatment were significantly lower at GD16 compared to GD10; the patterns of silver tissue distribution were similar at both time points. Silver nitrate and 10 nm AgNP treatments resulted in similar tissue concentrations in GD10 tissues with the exception of visceral yolk sac, for which the silver concentration was significantly higher after silver nitrate treatment. Silver distribution patterns were generally similar between 10 nm AgNP and silver nitrate treatments. No histological abnormalities were noted in maternal tissues, extra-embryonic tissues, or embryos. A significantly increased incidence of developmentally young (for gestational age) GD10 embryos was seen following 10 nm AgNP treatment; no significant morphological effects were observed in embryos or maternal tissues. Further research will be needed to fully evaluate potential effects of prenatal AgNP exposure on embryos. (Abstract shortened by UMI.)
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Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny
2016-02-01
Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.
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Development of a large commercial camel embryo transfer program: 20 years of scientific research.
Anouassi, Abdelhaq; Tibary, Ahmed
2013-01-10
Embryo transfer in camels was initiated to respond to demand from the camel industry particularly in the United Arab Emirates since 1990. This paper reviews the research performed in critical areas of reproductive physiology and reproductive function evaluation that constitute a pre-requisite for a successful embryo transfer program. A description of donor and recipient management as well as a retrospective evaluation of calf production in the embryo transfer program at Sweihan, UAE is provided. The program utilized two management systems for donors, with and without ovarian superstimulation. Non-stimulated donors are flushed every 14-15 days with a mean embryo production per year per female of 8.5±3.1 (mean±SEM). Response to gonadotropin stimulation is extremely variable. FSH doses and frequency of administration is often adjusted to a specific female. In the period of 1990-2010, 11,477 embryos were transferred to recipients. Transfers from 1990 to 2009 (n=10,600) resulted in 2858 weaned calves, representing an overall efficiency (% weaned calves/transfer) of 27%. Pregnancy rates at 60 days post transfer varied from 19 to 44%. Pregnancy length following transfer is extremely variable. A major challenge in a large embryo transfer program is finding good quality recipients. Causes of pregnancy and neonatal losses are under study. Copyright © 2012 Elsevier B.V. All rights reserved.
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Current knowledge and future challenges in camelid reproduction.
Tibary, A; Anouassi, A; Sghiri, A; Khatir, H
2007-01-01
Reproductive biology research on camelids offers some interesting peculiarities and challenges to scientists and animal production specialists. The objective of this paper is to review camelid reproduction, advances in reproductive physiology and reproductive biotechnologies in camelids and discuss some areas for further research. In the female, the focus has been on understanding follicular dynamics. This has allowed development of synchronization and superovulation strategies to support embryo transfer technologies which are now commonly used in camels. Some advances have been achieved in preservation of embryos by vitrification. Fertilization, early embryo development and embryo signaling for maternal recognition of pregnancy are still not fully understood. New information on the interaction of the developing embryo and the endometrium may shed some light on this signaling as well as the mechanism of prevention of luteolysis. The presence of a seminal ovulation-inducing factor (OIF) was confirmed in llamas and alpacas. Chronology of oocytes maturation has been described. In vitro production of embryos has been achieved resulting in successful pregnancies and births in the dromedary. These techniques offer a new tool for the production and study of interspecies/cross-species embryos and their effect on pregnancy. Male reproductive function remains poorly studied. Semen preservation and artificial insemination still present many challenges and are not used in production at the moment. The involvement of climatic and nutritional conditions as well as the role of leptin in the regulation of reproductive function need to be evaluated.
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The ethics of cloning and human embryo research.
Saran, Madeleine
2002-01-01
The successful cloning experiments that led to Dolly in 1997 have raised many ethical and policy questions. This paper will focus on cloning research in human embryonic cells. The possible gains of the research will be judged against the moral issues of doing research on a person. This paper concludes that while the embryo has some moral status, its moral status is outweighed by the multitude of benefits that embryonic stem cell research will bring to humanity. Policy suggestions are given for dealing with this new and developing field of stem cell research.
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Why scientific details are important when novel technologies encounter law, politics, and ethics.
Goldstein, Lawrence
2010-01-01
This paper focuses on the issue of what to do if a couple who generates embryos chooses to lawfully, and in their (and my) view, ethnically discard those embryos. Specifically, is it appropriate to use the cells that come from "excess" embryos in medical research instead of discarding them when a couple has ceased trying to have any additional children?
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Sotis, J J
2000-01-01
Since the human embryonic stem cell research involves destruction of human embryos and, therefore, hinges on the fundamental question of the status of the embryo, it is essential to examine this status carefully in order to establish fitting guidelines for research. The US National Institutes of Health has proposed its own guidelines on the matter recently (1999). The document, rooted in current pluralistic perspectives in moral philosophy (or bioethics), is criticised in this paper as morally inadequate. The argumentation of the criticism stems from the theological perspective on human personhood, which focuses on a continuity of personal identity from embryos to adult human beings. An additional concern for the author is the moral complicity in which the research dependent upon the destruction of human embryonic life is sanctioned.
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Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.
George, Robert P
2004-01-01
The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.
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Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface
Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro
2015-01-01
An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID:25768929
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Clarke, M
The British Medical Research Council (MRC), in association with the Royal College of Obstetricians and Gynaecologists, has responded to the Warnock report on human fertilization and embryology by setting up a voluntary authority to license such research. MRC also seeks to define the term "embryo" and to include as research "new and untried treatment." Possible lines of investigation include studies on infertility, genetic and congenital diseases, and contraceptive methods. However, if Enoch Powell's Parliamentary bill should become law, all research on human embryos would be illegal.
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Characterization of embryo-specific genes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sung, Z.R.
1988-01-01
The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that are not expressed in mature tissues -- the embryogenic genes. In order to isolate these genes, we immunized a rabbit with total extracts of somatic embryos of carrot, and enriched the anti-embryo antiserum for antibodies reacting with extracts of carrot somatic embryos. Using this enriched antiserum, we screened a lambda gt11 cDNA library constructed from embryo poly A{sup +} RNA, and isolated 10 cDNA clones that detect embryogenic mRNAs. Monospecific antibodies have beenmore » purified for proteins corresponding to each cDNA sequence. Four cDNA clones were further characterized in terms of the expression of their corresponding mRNA and protein in somatic embryos of carrot. In some cases, comparable gene sequences or products have been detected in somatic and zygotic embryos of other plant species. The characteristics of these 4 cDNA clones -- clone Nos. 8, 59, and 66 -- are described in this report. 3 figs.« less
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Human dignity in international policy documents: a useful criterion for public policy?
de Melo-Martín, Inmaculada
2011-01-01
Current developments in biomedicine are presenting us with difficult ethical decisions and raising complex policy questions about how to regulate these new developments. Particularly vexing for governments have been issues related to human embryo experimentation. Because some of the most promising biomedical developments, such as stem cell research and nuclear somatic transfer, involve such experimentation, several international bodies have drafted documents aimed to provide guidance to governments when developing biomedical science policy. Here I focus on two such documents: the Council of Europe's Convention for the Protection of Human Rights and Dignity of the Human Being and the Additional Protocol to the Convention for the Protection of Human Rights and Dignity of the Human Being. I argue that by using human dignity as a criterion to determine the permissibility of particular human embryo research practices, these documents cannot aid in identifying research that would be contrary to human dignity. Thus, they fail to guide public policy on embryo experimentation. Their use of human dignity as a criterion makes their task of offering guidance unfeasible because the concept as used in these documents is too vague and is applied in contradictory ways. I discuss the main goals of these documents and their claims in relation to human embryo research. I then discuss how they have influenced public policy in several countries. Finally, I show that although these Council of Europe treaties attempt to serve as public policy guides in the area of embryo research, they fail to do so. © 2009 Blackwell Publishing Ltd.
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Influence of embryo handling and transfer method on pig cloning efficiency.
Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong
2015-03-01
The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.
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Smith, Gary D.; Takayama, Shuichi; Swain, Jason E.
2011-01-01
ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future. PMID:21998170
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Pressure for a select committee on human embryo research and genetic engineering.
McKie, David
1985-11-02
By a commanding majority of almost five million votes, this year's Labour Party conference agreed that Labour Members of Parliament should not be permitted to let their consciences decide their votes on "issues affecting the reproductive rights of women." The targets for this censure were the 44 Labour MPs who backed Enoch Powell's bill to outlaw experiments on embryos. Conservative supporters of the Powell bill are countering their defeat by advocating a Parliamentary select committee to examine "matters of human embryo research and human genetic engineering." McKie comments that they are thus shifting emphasis from "fertility," which has public support, to genetic engineering, which generates fear.
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Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S
2014-01-01
In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575
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Ehrich, Kathryn; Farsides, Bobbie; Williams, Clare; Scott, Rosamund
2011-06-01
An Ethics & Policy Workshop was held with 20 invited UK stakeholders to consider whether embryo donors should be able to restrict the future use of human embryonic stem cells (hESCs) created from their embryos. Participants cited tensions between pure altruism and a more reciprocal basis for donation; and between basic research (in which genetic material would never form part of another living being) and treatment applications. Two restriction models were suggested to acknowledge specific ethical issues raised by hESCs' use in research and treatments: (1) a two tier system: hESCs with unrestricted consent could go to the UK Stem Cell Bank; those with restricted consent could be used in individual labs which could guarantee to honour the restrictions, and Bank deposit would not be required. (2) a three category system: restrictions could include (i) basic hESC research; (ii) hESC research and treatment; no gamete derivation (iii) 'unrestricted' hESC research and treatment.
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NASA Astrophysics Data System (ADS)
Zhang, Le; Zhang, Shaoxiang
2017-03-01
A body of research [1-7] has already shown that epigenetic reprogramming plays a critical role in maintaining the normal development of embryos. However, the mechanistic quantitation of the epigenetic interactions between sperms and oocytes and the related impact on embryo development are still not clear [6,7]. In this study, Wang et al., [8] develop a modeling framework that addresses this question by integrating game theory and the latest discoveries of the epigenetic control of embryo development.
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What justifies the United States ban on federal funding for nonreproductive cloning?
Cunningham, Thomas V
2013-11-01
This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.
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Practical applications of new research information in the practice of bovine embryo transfer.
Looney, C R; Pryor, J H
2010-01-01
For more than 40 years, practitioners have sought to improve all aspects of commercial bovine embryo transfer. The development of new technologies for this industry has been substantial, with recent focus on cryopreservation techniques and the in vitro production of embryos fertilised with sexed spermatozoa. When these and other new technologies are developed, the following questions remain: (1) is said technology regulated or does it require licensing; and (2) is it applicable and, if so, is it financially feasible? Computer access to published research and the advancement of data software programs conducive to the industry for data procurement have been essential for helping practitioners answer these questions by enhancing their ability to analyse and apply data. The focus of the present paper is to aid commercial embryo transfer practitioners in determining new technologies that are available and whether they can be implemented effectively, benefiting their programs.
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Sutton-McDowall, Melanie L; Gosnell, Martin; Anwer, Ayad G; White, Melissa; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; Thompson, Jeremy G
2017-10-01
Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
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Addressing the ethical issues raised by synthetic human entities with embryo-like features
Aach, John; Lunshof, Jeantine; Iyer, Eswar; Church, George M
2017-01-01
The "14-day rule" for embryo research stipulates that experiments with intact human embryos must not allow them to develop beyond 14 days or the appearance of the primitive streak. However, recent experiments showing that suitably cultured human pluripotent stem cells can self-organize and recapitulate embryonic features have highlighted difficulties with the 14-day rule and led to calls for its reassessment. Here we argue that these and related experiments raise more foundational issues that cannot be fixed by adjusting the 14-day rule, because the framework underlying the rule cannot adequately describe the ways by which synthetic human entities with embryo-like features (SHEEFs) might develop morally concerning features through altered forms of development. We propose that limits on research with SHEEFs be based as directly as possible on the generation of such features, and recommend that the research and bioethics communities lead a wide-ranging inquiry aimed at mapping out solutions to the ethical problems raised by them. DOI: http://dx.doi.org/10.7554/eLife.20674.001 PMID:28494856
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A step-wise approach for analysis of the mouse embryonic heart using 17.6 Tesla MRI
Gabbay-Benziv, Rinat; Reece, E. Albert; Wang, Fang; Bar-Shir, Amnon; Harman, Chris; Turan, Ozhan M.; Yang, Peixin; Turan, Sifa
2018-01-01
Background The mouse embryo is ideal for studying human cardiac development. However, laboratory discoveries do not easily translate into clinical findings partially because of histological diagnostic techniques that induce artifacts and lack standardization. Aim To present a step-wise approach using 17.6 T MRI, for evaluation of mice embryonic heart and accurate identification of congenital heart defects. Subjects 17.5-embryonic days embryos from low-risk (non-diabetic) and high-risk (diabetic) model dams. Study design Embryos were imaged using 17.6 Tesla MRI. Three-dimensional volumes were analyzed using ImageJ software. Outcome measures Embryonic hearts were evaluated utilizing anatomic landmarks to locate the four-chamber view, the left- and right-outflow tracts, and the arrangement of the great arteries. Inter- and intra-observer agreement were calculated using kappa scores by comparing two researchers’ evaluations independently analyzing all hearts, blinded to the model, on three different, timed occasions. Each evaluated 16 imaging volumes of 16 embryos: 4 embryos from normal dams, and 12 embryos from diabetic dams. Results Inter-observer agreement and reproducibility were 0.779 (95% CI 0.653–0.905) and 0.763 (95% CI 0.605–0.921), respectively. Embryonic hearts were structurally normal in 4/4 and 7/12 embryos from normal and diabetic dams, respectively. Five embryos from diabetic dams had defects: ventricular septal defects (n = 2), transposition of great arteries (n = 2) and Tetralogy of Fallot (n = 1). Both researchers identified all cardiac lesions. Conclusion A step-wise approach for analysis of MRI-derived 3D imaging provides reproducible detailed cardiac evaluation of normal and abnormal mice embryonic hearts. This approach can accurately reveal cardiac structure and, thus, increases the yield of animal model in congenital heart defect research. PMID:27569369
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2012-01-01
Background Previous studies have found that the decision-making process for stored unused frozen embryos involves much emotional burden influenced by socio-cultural factors. This study aims to ascertain how Japanese patients make a decision on the fate of their frozen embryos: whether to continue storage discard or donate to research. Methods Ten Japanese women who continued storage, 5 who discarded and 16 who donated to research were recruited from our infertility clinic. Tape-recorded interviews were transcribed and analyzed for emergent themes. Results A model of patients’ decision-making processes for the fate of frozen embryos was developed, with a common emergent theme, “coming to terms with infertility” resulting in either acceptance or postponing acceptance of their infertility. The model consisted of 5 steps: 1) the embryo-transfer moratorium was sustained, 2) the “Mottainai”- embryo and having another child were considered; 3) cost reasonability was taken into account; 4) partner’s opinion was confirmed to finally decide whether to continue or discontinue storage. Those discontinuing, then contemplated 5): the effect of donation. Great emotional conflict was expressed in the theme, steps 2, 4, and 5. Conclusions Patients’ 5 step decision-making process for the fate of frozen embryos was profoundly affected by various Japanese cultural values and moral standards. At the end of their decision, patients used culturally inherent values and standards to come to terms with their infertility. While there is much philosophical discussion on the moral status of the embryo worldwide, this study, with actual views of patients who own them, will make a significant contribution to empirical ethics from the practical viewpoint. PMID:22607034
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NASA Ames Summer High School Apprenticeship Research Program
1988-09-01
microorganisms, or by producing mutant bacteria and new breeds, interfering with the original destiny of Jovian evolution. And how would we know, many years...Spacelab mission, Spacelab Japan (SL-J), frog embryos will be fertilized in space in the frog embryology unit (FEU--fig. 3). Knowledge of embryology in...limited to animal embryos that develop quickly, which is necessary in a seven-to-ten-day shuttle flight. The rapid dovelopment of frog embryos makes
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In vitro production of small ruminant embryos: late improvements and further research.
de Souza-Fabjan, Joanna Maria Gonçalves; Panneau, Barbara; Duffard, Nicolas; Locatelli, Yann; de Figueiredo, José Ricardo; Freitas, Vicente José de Figueirêdo; Mermillod, Pascal
2014-06-01
Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination. Copyright © 2014 Elsevier Inc. All rights reserved.
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Hook, C Christopher
2010-07-01
The challenges before science and medicine are these: science must explore the natural world as thoroughly as possible, while still honoring, protecting, serving and preserving the subject of its investigations, and the human beings for whom it is a tool; medicine must confront disease and disability as effectively as possible, while also honoring, protecting, and preserving those beings for whom it serves - all of those beings, not just some, or even most, at the potential expense of others. These goals are challenged by embryo-destructive human embryonic stem cell research. The human embryo is a human being as clearly defined by embryology, and as such should be protected by the codes governing human subject research. However, because of the "potential" benefits offered by pluripotent stem cells, coupled with abortion politics and a very poorly regulated infertility industry, United States governmental advisory commissions and the scientific, medical, and political communities have attempted to define away the humanity of the human embryo, with a few notable exceptions. Because infertility treatments in the United States are poorly regulated, there are large numbers of supernumerary embryos in cryopreservation. However, only a tiny portion of these will ever be potentially available for research, and thus are not a realistic source of the cells necessary to provide treatments to the millions who might benefit from proposed stem cell based therapies. Cloning will not be the answer either, given the millions of women who must be exploited to provide sufficient numbers of eggs to generate the cloned cell lines. Moreover, the disposition decisions parents must make for their extra embryos are often agonizing, and not uncommonly change. The use of supernumerary embryos as a source for human embryonic stem cells is unethical, will never be a sufficient source for the medical treatments expected from stem cell research, and is often a source of great distress for the conceiving parents. The United States experience is not a positive model for other countries to emulate.
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Shaping the norms that regulate international commerce of embryos.
Gard, Julie A; Stringfellow, David A
2014-01-01
As various embryo technologies in livestock were developed and evolved to a state of usefulness over the past 40 years, scientists with a specific interest in infectious diseases sought to determine the epidemiologic consequences of movement, especially international movement, of increasing numbers of embryos. Many of the foundational studies in this area were reported in Theriogenology, beginning in the 1970s and especially throughout the 1980s and 1990s. Unquestionably, Theriogenology has been a widely used venue for dissemination of basic information on this subject, which ultimately led to the development of the now universally accepted techniques for certification of embryo health. Today it is well-recognized that movement in commerce of embryos, especially in vivo-derived embryos, is a very low-risk method for exchange of animal germ plasm. This paper chronicles the evolution of strategies for health certification of embryos. An overview is provided of the calculated efforts of practitioners, scientists, and regulators to organize, forge necessary partnerships, stimulate needed research, provide purposeful analysis of the results, and, through these processes, guarantee the universal acceptance of efficient protocols for certifying the health of embryos intended for movement in international commerce. Copyright © 2014 Elsevier Inc. All rights reserved.
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Glassfrog embryos hatch early after parental desertion.
Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle
2014-06-22
Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.
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Glassfrog embryos hatch early after parental desertion
Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle
2014-01-01
Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892
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A Marble Embryo: Meanings of a Portrait from 1900
Hopwood, Nick
2012-01-01
Portraits of scientists use attributes of discovery to construct identities; portraits that include esoteric accessories may fashion identities for these too. A striking example is a marble bust of the anatomist Wilhelm His by the Leipzig sculptor Carl Seffner. Made in 1900, it depicts the founder of modern human embryology looking down at a model embryo in his right hand. This essay reconstructs the design and viewing of this remarkable portrait in order to shed light on private and public relations between scientists, research objects and audiences. The bust came out of a collaboration to model the face of the composer Johann Sebastian Bach and embodies a shared commitment to anatomical exactitude in three dimensions. His’s research agendas and public character explain the contemplative pose and unprecedented embryo model, which he had laboriously constructed from material a midwife supplied. The early contexts of display in the His home and art exhibitions suggest interpretive resources for viewers and hence likely meanings. Seffner’s work remains exceptional, but has affinities to portraits of human embryologists and embryos produced since 1960. Embryo images have acquired such controversial prominence that the model may engage us more strongly now than it did exhibition visitors around 1900. PMID:22606754
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Syed, Saba H; Coughlin, Andrew J; Garcia, Monica D; Wang, Shang; West, Jennifer L; Larin, Kirill V; Larina, Irina V
2015-05-01
The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research.
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Syed, Saba H.; Coughlin, Andrew J.; Garcia, Monica D.; Wang, Shang; West, Jennifer L.; Larin, Kirill V.; Larina, Irina V.
2015-01-01
Abstract. The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research. PMID:25581495
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Julesz, Máté
2015-03-01
Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.
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Bahadur, G; Morrison, M; Machin, L
2010-12-01
Discussion about the ethics of human embryonic stem cell (ESC) research in the UK tends to be dominated by the divisive and potentially intractable issue of the moral status of the embryo. This can have the effect of silencing or marginalizing other concerns, especially in the context of public engagement with science in this field. One such area of potential public concern is the donation of oocytes and embryos to stem cell research. Contemporary research on the views of donors and potential donors about a wide range of biomaterials, from solid organs to gametes and bone marrow, is reviewed and used to illustrate the range and types of ethical concerns articulated by this important group of stakeholders. Attitudes to donation are found to vary according to the type of tissue being donated or collected, the purpose for which donation is being sought and the nature of the recipient of the donation. Pertinently, attitudes towards donating oocytes are found to differ in some respects from donation of embryos or fetal tissue. The implications of these findings for ensuring ethically robust informed consent and publicly acceptable sourcing of human biomaterials for stem cell research are then considered. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo
2016-10-01
Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Hasler, John F
2014-01-01
After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses. Copyright © 2014 Elsevier Inc. All rights reserved.
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Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle
2009-04-01
Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.
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Ice nucleating agents allow embryo freezing without manual seeding.
Teixeira, Magda; Buff, Samuel; Desnos, Hugo; Loiseau, Céline; Bruyère, Pierre; Joly, Thierry; Commin, Loris
2017-12-01
Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy). Copyright © 2017 Elsevier Inc. All rights reserved.
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Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?
Rocha, José C; Passalia, Felipe; Matos, Felipe D; Maserati, Marc P; Alves, Mayra F; Almeida, Tamie G de; Cardoso, Bruna L; Basso, Andrea C; Nogueira, Marcelo F G
2016-08-01
Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.
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Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?
Rocha, José C.; Passalia, Felipe; Matos, Felipe D.; Maserati Jr, Marc P.; Alves, Mayra F.; de Almeida, Tamie G.; Cardoso, Bruna L.; Basso, Andrea C.; Nogueira, Marcelo F. G.
2016-01-01
Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment. PMID:27584609
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Embryo malposition as a potential mechanism for mercury-induced hatching failure in bird eggs
Herring, G.; Ackerman, Joshua T.; Eagles-Smith, Collin A.
2010-01-01
We examined the prevalence of embryo malpositions and deformities in relation to total mercury (THg) and selenium (Se) concentrations in American avocet (Recurvirostra americana), black-necked stilt (Himantopus mexicanus), and Forster's tern (Sterna forsteri) eggs in San Francisco Bay (CA, USA) during 2005 to 2007. Overall, 11% of embryos were malpositioned in eggs ???18 d of age (n=282) and 2% of embryos were deformed in eggs ???13 d of age (n=470). Considering only those eggs that failed to hatch (n=62), malpositions occurred in 24% of eggs ???18 d of age and deformities occurred in 7% of eggs ???13 d of age. The probability of an embryo being malpositioned increased with egg THg concentrations in Forster's terns, but not in avocets or stilts. The probability of embryo deformity was not related to egg THg concentrations in any species. Using a reduced dataset with both Se and THg concentrations measured in eggs (n=87), we found no interaction between Se and THg on the probability of an embryo being malpositioned or deformed. Results of the present study indicate that embryo malpositions were prevalent in waterbird eggs that failed to hatch and the likelihood of an embryo being malpositioned increased with egg THg concentrations in Forster's terns. We hypothesize that malpositioning of avian embryos may be one reason for mercury-related hatching failure that occurs late in incubation, but further research is needed to elucidate this potential mechanism. ?? 2010 SETAC.
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Embryo dignity: the status and juridical protection of the in vitro embryo.
Raposo, Vera Lúcia; Osuna, Eduardo
2007-12-01
In the context of research and reproduction, the status of the human in vitro embryo ranges from being regarded as a person to being regarded as mere property. As regards the first view, one extreme of the spectrum for offering possible legal protection considers that the embryo constitutes a legal person from the moment of conception. For opponents of this view life is a continuum that runs from conception until death. In this process one of the most important stages is birth, the reason being that birth represents the transition between a potential person and a person. The term "embryo" is used to express the being that exists after fusion of the egg and a spermatozoon during the process of embryogenesis until it reaches eight weeks, after which time it is termed a foetus. The embryo's life is recognized as a constitutional value which deserves juridical protection, but not as a person. It only becomes a person with birth.
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EFFECT OF METHYLENE BLUE ON DEVELOPING ZEBRAFISH EMBRYOS Danio rerio
EFFECT OF METHYLENE BLUE ON DEVELOPING ZEBRAFISH EMBRYOS Danio rerioJoan M. Hedge*, Erik Sanders, Kimberly A. Jarema, Deborah Hunter, and Stephanie PadillaIntegrated Systems Toxicology Division, NHEERL, US EPA, Research Triangle Park, NC 27709hedge.joan@epa.govOur laboratory rout...
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The VIRTUAL EMBRYO. A Computational Framework for Developmental Toxicity
EPA’s ‘Virtual Embryo Project’ (v-Embryo™) is focused on the predictive toxicology of children’s health and developmental defects following prenatal exposure to environmental chemicals. The research is motivated by scientific principles in systems biology as a framework for the g...
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The v-Embryo™ is a far reaching new research program at the US EPA to develop a working computer model of a mammalian embryo that can be used to better understand the prenatal risks posed by environmental chemicals and to eventually predict a chemical’s potential developmental to...
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Human embryo cloning prohibited in Hong Kong.
Liu, Athena
2005-12-01
Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.
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Ernst, Stephan
2004-01-01
In the debate on the ethical evaluation of the stem cell research three levels can be differentiated. The first level of argumentation is that of weighing up goods: The possible therapeutical success for thousands of humans seems to justify the consumption of a few embryos. It is show, that this, however, already presupposes - on a second argumentation level - a judgement on the moral status of the embryo. Different moments of time, when human dignity and life protection are ascribe to the embryo, have already been discussed, but in spite of all rationality of the arguments a consensus has not been reached. On this third level of argumentation two fundamental meanings of reality can be differentiated. The empirical-observing and the communicative-participating view. These lead to a different moral evaluation of the embryo. This contribution votes for the priority of the communicative-participating view. It receives addition support by theology and Christian faith.
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A journey through people, places, and projects in equine assisted reproduction.
Hinrichs, Katrin
2016-07-01
A research study is a product of not only a question and its pursuit but also the people, places, and facilities available at the time. My work in equine assisted reproduction has progressed from embryo transfer to oocyte maturation, oocyte transfer, intracytoplasmic sperm injection, embryo biopsy, embryo vitrification, and cloning, as a result of collaborations with an array of remarkable people. This is a summary of some of the stories behind the studies. Copyright © 2016 Elsevier Inc. All rights reserved.
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Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel
2017-11-01
Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P < 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established pregnancies which survived to term (P < 0.05). These results suggest NEAT can identify which blastocysts survive cryopreservation, thus significantly reduce the transfer of non-viable embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.
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Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR.
Blanes, Milena S; Tsoi, Stephen C M; Dyck, Michael K
2016-02-14
Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.
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Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR
Dyck, Michael K.
2016-01-01
Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing. PMID:26966900
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Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik
2012-01-01
Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.
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Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun
2016-10-01
Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Formation of reactive oxygen species (ROS) is an indirect indicator of ETC activity of cells. The ROS levels of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Collectively, rhesus monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.
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[Therapeutic cloning in debate].
de Wert, G
2001-11-03
Human embryos can be conceived by cell nuclear transfer in order to isolate human embryonic stem cells (hES cells) for research into autologous cell therapy (therapeutic cloning). However, this technique broaches the major ethical problem concerning the instrumental use of human preimplantation embryos. From the viewpoint of subsidiarity, it is argued that various potential alternatives for therapeutic cloning should first be investigated further. The question as to whether therapeutic cloning should be allowed only becomes apparent when research with surplus embryos obtained in the course of in-vitro fertilization suggests that usable transplants can be obtained in vitro from hES cells, and when the potential alternatives for therapeutic cloning are either less promising or need more time for development than is currently expected.
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Zhang, Le; Zhang, Shaoxiang
2017-03-01
A body of research [1-7] has already shown that epigenetic reprogramming plays a critical role in maintaining the normal development of embryos. However, the mechanistic quantitation of the epigenetic interactions between sperms and oocytes and the related impact on embryo development are still not clear [6,7]. In this study, Wang et al., [8] develop a modeling framework that addresses this question by integrating game theory and the latest discoveries of the epigenetic control of embryo development. Copyright © 2017 Elsevier B.V. All rights reserved.
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Embryonic stem cell research: one small step for science or one giant leap back for mankind?
Erwin, Consuelo G
2003-01-01
At the forefront of modern debate over the ethical use of biotechnology is embryonic stem cell research. In this poignant analysis of its legitimacy, the author examines the history of this research in light of the United States' policy favoring the protection of human beings over scientific progress. Stem cells, which can divide in culture to create specialized cells in the human body, possess significant potential for curing disease, particularly when taken from human embryos. However, as evidenced by the research atrocities committed under the Nazi regime, the benefits of human research do not come without a cost to humanity. Recognizing this, the later trial of these scientists produced the Nuremberg Code, a set of natural law principles guiding future research on humans that continues to influence health policy decisions. Drawing on this background, the author first considers the appropriate legal status for a human embryo. Biologically, the characteristics of a human embryo place it between human tissue and a constitutional person. Judicially, the answer is even less clear. The author analyzes case law in the context of abortion and in vitro fertilization, as well as classifications by the common law, state legislation, and the National Bioethics Advisory Commission, to conclude that a human embryo should be subject to the same legal and ethical restrictions as any other "human subject." Accordingly, the author argues that embryonic stem cell research violates the ethical standards and purposes of the Nuremberg Code and should be banned by federal legislation. Such a prohibition will fulfill the societal policy choice of protecting potential life and vulnerable human subjects.
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Sleeboom-Faulkner, Margaret
2010-01-01
This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.
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Hwang, Young Sun; Seo, Minseok; Choi, Hee Jung; Kim, Sang Kyung; Kim, Heebal; Han, Jae Yong
2018-04-01
The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos. Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations. The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.
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The Molecular Basis of Development.
ERIC Educational Resources Information Center
Gehring, Walter J.
1985-01-01
Basic architecture of embryo development appears to be under homeobox control (a short stretch of DNA). Outlines research on this genetic segment in fruit flies which led to identification of this control on the embryo's spatial organization. Indicates that molecular mechanisms underlying development may be much more universal than previously…
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Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro.
Zhan, Shaoquan; Cao, Shanbo; Du, Hongzi; Sun, Yuan; Li, Li; Ding, Chenhui; Zheng, Haiyan; Huang, Junjiu
2018-04-21
Hatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear. The effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration. Firstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O 2 concentrations. 5% O 2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O 2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O 2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O 2 , and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O 2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences. Both parental genetic material and O 2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.
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Toxicity of chlorine to zebrafish embryos
Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.
2014-01-01
Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474
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Gaudenzi, Germano; Albertelli, Manuela; Dicitore, Alessandra; Würth, Roberto; Gatto, Federico; Barbieri, Federica; Cotelli, Franco; Florio, Tullio; Ferone, Diego; Persani, Luca; Vitale, Giovanni
2017-08-01
Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) y1 zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.
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In vitro culture of coconut (Cocos nucifera L.) zygotic embryos.
Engelmann, Florent; Malaurie, Bernard; N'Nan, Oulo
2011-01-01
Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Côte d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.
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A Heideggerian defense of therapeutic cloning.
Svenaeus, Fredrik
2007-01-01
Debates about the legitimacy of embryonic stem-cell research have largely focused on the type of ethical value that should be accorded to the human embryo in vitro. In this paper, I try to show that, to broaden the scope of these debates, one needs to articulate an ontology that does not limit itself to biological accounts, but that instead focuses on the embryo's place in a totality of relevance surrounding and guiding a human practice. Instead of attempting to substantiate the ethical value of the embryo exclusively by pointing out that it has potentiality for personhood, one should examine the types of practices in which the embryo occurs and focus on the ends inherent to these practices. With this emphasis on context, it becomes apparent that the embryo's ethical significance can only be understood by elucidating the attitudes that are established towards it in the course of specific activities. The distinction between fertilized embryos and cloned embryos proves to be important in this contextual analysis, since, from the point of view of practice, the two types of embryos appear to belong to different human practices: (assisted) procreation and medical research, respectively. In my arguments, I highlight the concepts of practice, technology, and nature, as they have been analyzed in the phenomenological tradition, particularly by Martin Heidegger. I come to the conclusion that therapeutic cloning should be allowed, provided that it turns out to be a project that benefits medical science in its aim to battle diseases. Important precautions have to be taken, however, in order to safeguard the practice of procreation from becoming perverted by the aims and attitudes of medical science when the two practices intersect. The threat in question needs to be taken seriously, since it concerns the structure and goal of practices which are central to our very self understanding as human beings.
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ENDOTHELIN-A RECEPTOR ANTAGONISM IN EMBRYO CULTURE: WINDOW OF SENSITIVITY AND TIMING OF DEFECT
BRANNEN, K.C., J.M. ROGERS, and E.S. HUNTER, Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina, and Reproductive Toxicology Division, NHEERL, U.S. EPA, Research Triangle Park, North Carolina. Endothelin-A receptor antagonism in embryo culture: w...
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MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE
ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.
MAP Kinase signal transduction is associated with a variety ... -
Farajkhoda, Tahmineh
2017-02-01
Conducting research on the stem cell lines might bring some worthy good to public. Human Stem Cells (hSCs) research has provided opportunities for scientific progresses and new therapies, but some complex ethical matters should be noticed to ensure that stem cell research is carried out in an ethically appropriate manner. The aim of this review article is to discuss the importance of stem cell research, code of ethics for stem cell research in Iran and ethical recommendation. Generation of stem cells for research from human embryo or adult stem cells, saving, maintenance and using of them are the main ethical, legal and jurisprudence concerns in Iran. Concerns regarding human reproduction or human cloning, breach of human dignity, genetic manipulation and probability of tumorogenisity are observed in adult/somatic stem cells. Destruction of embryo to generate stem cell is an important matter in Iran. In this regards, obtaining stem cell from donated frozen embryos through infertility treatment that would be discarded is an acceptable solution in Iran for generation of embryo for research. Ethical, legal, and jurisprudence strategies for using adult/somatic stem cells are determination of ownership of stem cells, trade prohibition of human body, supervision on bio banks and information of Oversight Committee on Stem Cell Research. Recommendations to handle ethical issues for conducting stem cell research are well-designed studies, compliance codes of ethics in biomedical research (specifically codes of ethics on stem cell research, codes of ethics on clinical trials studies and codes of ethics on animals studies), appropriate collaboration with ethics committees and respecting of rights of participants (including both of human and animal rights) in research. In addition, there is a necessity for extending global networks of bioethics for strengthening communications within organizations at both the regional and international level, strengthening legislation systems, designing and establishing convenient collaborative educational courses at different levels.
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Farajkhoda, Tahmineh
2017-01-01
Conducting research on the stem cell lines might bring some worthy good to public. Human Stem Cells (hSCs) research has provided opportunities for scientific progresses and new therapies, but some complex ethical matters should be noticed to ensure that stem cell research is carried out in an ethically appropriate manner. The aim of this review article is to discuss the importance of stem cell research, code of ethics for stem cell research in Iran and ethical recommendation. Generation of stem cells for research from human embryo or adult stem cells, saving, maintenance and using of them are the main ethical, legal and jurisprudence concerns in Iran. Concerns regarding human reproduction or human cloning, breach of human dignity, genetic manipulation and probability of tumorogenisity are observed in adult/somatic stem cells. Destruction of embryo to generate stem cell is an important matter in Iran. In this regards, obtaining stem cell from donated frozen embryos through infertility treatment that would be discarded is an acceptable solution in Iran for generation of embryo for research. Ethical, legal, and jurisprudence strategies for using adult/somatic stem cells are determination of ownership of stem cells, trade prohibition of human body, supervision on bio banks and information of Oversight Committee on Stem Cell Research. Recommendations to handle ethical issues for conducting stem cell research are well-designed studies, compliance codes of ethics in biomedical research (specifically codes of ethics on stem cell research, codes of ethics on clinical trials studies and codes of ethics on animals studies), appropriate collaboration with ethics committees and respecting of rights of participants (including both of human and animal rights) in research. In addition, there is a necessity for extending global networks of bioethics for strengthening communications within organizations at both the regional and international level, strengthening legislation systems, designing and establishing convenient collaborative educational courses at different levels. PMID:28462397
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Wu, Tong; Yu, Gui-Yuan; Xiao, Jia; Yan, Chang; Kurihara, Hiroshi; Li, Yi-Fang; So, Kwok-Fai; He, Rong-Rong
2018-04-19
Efficacy and safety assessments are essential thresholds for drug candidates from preclinical to clinical research. Conventional mammalian in vivo models cannot offer rapid pharmacological and toxicological screening, whereas cell-based or cell-free in vitro systems often lead to inaccurate results because of the lack of physiological environment. Within the avian species, gallus gallus is the first bird to have its genome sequencing. Meantime, chick embryo is an easily operating, relatively transparent and extensively accessible model, whose physiological and pathological alterations can be visualized by egg candler, staining and image technologies. These features facilitate chick embryo as a high-throughput screening platform bridging in vivo and in vitro gaps in the pharmaceutical research. Due to the complicated ingredients and multiple-targets natures of traditional Chinese medicine (TCM), testing the efficacy and safety of TCM by in vitro methods are laborious and inaccurate, while testing in mammalian models consume massive cost and time. As such, the productive living organism chick embryo serves as an ideal biological system for pharmacodynamics studies of TCM. Herein, we comprehensively update recent progresses on the specialty of chick embryo in evaluation of efficacy and toxicity of drugs, with special concerns of TCM. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Embryo donation and understanding of kinship: the impact of law and policy.
Millbank, Jenni; Stuhmcke, Anita; Karpin, Isabel
2017-01-01
What is the impact of law and policy upon the experience of embryo donation for reproductive use? Access to, and experience of, embryo donation are influenced by a number of external factors including laws that impose embryo storage limits, those that frame counselling and approval requirements and allow for, or mandate, donor identity disclosure. To date only three qualitative studies in Australia and New Zealand have been completed on the experience of embryo donation for reproductive purposes, each with a small cohort of interviewees and divergent findings. Embryo donors, recipients, and would-be donors were interviewed between July 2010 and July 2012, with three additional interviews between September 2015 and September 2016, on their experiences of embryo donation. The sampling protocol had the advantage of addressing donation practices across multiple clinical sites under distinct legal frameworks. Participants were recruited from five Australian jurisdictions and across 11 clinical sites. Twenty-six participants were interviewed, comprising: 11 people who had donated embryos for the reproductive use of others (nine individuals and one couple), six recipients of donated embryos (four individuals and one couple) and nine individuals who had attempted to donate, or had a strong desire to donate, but had been prevented from doing so. In total, participants reported on 15 completed donation experiences; of which nine had resulted in offspring to the knowledge of the donor. Donors positively desired donation and did not find the decision difficult. Neither donors nor recipients saw the donation process as akin to adoption . The process and practice of donation varied considerably across different jurisdictions and clinical sites. Because the pool of donors and recipients is small, caution must be exercised over drawing general conclusions. Saturation was not reached on themes of counselling models and future contact. The differences between our findings and those of a previous study are attributable to varied legal and counselling regimes. Therefore, law and policy governing embryo storage limits, counselling protocols and identity disclosure shape the donation experience and how it is described. This research was supported by Discovery Project Grants DP 0986213 and 15010157 from the Australian Research Council and additional funding from UTS: Law. There are no conflicts of interest to declare. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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The presence of concanavalin A and canatoxin in Canavalia ensiformis DC tissue culture.
Sato, A; Barcellos, G B; Riedel, E C; Carneiro, J A; Carlini, C R; Esquibel, M A
1993-02-01
Isolated embryos, cotyledons and embryos plusa fragment of cotyledon from seeds of Canavalia ensiformis (jack bean) were cultured in vitro. Concanavalin A and canatoxin cross-reactive material were detected by double immunodiffusion tests. Canatoxin was detectable until 30 days in cultures of embryos, embryos plus cotyledons and hypocotyls. Concanavalin A was also present in all cultures being detected until 90 days in cultures treated with 6-benzylaminopurine. No concanavalin A was detected in root cultures. Concanavalin A was present in cell suspensions until 45 days of culture; the culture medium contained neither concanavalin A nor canatoxin. Tissue cultures thus can produce Con A and CNTX and will be an important research tool for studying the biosynthesis of such substances.
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Methods for conducting bioassays using embryos and larvae of Pacific herring, Clupea pallasi.
Dinnel, Paul A; Middaugh, Douglas P; Schwarck, Nathan T; Farren, Heather M; Haley, Richard K; Hoover, Richard A; Elphick, James; Tobiason, Karen; Marshall, Randall R
2011-02-01
The rapid decrease of several stocks of Pacific herring, Clupea pallasi, in Puget Sound, Washington, has led to concerns about the effects of industrial and nonpoint source contamination on the embryo and larval stages of this and related forage fish species. To address these concerns, the state of Washington and several industries have funded efforts to develop embryo and larval bioassay protocols that can be used by commercial laboratories for routine effluent testing. This article presents the results of research to develop herring embryo and larval bioassay protocols. Factors evaluated during protocol development included temperature, salinity, dissolved oxygen (DO), light intensity, photoperiod, larval feeding regimes, use of brine and artificial sea salts, gonad sources, collection methods, and egg quality.
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The Effect of Reactive Oxygen Species on Embryo Quality in IVF.
Siristatidis, Charalampos; Vogiatzi, Paraskevi; Varounis, Christos; Askoxylaki, Marily; Chrelias, Charalampos; Papantoniou, Nikolaos
2016-01-01
BACKROUND/AIM: Reactive oxygen species (ROS) are involved in critical biological processes in human reproduction. The aim of this study was to evaluate the association of embryo quality following in vitro fertilization (IVF), with ROS levels in the serum and follicular fluid (FF). Eighty-five participants underwent ovarian stimulation and IVF; ROS levels were measured in blood samples on the day of oocyte retrieval and in the FF from follicular aspirates using enzyme-linked immunosorbent assay. These values were associated with the quality of embryos generated. Univariable zero-inflated Poisson model revealed that ROS levels at both oocyte retrieval and in FF were not associated with the number of grade I, II, III and IV embryos (p>0.05). Age, body mass index, stimulation protocol and smoking status were not associated with the number of embryos of any grade (p>0.05). Neither ROS levels in serum nor in FF are associated with the quality of embryos produced following IVF. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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NASA Astrophysics Data System (ADS)
Cornaglia, Matteo; Mouchiroud, Laurent; Marette, Alexis; Narasimhan, Shreya; Lehnert, Thomas; Jovaisaite, Virginija; Auwerx, Johan; Gijs, Martin A. M.
2015-05-01
Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population’s statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPRmt) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.
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Qin, Zu-Xing; Huang, Gao-Bo; Luo, Jun; Ning, Shu-Fang; Lu, Sheng-Sheng; Lu, Ke-Huan
2012-03-01
Long-tailed macaque-pig interspecies somatic cell nuclear transfer (iSCNT) is beneficial to yield embryonic stem cells from iSCNT embryos with similar genetic background as human, which can be used as materials for medical and basic research. The primary objective of this study was to investigate the effects of concentrations and treatment duration of two histone deacetylase inhibitors-Trichostatin A (TSA) and Valproic acid (VPA) and two different embryo culture media (PZM-3 and HECM-10) on the in vitro development of iSCNT embryos. The results suggested that when PZM-3 was used as the embryo culture medium, the blastocyst rate of 10 nmol/L TSA treatment for 48 h was significantly higher than the control group (22.78% vs 9.86%, P< 0.05). However, neither in PZM-3 nor in HECM-10, 2-10 mmol/L VPA treatment did not increase the in vitro developmental potential of iSCNT embryos. It was concluded that TSA treatment could enhance the in vitro developmental potential of long-tailed macaque-pig iSCNT embryos.
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Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi
2012-11-01
Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.
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In vitro sterilization technique on embryo of black Toraja rice
NASA Astrophysics Data System (ADS)
Haring, F.; Riadi, M.; Rafiuddin; Sjahril, R.; Muchlis, A. R.
2018-05-01
Toraja black rice has a high anthocyanin content, a water-soluble pigments, with antioxidant activity. Toraja black rice has a variety of seeds colour in one panicles such as full black (the outside and inside the rice), medium black (the outside and slightly inside rice) and a little black (only the outside of rice). Embryo culture in vitro is one way to grow plants in sterile conditions. The presence of contamination and the death of the embryo require in vitro embryo culture. The sterilization technique is a very important first step to eliminate contamination and the death of embryos. This research aims to determine the right material composition for sterilization of black rice’s embryo. The experiment was done by growing black rice on half strength MS media with the treatment of three method of sterilization, i.e.: S1 (70% alcohol for 5 minutes, 3% and 2% Chlorox each for 10 minutes,), S2 (70% alcohol for 3 minutes, 2% Clorox for 10 minutes) and S3 (70% alcohol for 3 minutes and 1% Clorox for 15 minutes). The materials used are rice seedlings that have been cut in two and opened the pericarp of paddy grain, leaving a piece of rice that has a complete embryo. The best sterilization for Toraja black rice embryo culture was using the S3 composition. Best germination was seen on the seeds with full and medium black color.
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Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik
2012-01-01
Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development. PMID:23227157
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Du, Wei-Guo; Shine, Richard
2015-02-01
Temperature profoundly affects the rate and trajectory of embryonic development, and thermal extremes can be fatal. In viviparous species, maternal behaviour and physiology can buffer the embryo from thermal fluctuations; but in oviparous animals (like most reptiles and all birds), an embryo is likely to encounter unpredictable periods when incubation temperatures are unfavourable. Thus, we might expect natural selection to have favoured traits that enable embryos to maintain development despite those fluctuations. Our review of recent research identifies three main routes that embryos use in this way. Extreme temperatures (i) can be avoided (e.g. by accelerating hatching, by moving within the egg, by cooling the egg by enhanced rates of evaporation, or by hysteresis in rates of heating versus cooling); (ii) can be tolerated (e.g. by entering diapause, by producing heat-shock proteins, or by changing oxygen use); or (iii) the embryo can adjust its physiology and/or developmental trajectory in ways that reduce the fitness penalties of unfavourable thermal conditions (e.g. by acclimating, by exploiting brief windows of favourable conditions, or by producing the hatchling phenotype best suited to those incubation conditions). Embryos are not simply passive victims of ambient conditions. Like free-living stages of the life cycle, embryos exhibit behavioural and physiological plasticity that enables them to deal with unpredictable abiotic challenges. © 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society.
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OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)
NASA Astrophysics Data System (ADS)
Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.
2016-03-01
The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.
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How should we assess the safety of IVF technologies?
Brison, Daniel R; Roberts, Stephen A; Kimber, Susan J
2013-12-01
Clinical IVF treatment was established over 30 years ago through pioneering work by Edwards and Steptoe and other teams around the world and is now considered routine treatment. However, the pace of scientific and technological advances means that IVF practitioners can now access an increasing array of new and invasive technologies. The examples are many but include: extended embryo culture, development of media to include growth factors, developments in genetic screening, use of time-lapse technology and the advent of vitrification of embryos and oocytes. In parallel, wider scientific and medical advances are raising our awareness of the potential impact of assisted reproduction technology on areas such as embryonic development, gene expression and genomic imprinting and the developmental origins of health and disease. A recently suggested paradigm for assessing new technologies in IVF includes development in animal models such as rodents and large animals, preclinical research with human gametes and embryos donated to research, prospective clinical trials in IVF and, finally, follow-up studies of IVF children. In this paper, we describe efforts to address key areas of this pathway, namely preclinical research using human gametes/embryos and long-term, follow-up studies of the health of assisted reproduction children. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Embryonic death and the creation of human embryonic stem cells.
Landry, Donald W; Zucker, Howard A
2004-11-01
The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.
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Research ethics in Canada: experience of a group operating a human embryo and fetal tissue bank.
Milos, N; Bamforth, S; Bagnall, K
1999-04-01
A Canadian research group is establishing a human embryo and fetal tissue bank. Its purpose is to provide researchers with frozen or fixed tissue specimens for use in protein and gene expression studies. Several legal and ethical issues have arisen, including questions about consent, use of these rare tissues, cost recovery, and profit-making. These issues are discussed here in light of the present lack of legislation in Canada. We make recommendations in these areas, and suggest that the bank's operations could legally fall under the jurisdiction of the Human Tissue Gift Act.
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Embry, Michelle R; Belanger, Scott E; Braunbeck, Thomas A; Galay-Burgos, Malyka; Halder, Marlies; Hinton, David E; Léonard, Marc A; Lillicrap, Adam; Norberg-King, Teresa; Whale, Graham
2010-04-15
Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.
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Berger, C; Boggavarapu, N R; Menezes, J; Lalitkumar, P G L; Gemzell-Danielsson, K
2015-04-01
Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and FGF2 (P = 0.023) were down-regulated with the exposure of UPA, compared with control group. This proof of concept study was conducted with a few human embryos, as their availability was limited. Although the 3D model used for this study is well established and the artificial endometrial luminal epithelium shown to express progesterone regulated markers of endometrial receptivity it is still an in vitro model, lacking all cell types that constitute the receptive endometrium in vivo. This study provides new insights on the mechanism of action of UPA on human embryo implantation, demonstrating that UPA in a dosage used for EC does not affect embryo viability and the implantation process of embryo. Progesterone receptor modulators (PRMs) hold the potential to be attractive estrogen- and gestagen-free contraceptives and thus may be made available to a larger proportion of women globally due to these findings. Swedish Research Council (K2010-54X-14212-09-3) and support provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska University Hospital. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J
2014-10-10
Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development. Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05). Due to the nature of this work, embryo development and implantation was only examined in the mouse. The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation. This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Characterization of embryo-specific genes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Not Available
1989-01-01
The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that is not expressed in mature tissues -- the embryonic genes. In the last two years, using cDNA clones, we have isolated 22 cDNA clones, and characterized the expression pattern of their corresponding RNA. At least 4 cDNA clones detect RNAs of embryonic genes. These cDNA clones detect RNAs expressed in somatic as well as zygotic embryos of carrot. Using the cDNA clones, we screened the genomic library of carrot embryo DNA, and isolatedmore » genomic clones for three genes. The structure and function of two genes DC 8 and DC 59 have been characterized and are reported in this paper.« less
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Alichniewicz, Anna; Michalowska, Monika
2014-02-01
The spectacular progress in assisted reproduction technology that has been witnessed for the past thirty years resulted in emerging new ethical dilemmas as well as the revision of some perennial ones. The paper aims at a feminist approach to oocyte and spare embryo donation for research. First, referring to different concepts of autonomy and informed consent, we discuss whether the decision to donate oocyte/embryo can truly be an autonomous choice of a female patient. Secondly, we argue the commonly adopted language of gift is misleading and that calling for altruism could put female patients at risk of exploitation. Finally, we point out that the presence of gender stereotypes in the procreative area casts doubt whether even a more robust notion of informed consent manages to overcome this risk.
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Reproductive technology: in Britain, the debate after the Warnock Report.
Gillon, Raanan
1987-06-01
Gillon contributes an article on Great Britain to the Hastings Center Report series on reproductive technologies outside the United States. In 1984 the Warnock Committee's report represented the first attempt by a national government to formulate a policy on reproductive issues such as artificial insemination, in vitro fertilization, surrogate mothers, and research on human embryos. Reaction to the Warnock report has focused on its recommendations to ban commercial surrogacy and to allow experimentation on embryos up to 14 days after fertilization. Legislation on surrogacy was passed in 1985, while bills banning embryo research failed in 1986. A 1986 government consultation paper called for discussion of other aspects of the Warnock report, including its recommendation that a statutory licensing authority to regulate reproductive technologies be established. Gillon predicts that no new legislation will be enacted under the present government.
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Ethical and legal controversies in cloning for biomedical research--a South African perspective.
Dhai, A; Moodley, J; McQuoid-Mason, D J; Rodeck, C
2004-11-01
Therapeutic embryonic stem cell research raises a number of ethical and legal issues. The promised benefits are new and important knowledge of human embryological development, gene action, and the production of transplantable tissue and organs that could be effective in reversing or curing currently irreversible disease processes. However, this research involves the deliberate production, use, and ultimate destruction of cloned embryos, hence re-awakening the debate on the moral status of the embryo. Other moral anxieties include the possibility that women (as donors of ova) would be exploited, that this research would land on the slippery slope of reproductive cloning, and that promises made too early could lead to false hope among sick patients. It also raises the question of intellectual and actual property rights in human cell lines and the techniques by which they are produced. Review of legal systems internationally reveals that there is no global consensus on therapeutic embryonic stem cell research. Legal considerations are very much influenced by ethical deliberations on the moral status of the embryo. The South African parliament is promulgating legislation permitting therapeutic cloning, thereby demonstrating a commitment by the state to act in the best interests of patients and of regenerative medicine.
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The embryo research debate in Brazil: from the National Congress to the Federal Supreme Court.
Cesarino, Letícia; Luna, Naara
2011-04-01
New forms of life produced by biomedical research, such as human embryonic stem cells (hESC), have been the object of public debate beyond the scientific fields involved. This article brings to light the case of Brazil, where recently passed federal legislation has authorized research with in vitro human embryos. It focuses on the legislative debate in the Brazilian National Congress between 2003 and 2005 on the Biosafety Bill of Law, which cleared for hESC research a certain share of supernumerary and unviable human embryos frozen in the country's assisted reproduction clinics. The passing of this Bill triggered other public reactions, chiefly a Direct Action of Unconstitutionality in Brazil's Federal Supreme Court. This study adopts an anthropological perspective for describing and analyzing the chief arguments in both debates, in terms of how the notion of 'life' was deployed and negotiated by contending parties. If, on the one hand, the definition of life appeared firmly attached to a conception of both the in vitro embryo and the fetus as a human person, on the other a movement towards breaking down life along utilitarian lines was found when the potential beneficiaries of stem cell therapy came into the equation. In all cases, however, notions of life were negotiated from a hybrid continuum of (biological) facts and (religious, moral and juridical) values, and resonated in different ways with the idea of the individual as privileged mode of constructing personhood in the context of modern nation states.
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Wilson, Robert; McGuire, Christina; Mohun, Timothy
2016-01-01
The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate. PMID:26519470
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The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages
Motaln, Helena; Turnšek, Tamara Lah
2015-01-01
Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma. PMID:26109632
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Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger
2016-01-01
Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP. PMID:27385131
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Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger
2016-07-07
Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.
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Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.
Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P
2013-04-01
Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.
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Research on Human Embryos and Reproductive Materials: Revisiting Canadian Law and Policy
Zarzeczny, Amy; Baltz, Jay; Bedford, Patrick; Du, Jenny; Hyun, Insoo; Jaafar, Yasmeen; Jurisicova, Andrea; Kleiderman, Erika; Koukio, Yonida; Knoppers, Bartha Maria; Leader, Arthur; Master, Zubin; Nguyen, Minh Thu; Noohi, Forough; Ravitsky, Vardit; Toews, Maeghan
2018-01-01
Research involving human embryos and reproductive materials, including certain forms of stem cell and genetic research, is a fast-moving area of science with demonstrated clinical relevance. Canada's current governance framework for this field of research urgently requires review and reconsideration in view of emerging applications. Based on a workshop involving ethics, legal, policy, scientific and clinical experts, we present a series of recommendations with the goal of informing and supporting health policy and decision-making regarding the governance of the field. With a pragmatic and principled governance approach, Canada can continue its global leadership in this field, as well as advance the long-term health and well-being of Canadians. PMID:29595433
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First live offspring born in superovulated sika deer (Cervus nippon) after embryo vitrification.
Wang, L; Zhou, G B; Shi, W Q; Shi, J M; Tian, X Z; Gao, C; Zhang, L; Zhu, S E; Zhang, T T; Zeng, S M; Liu, G S
2012-10-15
The rapid growth in sika deer (Cervus nippon) farming and interest in their conservation is an impetus for development of embryo transfer (ET) procedures. However, a paucity of research has prevented widespread application of ET in this species. The objective of the present study was to establish a multiple ovulation and ET procedure with both fresh and vitrified embryos in sika deer. Multiparous weaned hinds (N = 18) were used as embryo donors during the reproductive season of 2008 at a local breeding farm in China. Estrus was synchronized in donors and recipients (N = 38) by inserting a controlled internal drug release for 12 days (insertion = Day 0). Superovulation was induced with a total of 320 mg of NIH-FSH-P1 (Folltropin-V; Bioniche, Belleville, ON, Canada) given as 40 mg im every 12 h from the afternoon of Day 9 to the morning of Day 13. After estrus was detected, donors were artificially inseminated using a transcervical technique. The embryo recovery rate was 76.8% (63/82), including 1.6% (1/63), 77.8% (49/63), and 1.6% (1/63) blastocysts, morula, and eight-cell embryos, respectively. After transfer of fresh and vitrified embryos, pregnancy rates were 85.7% and 61.6% and birth rates were 64.3% and 53.9% (P > 0.05). In conclusion, we developed a satisfactory multiple ovulation and ET procedure in farmed sika deer using vitrified embryos. Copyright © 2012 Elsevier Inc. All rights reserved.
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Donoughe, Seth; Kim, Chiyoung; Extavour, Cassandra G
2018-04-30
High-throughput live-imaging of embryos is an essential technique in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device to easily mount dozens of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be fabricated by cutting pieces from a sheet of acrylic using a laser cutter or by making them with a 3D printer. We demonstrate how to design a custom mold and use it to live-image dozens of embryos at a time. We include descriptions, schematics, and design files for 13 additional molds for nine animal species, including most major traditional laboratory models and a number of emerging model systems. Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein. © 2018. Published by The Company of Biologists Ltd.
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Yu, Hui-Ting; Yang, Qing; Sun, Xiao-Xi; Chen, Guo-Wu; Qian, Nai-Si; Cai, Ren-Zhi; Guo, Han-Bing; Wang, Chun-Fang
2018-05-01
To evaluate the impact of assisted reproductive technology (ART) on the offspring of Chinese population. Retrospective, data-linkage cohort. Not applicable. Live births resulting from ART or natural conception. None. Birth defects coded according to ICD-10. Births after ART were more likely to be female and multiple births, especially after intracytoplasmic sperm injection (ICSI). ART was associated with a significantly increased risk of birth defects, especially, among singleton births, a significantly increased risk in fresh-embryo cycles after in vitro fertilization (IVF) and frozen-embryo cycles after ICSI. Associations between ART and multiple defects, between ART and gastrointestinal malformation, genital organs malformation, and musculoskeletal malformation among singleton births, and between ART and cardiac septa malformation among multiple births were observed. This study suggests that ART increases the risk of birth defects. Subgroup analyses indicate higher risk for both fresh and frozen embryos, although nonsignificantly for frozen embryos after IVF and for fresh embryos were presented with low power. Larger sample size research is needed to clarify effects from fresh- or frozen-embryo cycles after IVF and ICSI. Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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Biosensors for detecting stress in developing embryos
NASA Astrophysics Data System (ADS)
Purdey, Malcolm S.; Saini, Avishkar; McLennan, Hanna J.; Pullen, Benjamin J.; Schartner, Erik P.; Sutton-McDowall, Melanie L.; Thompson, Jeremy G.; Monro, Tanya M.; Nicholls, Stephen J.; Abell, Andrew D.
2016-12-01
Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.
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Developmental imaging: the avian embryo hatches to the challenge.
Kulesa, Paul M; McKinney, Mary C; McLennan, Rebecca
2013-06-01
The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Copyright © 2013 Wiley Periodicals, Inc.
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Time lapse imaging: is it time to incorporate this technology into routine clinical practice?
Bhide, Priya; Maheshwari, Abha; Cutting, Rachel; Seenan, Susan; Patel, Anita; Khan, Khalid; Homburg, Roy
2017-06-01
Time-lapse imaging (TLI) systems for embryo incubation, assessment and selection are a novel technology available to in vitro fertilization (IVF) clinics. However, there is uncertainty about their clinical and cost-effectiveness and insufficient good quality evidence to warrant their routine use. Despite this, enthusiastic commercial marketing and slipping clinical equipoise have led to the widespread hasty introduction of this technology into practice, often at a considerable expense to the patient. We have reviewed the published literature and aim to summarize the strengths, weaknesses, opportunities and threats of these systems. These specialized incubators provide undisturbed embryo culture conditions and, by almost continuous monitoring of embryo development, generate morphokinetic parameters to aid embryo selection. They are thus hypothesized to improve outcomes following IVF. Although literature reports improved reproductive outcomes, these outcomes are largely surrogate and there is a paucity of studies reporting live births. The use of time lapse systems may reduce early pregnancy loss, increase elective single embryo transfers and limit multiple pregnancies through better embryo selection. However, the quality of the studies and hence the evidence so far, is low to moderate quality. We recommend further research producing robust high-quality evidence for and against the use of these systems.
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Hammond, Elizabeth R; Shelling, Andrew N; Cree, Lynsey M
2016-08-01
The ability to screen embryos for aneuploidy or inherited disorders in a minimally invasive manner may represent a major advancement for the future of embryo viability assessment. Recent studies have demonstrated that both blastocoele fluid and embryo culture medium contain genetic material, which can be isolated and subjected to downstream genetic analysis. The blastocoele fluid may represent an alternative source of nuclear DNA for aneuploidy testing, although the degree to which the isolated genetic material is solely representative of the developing embryo is currently unclear. In addition to nuclear DNA, mitochondrial DNA (mtDNA) can be detected in the embryo culture medium. Currently, the origin of this nuclear and mtDNA has not been fully evaluated and there are several potential sources of contamination that may contribute to the genetic material detected in the culture medium. There is however evidence that the mtDNA content of the culture medium is related to embryo fragmentation levels and its presence is predictive of blastulation, indicating that embryo development may influence the levels of genetic material detected. If the levels of genetic material are strongly related to aspects of embryo quality, then this may be a novel biomarker of embryo viability. If the genetic material does have an embryo origin, the mechanisms by which DNA may be released into the blastocoele fluid and embryo culture medium are unknown, although apoptosis may play a role. While the presence of this genetic material is an exciting discovery, the DNA in the blastocoele fluid and embryo culture medium appears to be of low yield and integrity, which makes it challenging to study. Further research aimed at assessing the methodologies used for both isolating and analysing this genetic material, as well as tracing its origin, are needed in order to evaluate its potential for clinical use. Should such methodologies prove to be routinely successful and the DNA recovered demonstrated to be embryonic in origin, then they may be used in a minimally invasive and less technical methodology for genetic analysis and embryo viability assessment than those currently available. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei
2014-01-01
ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661
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Embryo donation and understanding of kinship: the impact of law and policy
Millbank, Jenni; Stuhmcke, Anita; Karpin, Isabel
2017-01-01
STUDY QUESTION What is the impact of law and policy upon the experience of embryo donation for reproductive use? SUMMARY ANSWER Access to, and experience of, embryo donation are influenced by a number of external factors including laws that impose embryo storage limits, those that frame counselling and approval requirements and allow for, or mandate, donor identity disclosure. WHAT IS KNOWN ALREADY To date only three qualitative studies in Australia and New Zealand have been completed on the experience of embryo donation for reproductive purposes, each with a small cohort of interviewees and divergent findings. STUDY DESIGN, SIZE, DURATION Embryo donors, recipients, and would-be donors were interviewed between July 2010 and July 2012, with three additional interviews between September 2015 and September 2016, on their experiences of embryo donation. The sampling protocol had the advantage of addressing donation practices across multiple clinical sites under distinct legal frameworks. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Participants were recruited from five Australian jurisdictions and across 11 clinical sites. Twenty-six participants were interviewed, comprising: 11 people who had donated embryos for the reproductive use of others (nine individuals and one couple), six recipients of donated embryos (four individuals and one couple) and nine individuals who had attempted to donate, or had a strong desire to donate, but had been prevented from doing so. In total, participants reported on 15 completed donation experiences; of which nine had resulted in offspring to the knowledge of the donor. MAIN RESULTS AND THE ROLE OF CHANCE Donors positively desired donation and did not find the decision difficult. Neither donors nor recipients saw the donation process as akin to adoption . The process and practice of donation varied considerably across different jurisdictions and clinical sites. LIMITATIONS, REASONS FOR CAUTION Because the pool of donors and recipients is small, caution must be exercised over drawing general conclusions. Saturation was not reached on themes of counselling models and future contact. WIDER IMPLICATIONS OF THE FINDINGS The differences between our findings and those of a previous study are attributable to varied legal and counselling regimes. Therefore, law and policy governing embryo storage limits, counselling protocols and identity disclosure shape the donation experience and how it is described. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by Discovery Project Grants DP 0986213 and 15010157 from the Australian Research Council and additional funding from UTS: Law. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER Not applicable. PMID:27927845
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Safari, Somayyeh; Khalili, Mohammad Ali; Barekati, Zeinab; Halvaei, Iman; Anvari, Morteza; Nottola, Stefania A
2017-09-01
The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n=32), sham (n=32) and control (n=32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P=0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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Liang, X W; Lu, Y Q; Chen, M T; Zhang, X F; Lu, S S; Zhang, M; Pang, C Y; Huang, F X; Lu, K H
2008-04-15
The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.
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Assisted reproductive technologies in rhesus macaques
Wolf, Don P
2004-01-01
The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized. PMID:15200674
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D'Angeli, S; Falasca, G; Matteucci, M; Altamura, M M
2013-01-01
FAD2 and FAD7 desaturases are involved in cold acclimation of olive (Olea europaea) mesocarp. There is no research information available on cold acclimation of seeds during mesocarp cold acclimation or on differences in the cold response of the seed coat and embryo. How FAD2 and FAD7 affect seed coat and embryo cold responses is unknown. Osmotin positively affects cold acclimation in olive tree vegetative organs, but its role in the seeds requires investigation. OeFAD2.1, OeFAD2.2, OeFAD7 and Oeosmotin were investigated before and after mesocarp acclimation by transcriptomic, lipidomic and immunolabelling analyses, and cytosolic calcium concentration ([Ca(2+)](cyt)) signalling, F-actin changes and seed development were investigated by epifluorescence/histological analyses. Transient [Ca(2+)](cyt) rises and F-actin disassembly were found in cold-shocked protoplasts from the seed coat, but not from the embryo. The thickness of the outer endosperm cuticle increased during drupe exposure to lowering of temperature, whereas the embryo protoderm always lacked cuticle. OeFAD2 transcription increased in both the embryo and seed coat in the cold-acclimated drupe, but linoleic acid (i.e. the product of FAD2 activity) increased solely in the seed coat. Osmotin was immunodetected in the seed coat and endosperm of the cold-acclimated drupe, and not in the embryo. The results show cold responsiveness in the seed coat and cold tolerance in the embryo. We propose a role for the seed coat in maintaining embryo cold tolerance by increasing endosperm cutinization through FAD2 and osmotin activities. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.
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Vallverdú-Queralt, Anna; Boix, Nuria; Piqué, Ester; Gómez-Catalan, Jesús; Medina-Remon, Alexander; Sasot, Gemma; Mercader-Martí, Mercè; Llobet, Juan M; Lamuela-Raventos, Rosa M
2015-08-15
The zebrafish embryo is a highly interesting biological model with applications in different scientific fields, such as biomedicine, pharmacology and toxicology. In this study, we used liquid chromatography/electrospray ionisation-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC/ESI-LTQ-Orbitrap-MS) to identify the polyphenol compounds in a red wine extract and zebrafish embryos. Phenolic compounds and anthocyanin metabolites were determined in zebrafish embryos previously exposed to the red wine extract. Compounds were identified by injection in a high-resolution system (LTQ-Orbitrap) using accurate mass measurements in MS, MS(2) and MS(3) modes. To our knowledge, this research constitutes the first comprehensive identification of phenolic compounds in zebrafish by HPLC coupled to high-resolution mass spectrometry. Copyright © 2015 Elsevier Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Licata, Nicholas; Clark, Aaron
2014-03-01
Aquatic microorganisms face a variety of challenges in the course of development. One central challenge is efficiently regulating the export of toxic molecules inside the developing embryo. The strategies employed should be robust with respect to the variable ocean environment and limit the chances that exported toxins are reabsorbed. In this talk we consider the first-passage problem for the uptake of exported toxins by a spherical embryo. A perturbative solution of the advection-diffusion equation reveals that a concentration boundary layer forms in the vicinity of the embryo, and that fluid flow enhances the effectiveness of toxin export. We highlight connections between the model results and recent experiments on the development of sea urchin embryos. We acknowledge financial support from the University of Michigan-Dearobrn CASL Faculty Summer Research Grant.
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From experimental imaging techniques to virtual embryology.
Weninger, Wolfgang J; Tassy, Olivier; Darras, Sébastien; Geyer, Stefan H; Thieffry, Denis
2004-01-01
Modern embryology increasingly relies on descriptive and functional three dimensional (3D) and four dimensional (4D) analysis of physically, optically, or virtually sectioned specimens. To cope with the technical requirements, new methods for high detailed in vivo imaging, as well as the generation of high resolution digital volume data sets for the accurate visualisation of transgene activity and gene product presence, in the context of embryo morphology, were recently developed and are under construction. These methods profoundly change the scientific applicability, appearance and style of modern embryo representations. In this paper, we present an overview of the emerging techniques to create, visualise and administrate embryo representations (databases, digital data sets, 3-4D embryo reconstructions, models, etc.), and discuss the implications of these new methods on the work of modern embryologists, including, research, teaching, the selection of specific model organisms, and potential collaborators.
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Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns.
Xiong, Xian-rong; Lan, Dao-liang; Li, Jian; Wang, Yong; Zhong, Jin-cheng
2015-02-01
Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear-cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine-yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and 'corrected' the gene expression patterns of yak iSCNT embryos.
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Borek, Sławomir; Paluch-Lubawa, Ewelina; Pukacka, Stanisława; Pietrowska-Borek, Małgorzata; Ratajczak, Lech
2017-02-01
The research was conducted on embryo axes of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), which were isolated from imbibed seeds and cultured for 96h in vitro under different conditions of carbon and nitrogen nutrition. Isolated embryo axes were fed with 60mM sucrose or were sugar-starved. The effect of 35mM asparagine (a central amino acid in the metabolism of germinating lupin seeds) and 35mM nitrate (used as an inorganic kind of nitrogen) on growth, storage lipid breakdown and autophagy was investigated. The sugar-starved isolated embryo axes contained more total lipid than axes fed with sucrose, and the content of this storage compound was even higher in sugar-starved isolated embryo axes fed with asparagine. Ultrastructural observations showed that asparagine significantly slowed down decomposition of autophagic bodies, and this allowed detailed analysis of their content. We found peroxisomes inside autophagic bodies in cells of sugar-starved Andean lupin embryo axes fed with asparagine, which led us to conclude that peroxisomes may be degraded during autophagy in sugar-starved isolated lupin embryo axes. One reason for the slower degradation of autophagic bodies was the markedly lower lipolytic activity in axes fed with asparagine. Copyright © 2016 The Author(s). Published by Elsevier GmbH.. All rights reserved.
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Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.
Glenn, David R J; McClure, Neil; Cosby, S Louise; Stevenson, Michael; Lewis, Sheena E M
2009-03-01
To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. Academic research environment. Male and female CBAB(6) mice. Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. Fertilization rates and numbers of embryos were evaluated after treatment. Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.
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Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian
2012-03-01
The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.
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Human embryonic stem cell lines: socio-legal concerns and therapeutic promise.
McLaren, Anne
2002-10-01
Stem cell lines would be very valuable for the repair of diseased or damaged organs. Stem cells derived from adult tissues raise few ethical problems, and would not be rejected if derived from the patient. They show considerable plasticity and might be appropriate for some clinical conditions, but they tend not to grow well in culture. Stem cells derived from the early human embryo proliferate indefinitely in culture and can give rise to many different tissues, but their derivation requires destruction of the embryo, which is not ethically acceptable in some countries. Other countries allow strictly regulated destructive research on human embryos, usually those that have been produced for infertile couples in infertility clinics. Embryos that are no longer required for the couple's own reproductive project could be donated for research rather than just discarded. Different approaches are being developed to avoid immunological rejection of embryonic stem cells used for therapy. Derivation of embryonic stem cell lines by somatic cell nuclear transfer ('cloning') from the patients themselves might be one possible approach, but is unlikely to be used in routine clinical practice if more cost-effective methods are available.
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Moutel, Grégoire; Gregg, Edna; Meningaud, Jean Paul; Hervé, Christian
2002-01-01
1985 witnessed the first transfers of frozen embryos resulting in live births in France. Since this time the number of embryos obtained by in vitro fertilisation (IVF) has increased each year. In 1999 each IVF attempt obtains, on average, 4.5 embryos that can be successfully implanted. In this paper we consider only those couples who have successfully obtained embryos (either by ICSI or traditional IVF techniques). The aims of the study are: To show how developments in embryo production and conservation have influenced the number of embryos stored. To address the socio-medical and ethical issues raised and to provide practitioners with some thoughts for reflection when consulting with couples based on the study findings To discuss the results of our findings in the light of those ethical questions raised by the imminent revision of the Laws of Bioethics. In the first instance we did a retrospective analysis of quantitative data that 17 storage centres had collected over a period of 5 years. This period was marked by the implementation in 1994 of Laws described as Bioethics' Laws in France. During a second period we conducted a qualitative study regarding the fate of stored embryos. In order to do this, we began an analysis of the "status" of embryos and the decisions of those couples whose embryos were still in storage. For this a questionnaire was used. The number of embryos that remain in storage in the 17 storage centres has increased reaching a total of 17,592 embryos involving 3,888 couples. The results show a consistent and persistent increase in the number of embryos stored before and after 1994. The qualitative study shows that: 51% of couples with embryos in storage can no longer be found, 23.6% request a continuance of storage, 12% would accept donating their embryos to medical research, 9.1% would wish for other couples to take eventual ownership of the embryo in 7.2% of cases the storage centre has can provide no information concerning the continuing of storage of such embryos. The Bioethics Laws have therefore not succeeded in limiting the inflation in the number of embryos stored despite that the fact that this was one of the major concerns of those involved in formulating the laws and the medical professionals involved. Our study shows that the guidelines provided by these Laws remain ambiguous and that the objectives defined therein are thus difficult to achieve. Our study highlights the impact of such developments on the possible eventual "destiny" of such embryos, the behaviour of and the advice provided to couples, and the management practice of the storage centres involved. We present these results in the light of the laws established in 1994 in order to define not only the benefits but also the potential limitations of such legislation. No guidelines were laid down regarding the future of embryos belonging to a couple that no longer wishes to embark upon a "parental project". As a result it is not possible to terminate the conservation of such embryos which therefore remain in storage awaiting the revision of the current laws. The major objectives of our study assess the impact of the Laws and their impact on practice and to contribute to the debate which has yet to take place in the social and political arena.
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Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj
2016-06-01
The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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NASA Astrophysics Data System (ADS)
Frederick, Rebecca A.; Hsieh, Sheng-Jen; Palomares, Benjamin Giron
2012-06-01
Monitoring development of chicken embryos allows determination of when an egg is not developing and when eggs are close to hatching for more efficient production. Research has been conducted on the effects of temperature fluctuations and light exposure on embryo development; similarities between chicken and mammal embryos; and the use of MRI, tomography, and ultrasound to view specific areas and processes within the embryo. However, there has been little exploration of the use of infrared imaging as a non-destructive method for analyzing and predicting embryonic development. In this study, we built an automated loading system for image acquisition. Pilot experiments were conducted to determine the overall scanning time and scanning frequency. A batch of fertilized eggs was scanned each day as the embryos continued to grow. The captured images were analyzed and categorized into three stages: Stage 1 (days 1 to 7), Stage 2 (days 8 to 14), and Stage 3 (days 15 to 21). The temperature data abstracted from the captured images were divided into two groups. Group 1, consisting of two-thirds of the data, was used to construct a model. Group 2, consisting of one-third of the data, was used to evaluate the predictive accuracy of the model. A three-layer artificial neural network model was developed to predict embryo development stage given a temperature profile. Results suggest that the neural network model is sufficient to predict embryo development stage with good accuracy of 75%. Accuracy can likely be improved if more data sets for each development stage are available.
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Ethical and legal issues in therapeutic cloning and the study of stem cells.
Lisker, Rubén
2003-01-01
Therapeutic cloning is a new technology with great medical potential, particularly in the area of transplantation medicine. It involves the transfer of the nucleus of a patient's cell into an enucleated donor oocyte for the purpose of generating an embryo. This embryo is allowed to grow until the blastocyst stage, at which time stem cells can be obtained and differentiated into the tissue needed. Stem cells can also be obtained from adult tissues, as they seem to have sufficient plasticity to use for the stated purpose. A literature review was performed, and it is clear that the main controversy regarding the use of stem cells is the origin. Few people would object to their use if obtained from adult tissues; however, many oppose harvesting them from embryos in the blastocyst stage regardless of whether 1) they are obtained from surplus embryos donated by couples after assisted reproductive techniques, or 2) they are specially manufactured for research purposes. The central reason is the consideration that embryos should be treated as full humans from the moment of fertilization. This argument is also at the bottom of an older discussion regarding the validity of abortion. There is no consensus at the present time in this regard, and it is unlikely one will be forthcoming in the future. Arguments on both sides of the issue are presented, but emphasis is made on the need for using this technology for research purposes because of its potential value as a therapeutic tool.
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Jugulam, M; Ziauddin, Asma; So, Kenny K Y; Chen, Shu; Hall, J Christopher
2015-01-01
Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.
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Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa
Liu, Wenting; Liang, Zongsuo; Wang, Xinhua; Sibbald, Susan; Hunter, David; Tian, Lining
2013-01-01
Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species. PMID:23690914
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Pandian, Z; Bhattacharya, S; Ozturk, O; Serour, G I; Templeton, A
2004-10-18
The traditional reliance on the transfer of multiple embryos during in vitro fertilisation (IVF) in order to maximise the chance of pregnancy, has resulted in increasing rates of multiple pregnancies. Women undergoing IVF had a 20 - fold increased risk of twins and 400 - fold increased risk of higher order pregnancies (Martin 1998). The maternal and perinatal morbidity and mortality as well as national health service costs associated with multiple pregnancies is significantly high in comparison with singleton births (Luke 1992; Callahan 1994; Goldfarb 1996). Single embryo transfer is now being considered as an effective means of reducing this iatrogenic complication. This systematic review evaluates the effectiveness of elective two embryo transfer in comparison with single and more than two embryo transfer following IVF and ICSI (intra cytoplasmic sperm injection) treatment. The aim of this review is to determine, whether in couples who undergo IVF/ICSI: (1) the elective transfer of two embryos improves the probability of livebirth compared with: (a) Single embryo transfer, (b) Three embryo transfer or (c) Four embryo transfer.(2) the elective transfer of three embryos improves the probability of livebirth compared with: (a) Single embryo transfer, or (b) Four embryo transfer, We searched the Cochrane Menstrual Disorders and Subfertility Group's trials register (searched June 2003), the Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 4, 2003), MEDLINE (1970 to 2003), EMBASE (1985 to 2003) and reference lists of articles. We also handsearched relevant conference proceedings and contacted researchers in the field. Only randomised controlled trials were included. Two reviewers independently assessed eligibility and quality of trials. We found no studies that compared a policy of transferring multiple embryos on one cycle versus a policy of cryo- preservation and transfer of a single embryo over multiple cycles. We also found no trials comparing transfer of two versus three embryos. Three small, poorly reported trials compared transfer of two versus one embryo in a single cycle, and one small, poorly reported trial compared transfer of two versus four embryos in a single cycle. The clinical pregnancy rate per woman/couple associated with two embryo transfer was significantly higher compared to single embryo transfer (OR 2.08, 95% CI 1.24 to 3.50; test for overall effect p = 0.006). The live birth rate per woman/couple associated with two embryo transfer was also significantly higher than that associated with single embryo transfer (OR 1.90, 95% CI 1.12 to 3.22, test for overall effect p=0.02). The multiple pregnancy rate was significantly lower in women who had single embryo transfer (OR 9.97, 95% CI 2.61 to 38.19; p = 0.0008). The effectiveness of double embryo transfer versus four embryo transfer was tested in a single trial. There was no statistically significant differences in the clinical pregnancy rate (OR 0.75, 95% CI 0.26 to 2.16; p=0.6), and multiple pregnancy rates (OR 0.44. 95% CI 0.10 to 1.97; p = 0.28) between the two groups. The livebirth rate in the four embryo transfer group was higher compared to the two embryo transfer group, but the results were not statistically significant (OR 0.35, 95% CI 0.11 to 1.05; p = 0.06). The results of this systematic review suggest that live birth and pregnancy rates following single embryo transfer are lower than those following double embryo transfer as are the chances of multiple pregnancy including twins. As such, it is unlikely that the conclusions are robust enough to catalyse a change in clinical practice. The studies included are limited by their small sample size, so that even large differences might be hidden. Cumulative livebirth rates are seldom reported. The data were inadequate to draw conclusions about single embryo transfer and first frozen single embryo transfer (1FZET) or subsequent single frozen embryo transfers. Until more evidence is available single embryo transfer may not be the preferred choice for all patients undergoing IVF/ICSI. Clinicians may need to individualise protocols for couples based on their risks of multiple pregnancy. A definitive pragmatic, large multi centre randomised controlled trial comparing single embryo versus double embryo transfer in terms of clinical and cost effectiveness as well as acceptability is required. The primary outcome measured should be cumulative livebirth per woman/couple.
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Busardò, Francesco Paolo; Napoletano, Simona; Zaami, Simona; Frati, Paola
2014-01-01
Medically Assisted Reproduction (MAR), involving in vitro fertilisation (IVF), and research on embryos have created expectation to many people affected by infertility; at the same time it has generated a surplus of laws and ethical and social debates. Undoubtedly, MAR represents a rather new medical field and constant developments in medicine and new opportunities continue to defy the attempt to respond to those questions. In this paper, the authors reviewed the current legislation in the 28 EU member states trying to evaluate the different legislation paths adopted over the last 15 years and highlighting those EU countries with no specific legislation in place and MAR is covered by a general health Law and those countries in which there are no laws in this field but only “guidelines.” The second aim of this work has been to compare MAR legislation and embryo research in EU countries, which derive from different origins ranging from an extremely prohibitive approach versus a liberal one, going through a cautious regulatory approach. PMID:25147794
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Embryo futures and stem cell research: the management of informed uncertainty
Ehrich, Kathryn; Williams, Clare; Farsides, Bobbie; Scott, Rosamund
2012-01-01
In the social worlds of assisted conception and stem cell science, uncertainties proliferate and particular framings of the future may be highly strategic. In this article we explore meanings and articulations of the future using data from our study of ethical and social issues implicated by the donation of embryos to human embryonic stem cell research in three linked assisted conception units and stem cell laboratories in the UK. Framings of the future in this field inform the professional management of uncertainty and we explore some of the tensions this involves in practice. The bifurcation of choices for donating embryos into accepting informed uncertainty or not donating at all was identified through the research process of interviews and ethics discussion groups. Professional staff accounts in this study contained moral orientations that valued ideas such as engendering patient trust by offering full information, the sense of collective ownership of the National Heath Service and publicly funded science and ideas for how donors might be able to give restricted consent as a third option. PMID:21812792
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Busardò, Francesco Paolo; Gulino, Matteo; Napoletano, Simona; Zaami, Simona; Frati, Paola
2014-01-01
Medically Assisted Reproduction (MAR), involving in vitro fertilisation (IVF), and research on embryos have created expectation to many people affected by infertility; at the same time it has generated a surplus of laws and ethical and social debates. Undoubtedly, MAR represents a rather new medical field and constant developments in medicine and new opportunities continue to defy the attempt to respond to those questions. In this paper, the authors reviewed the current legislation in the 28 EU member states trying to evaluate the different legislation paths adopted over the last 15 years and highlighting those EU countries with no specific legislation in place and MAR is covered by a general health Law and those countries in which there are no laws in this field but only "guidelines." The second aim of this work has been to compare MAR legislation and embryo research in EU countries, which derive from different origins ranging from an extremely prohibitive approach versus a liberal one, going through a cautious regulatory approach.
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3D confocal reconstruction of gene expression in mouse.
Hecksher-Sørensen, J; Sharpe, J
2001-01-01
Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.
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Germán Zurriaráin, Roberto
2009-01-01
This article examines the Laws on Human Assisted Reproduction and Biomedical Research in Spain. The Laws permit the use of human ovules, embryos and fetuses. Close to the technical and ethical problems that carry the research on embryonic stem cells, the detection of induced reprogramming of adult cells to an embryonic stage (iPS) opens up new perspectives in regenerative medicine. It makes unnecessary the use of frozen embryos or produced by nuclear transfer. These reasons would involve a review of the Spanish Legislation in this matter, in order that the human life is an ethical barrier and a fundamental to actual biomedical research.
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Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi
2010-01-01
Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828
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Effects of temperature on gene expression in embryos of the coral Montastraea faveolata
2009-01-01
Background Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5°C, 29.0°C, and 31.5°C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours. Results Analysis of differentially expressed genes indicated that increased temperatures may lead to oxidative stress, apoptosis, and a structural reconfiguration of the cytoskeletal network. Metabolic processes were downregulated, and the action of histones and zinc finger-containing proteins may have played a role in the long-term regulation upon heat stress. Conclusions Embryos responded differently depending on exposure time and temperature level. Embryos showed expression of stress-related genes already at a temperature of 29.0°C, but seemed to be able to counteract the initial response over time. By contrast, embryos at 31.5°C displayed continuous expression of stress genes. The genes that played a role in the response to elevated temperatures consisted of both highly conserved and coral-specific genes. These genes might serve as a basis for research into coral-specific adaptations to stress responses and global climate change. PMID:20030803
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Storr, Ashleigh; Venetis, Christos A; Cooke, Simon; Kilani, Suha; Ledger, William
2017-02-01
What is the inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer? The inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer was generally good, although not optimal, even among experienced embryologists. Previous research on the morphological assessment of early stage (two pronuclei to Day 3) embryos has shown varying levels of inter-observer and intra-observer agreement. However, single blastocyst transfer is now becoming increasingly popular and there are no published data that assess inter-observer and intra-observer agreement when selecting a single embryo for Day 5 transfer. This was a prospective study involving 10 embryologists working at five different IVF clinics within a single organization between July 2013 and November 2015. The top 10 embryologists were selected based on their yearly Quality Assurance Program scores for blastocyst grading and were asked to morphologically grade all Day 5 embryos and choose a single embryo for transfer in a survey of 100 cases using 2D images. A total of 1000 decisions were therefore assessed. For each case, Day 5 images were shown, followed by a Day 3 and Day 5 image of the same embryo. Subgroup analyses were also performed based on the following characteristics of embryologists: the level of clinical embryology experience in the laboratory; amount of research experience; number of days per week spent grading embryos. The agreement between these embryologists and the one that scored the embryos on the actual day of transfer was also evaluated. Inter-observer and intra-observer variability was assessed using the kappa coefficient to evaluate the extent of agreement. This study showed that all 10 embryologists agreed on the embryo chosen for transfer in 50 out of 100 cases. In 93 out of 100 cases, at least 6 out of the 10 embryologists agreed. The inter-observer and intra-observer agreement among embryologists when selecting a single Day 5 embryo for transfer was generally good as assessed by the kappa scores (kappa = 0.734, 95% CI: 0.665-0.791 and 0.759, 95% CI: 0.622-0.833, respectively). The subgroup analyses did not substantially alter the inter-observer and intra-observer agreement among embryologists. The agreement when Day 3 images were included alongside Day 5 images of the same embryos resulted in a change of mind at least three times by each embryologist (on average for <10% of cases) and resulted in a small decrease in inter-observer and intra-observer agreement between embryologists (kappa = 0.676, 95% CI: 0.617-0.724 and 0.752, 95% CI: 0.656-808, respectively).The assessment of the inter-observer agreement with regard to morphological grading of Day 5 embryos showed only a fair-to-moderate agreement, which was observed across all subgroup analyses. The highest overall kappa coefficient was seen for the grading of the developmental stage of an embryo (0.513; 95% CI: 0.492-0.538). The findings were similar when the individual embryologists were compared with the embryologist who made the morphological assessments of the available embryos on the actual day of transfer. All embryologists had already completed their training and were working under one organization with similar policies between the five clinics. Therefore, the inter-observer agreement might not be as high between embryologists working in clinics with different policies or with different levels of training. The generally good, although not optimal uniformity between participating embryologists when selecting a Day 5 embryo for transfer, as well as, the surprisingly low agreement when morphologically grading Day 5 embryos could be improved, potentially resulting in increased pregnancy rates. Future studies need to be directed toward technologies that can help achieve this. None declared. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Association of abnormal morphology and altered gene expression in human preimplantation embryos.
Wells, Dagan; Bermúdez, Mercedes G; Steuerwald, Nury; Malter, Henry E; Thornhill, Alan R; Cohen, Jacques
2005-08-01
We set out to characterize the expression of nine genes in human preimplantation embryos and determine whether abnormal morphology is associated with altered gene activity. Reverse transcription and real-time polymerase chain reaction were used to quantify the expression of multiple genes in each embryo. The genes studied have various important cellular roles (e.g., cell cycle regulation, DNA repair, and apoptosis). Research laboratory working closely with a clinical IVF practice. Over 50 embryos were donated by infertile patients (various etiologies). Among these, all major stages of preimplantation development and a variety of common morphologic abnormalities were represented. None. Quantification of mRNA transcripts. We detected an association between certain forms of abnormal morphology and disturbances of gene activity. Cellular fragmentation was associated with altered expression of several genes, including TP53, suggesting that fragmenting blastomeres are suffering stress of a type monitored by p53, possibly as a consequence of suboptimal culture conditions. Appropriate gene expression is vital for the regulation of metabolic pathways and key developmental events. Our data indicates a possible causal relationship between changes in gene expression and the formation of clinically relevant abnormal embryo morphologies. We hypothesize that embryos with expression profiles characteristic of good morphology and appropriate for their developmental stage have the greatest potential for implantation. If confirmed, this could lead to a new generation of preimplantation genetic diagnosis (PGD) tests for assessing embryo viability and predicting implantation potential.
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DuRant, S E; Hopkins, W A; Hepp, G R
2011-01-01
Recent research in birds has demonstrated that incubation temperature influences a suite of traits important for hatchling development and survival. We explored a possible mechanism for the effects on hatchling quality by determining whether incubation temperature influences embryonic energy expenditure of wood ducks (Aix sponsa). Because avian embryos are ectothermic, we hypothesized that eggs incubated at higher temperatures would have greater energy expenditure at any given day of incubation. However, because eggs incubated at lower temperatures take longer to hatch than embryos incubated at higher temperatures, we hypothesized that the former would expend more energy during incubation. We incubated eggs at three temperatures (35.0°, 35.9°, and 37.0°C) that fall within the range of temperatures of naturally incubated wood duck nests. We then measured the respiration of embryos every 3 d during incubation, immediately after ducks externally pipped, and immediately after hatching. As predicted, embryos incubated at the highest temperature had the highest metabolic rates on most days of incubation, and they exhibited faster rates of development. Yet, because of greater energy expended during the hatching process, embryos incubated at the lowest temperature expended 20%-37% more energy during incubation than did embryos incubated at the higher temperatures. Slower developmental rates and greater embryonic energy expenditure of embryos incubated at the lowest temperature could contribute to their poor physiological performance as ducklings compared with ducklings that hatch from eggs incubated at higher temperatures.
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Sills, Eric Scott; Murphy, Sarah Ellen
2009-01-01
The development of in vitro fertilisation (IVF) as a treatment for human infertilty was among the most controversial medical achievements of the modern era. In Ireland, the fate and status of supranumary (non-transferred) embryos derived from IVF brings challenges both for clinical practice and public health policy because there is no judicial or legislative framework in place to address the medical, scientific, or ethical uncertainties. Complex legal issues exist regarding informed consent and ownership of embryos, particularly the use of non-transferred embryos if a couple separates or divorces. But since case law is only beginning to emerge from outside Ireland and because legislation on IVF and human embryo status is entirely absent here, this matter is poised to raise contractual, constitutional and property law issues at the highest level. Our analysis examines this medico-legal challenge in an Irish context, and summarises key decisions on this issue rendered from other jurisdictions. The contractual issues raised by the Roche case regarding informed consent and the implications the initial judgment may have for future disputes over embryos are also discussed. Our research also considers a putative Constitutional 'right to procreate' and the implications EU law may have for an Irish case concerning the fate of frozen embryos. Since current Medical Council guidelines are insufficient to ensure appropriate regulation of the advanced reproductive technologies in Ireland, the report of the Commission on Assisted Human Reproduction is most likely to influence embryo custody disputes. Public policy requires the establishment and implementation of a more comprehensive legislative framework within which assisted reproductive medical services are offered. PMID:19589140
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Cheng, Christina N; Li, Yue; Marra, Amanda N; Verdun, Valerie; Wingert, Rebecca A
2014-07-17
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.
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Cheng, Christina N.; Li, Yue; Marra, Amanda N.; Verdun, Valerie; Wingert, Rebecca A.
2014-01-01
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples. PMID:25078510
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Decisional conflict and the disposition of frozen embryos: implications for informed consent
Lyerly, A.D.; Nakagawa, S.; Kuppermann, M.
2011-01-01
BACKGROUND Fertility patients often struggle with decisions about disposition of embryos remaining after fertility treatment. We aimed to identify predictors and correlates of decisional conflict among patients facing these decisions. METHODS We analyzed results from a survey of 2210 patients from nine geographically diverse US fertility clinics. The main outcome measure was decisional conflict about embryo disposition, as measured by the decisional conflict scale (DCS). RESULTS Of 1244 respondents who returned the survey, 1005 with cryopreserved embryos and DCS scores were included in the analysis. Of the respondents, 39% reported high decisional conflict (DCS ≥ 37.5). Thoughts about future childbearing were associated with high decisional conflict: respondents who were either uncertain about whether to have a baby in the future or sure they did not want to have a baby were at higher odds of high decisional conflict than participants who desired a baby [adjusted odds ratio (aOR) = 3.93, P < 0.001 and aOR = 1.69, P = 0.04, respectively]. Also associated with high decisional conflict were being likely to have embryos thawed and discarded (aOR = 2.08, P < 0.001), donated for research (aOR = 1.66, P = 0.01) or frozen ‘forever’ (aOR = 1.90, P = 0.01); being likely to choose compassionate transfer if it were available (aOR = 1.65, P = 0.03); attributing high, but not full, moral status to human embryos; not having enough information; and not being satisfied with the informed consent process. CONCLUSIONS Decisional conflict about frozen embryo disposition differs according to reproductive preferences that may vary according to stage of treatment. Informed consent for embryo disposition should be revisited periodically, with serious discussions about disposition after childbearing is complete. PMID:21216789
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Non-induction of radioadaptive response in zebrafish embryos by neutrons.
Ng, Candy Y P; Kong, Eva Y; Kobayashi, Alisa; Suya, Noriyoshi; Uchihori, Yukio; Cheng, Shuk Han; Konishi, Teruaki; Yu, Kwan Ngok
2016-06-01
In vivo neutron-induced radioadaptive response (RAR) was studied using zebrafish (Danio rerio) embryos. The Neutron exposure Accelerator System for Biological Effect Experiments (NASBEE) facility at the National Institute of Radiological Sciences (NIRS), Japan, was employed to provide 2-MeV neutrons. Neutron doses of 0.6, 1, 25, 50 and 100 mGy were chosen as priming doses. An X-ray dose of 2 Gy was chosen as the challenging dose. Zebrafish embryos were dechorionated at 4 h post fertilization (hpf), irradiated with a chosen neutron dose at 5 hpf and the X-ray dose at 10 hpf. The responses of embryos were assessed at 25 hpf through the number of apoptotic signals. None of the neutron doses studied could induce RAR. Non-induction of RAR in embryos having received 0.6- and 1-mGy neutron doses was attributed to neutron-induced hormesis, which maintained the number of damaged cells at below the threshold for RAR induction. On the other hand, non-induction of RAR in embryos having received 25-, 50- and 100-mGy neutron doses was explained by gamma-ray hormesis, which mitigated neutron-induced damages through triggering high-fidelity DNA repair and removal of aberrant cells through apoptosis. Separate experimental results were obtained to verify that high-energy photons could disable RAR. Specifically, 5- or 10-mGy X-rays disabled the RAR induced by a priming dose of 0.88 mGy of alpha particles delivered to 5-hpf zebrafish embryos against a challenging dose of 2 Gy of X-rays delivered to the embryos at 10 hpf. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.
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Recent microfluidic devices for studying gamete and embryo biomechanics.
Lai, David; Takayama, Shuichi; Smith, Gary D
2015-06-25
The technical challenges of biomechanic research such as single cell analysis at a high monetary cost, labor, and time for just a small number of measurements is a good match to the strengths of microfluidic devices. New scientific discoveries in the fertilization and embryo development process, of which biomechanics is a major subset of interest, is crucial to fuel the continual improvement of clinical practice in assisted reproduction. The following review will highlight some recent microfluidic devices tailored for gamete and embryo biomechanics where biomimicry arises as a major theme of microfluidic device design and function, and the application of fundamental biomechanic principles are used to improve outcomes of cryopreservation. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Cell adhesion molecules and in vitro fertilization.
Simopoulou, Maria; Nikolopoulou, Elena; Dimakakos, Andreas; Charalabopoulos, Konstantinos; Koutsilieris, Michael
2014-01-01
This review addresses issues regarding the need in the in vitro fertilization (IVF) field for further predictive markers enhancing the standing embryo selection criteria. It aims to serve as a source of defining information for an audience interested in factors related to the wide range of multiple roles played by cell adhesion molecules (CAMs) in several aspects of IVF ultimately associated with the success of an IVF cycle. We begin by stressing the importance of enriching the standing embryo selection criteria available aiming for the golden standard: "extract as much information as possible focusing on non-invasive techniques" so as to guide us towards selecting the embryo with the highest implantation potential. We briefly describe the latest trends on how to best select the right embryo, moving closer towards elective single embryo transfer. These trends are: frozen embryo transfer for all, preimplantation genetic screening, non-invasive selection criteria, and time-lapse imaging. The main part of this review is dedicated to categorizing and presenting published research studies focused on the involvement of CAMs in IVF and its final outcome. Specifically, we discuss the association of CAMs with conditions and complications that arise from performing assisted reproductive techniques, such as ovarian hyperstimulation syndrome, the state of the endometrium, and tubal pregnancies, as well as the levels of CAMs in biological materials available in the IVF laboratory such as follicular fluid, trophectoderm, ovarian granulosa cells, oocytes, and embryos. To conclude, since CAMs have been successfully employed as a diagnostic tool in several pathologies in routine clinical work, we suggest that their multi-faceted nature could serve as a prognostic marker in assisted reproduction, aiming to enrich the list of non-invasive selection and predictive criteria in the IVF setting. We propose that in light of the well-documented involvement of CAMs in the developmental processes of fertilization, embryogenesis, implantation, placentation, and embryonic development, further studies could contribute significantly to achieving a higher quality of treatment and management of infertility. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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NASA Astrophysics Data System (ADS)
Miller, James Henry
This report describes the research effort that was undertaken to develop and understand processing techniques for the deposition of both low and high density SiC coatings from a non-halide precursor, in support of the Generation IV Gas-Cooled Fast Reactor (GFR) fuel development program. The research was conducted in two phases. In the first phase, the feasibility of producing both porous SiC coatings and dense SiC coatings on surrogate fuel particles by fluidized bed chemical vapor deposition (FBCVD) using gas mixtures of methylsilane and argon was demonstrated. In the second phase, a combined experimental and modeling effort was carried out in order to gain an understanding of the deposition mechanisms that result in either porous or dense SiC coatings, depending on the coating conditions. For this second phase effort, a simplified (compared to the fluid bed) single-substrate chemical vapor deposition (CVD) system was employed. Based on the experimental and modeling results, the deposition of SiC from methylsilane is controlled by the extent of gas-phase reaction, and is therefore highly sensitive to temperature. The results show that all SiC coatings are due to the surface adsorption of species that result from gas-phase reactions. The model terms these gas-borne species embryos, and while the model does not include a prediction of coating morphology, a comparison of the model and experimental results indicates that the morphology of the coatings is controlled by the nucleation and growth of the embryos. The coating that results from small embryos (embryos with only two Si-C pairs) appears relatively dense and continuous, while the coating that results from larger embryos becomes less continuous and more nodular as embryo size increases. At some point in the growth of embryos they cease to behave as molecular species and instead behave as particles that grow by either agglomeration or by incorporation of molecular species on their surface. As these particles adhere to the substrate surface and become fixed in place by surface deposition in the interstices between adjacent particles, a low density coating consisting of these particles results.
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From Fertilization to Birth: Representing Development in High School Biology Textbooks
NASA Astrophysics Data System (ADS)
Wellner, Karen L.
Biology textbooks are everybody's business. In accepting the view that texts are created with specific social goals in mind, I examined 127 twentieth-century high school biology textbooks for representations of animal development. Paragraphs and visual representations were coded and placed in one of four scientific literacy categories: descriptive, investigative, nature of science, and human embryos, technology, and society (HETS). I then interpreted how embryos and fetuses have been socially constructed for students. I also examined the use of Haeckel's embryo drawings to support recapitulation and evolutionary theory. Textbooks revealed that publication of Haeckel's drawings was influenced by evolutionists and anti-evolutionists in the 1930s, 1960s, and the 1990s. Haeckel's embryos continue to persist in textbooks because they "safely" illustrate similarities between embryos and are rarely discussed in enough detail to understand comparative embryology's role in the support of evolution. Certain events coincided with changes in how embryos were presented: (a) the growth of the American Medical Association (AMA) and an increase in birth rates (1950s); (b) the Biological Sciences Curriculum Study (BSCS) and public acceptance of birth control methods (1960s); (c) Roe vs. Wade (1973); (d) in vitro fertilization and Lennart Nilsson's photographs (1970s); (e) prenatal technology and fetocentrism (1980s); and (f) genetic engineering and Science-Technology-Society (STS) curriculum (1980s and 1990s). By the end of the twentieth century, changing conceptions, research practices, and technologies all combined to transform the nature of biological development. Human embryos went from a highly descriptive, static, and private object to that of sometimes contentious public figure. I contend that an ignored source for helping move embryos into the public realm is schoolbooks. Throughout the 1900s, authors and publishers accomplished this by placing biology textbook embryos and fetuses in several different contexts--biological, technological, experimental, moral, social, and legal.
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Macroevolutionary developmental biology: Embryos, fossils, and phylogenies.
Organ, Chris L; Cooper, Lisa Noelle; Hieronymus, Tobin L
2015-10-01
The field of evolutionary developmental biology is broadly focused on identifying the genetic and developmental mechanisms underlying morphological diversity. Connecting the genotype with the phenotype means that evo-devo research often considers a wide range of evidence, from genetics and morphology to fossils. In this commentary, we provide an overview and framework for integrating fossil ontogenetic data with developmental data using phylogenetic comparative methods to test macroevolutionary hypotheses. We survey the vertebrate fossil record of preserved embryos and discuss how phylogenetic comparative methods can integrate data from developmental genetics and paleontology. Fossil embryos provide limited, yet critical, developmental data from deep time. They help constrain when developmental innovations first appeared during the history of life and also reveal the order in which related morphologies evolved. Phylogenetic comparative methods provide a powerful statistical approach that allows evo-devo researchers to infer the presence of nonpreserved developmental traits in fossil species and to detect discordant evolutionary patterns and processes across levels of biological organization. © 2015 Wiley Periodicals, Inc.
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An automated assay for the assessment of cardiac arrest in fish embryo.
Puybareau, Elodie; Genest, Diane; Barbeau, Emilie; Léonard, Marc; Talbot, Hugues
2017-02-01
Studies on fish embryo models are widely developed in research. They are used in several research fields including drug discovery or environmental toxicology. In this article, we propose an entirely automated assay to detect cardiac arrest in Medaka (Oryzias latipes) based on image analysis. We propose a multi-scale pipeline based on mathematical morphology. Starting from video sequences of entire wells in 24-well plates, we focus on the embryo, detect its heart, and ascertain whether or not the heart is beating based on intensity variation analysis. Our image analysis pipeline only uses commonly available operators. It has a low computational cost, allowing analysis at the same rate as acquisition. From an initial dataset of 3192 videos, 660 were discarded as unusable (20.7%), 655 of them correctly so (99.25%) and only 5 incorrectly so (0.75%). The 2532 remaining videos were used for our test. On these, 45 errors were made, leading to a success rate of 98.23%. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development
Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.
2014-01-01
The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108
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Sell, Stewart; Nicolini, Andrea; Ferrari, Paola; Biava, Pier M
2016-01-01
Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of "cancer stem cells theory". In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the "maturation arrest" of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer can be considered a problem of developmental biology and that one of the most important hallmarks of cancer is the loss of differentiation as already described by us in other articles.
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Frati, Paola; Pacchiarotti, Arianna; D'Errico, Stefano
2014-01-01
To evaluate the Italian physicians' knowledge/information level about the therapeutic potential of stem cells, the research choice between embryonic and cordonal stem cells, and the preference between autologous and heterologous storage of cordonal stem cells, we performed a national survey. The questionnaire—distributed to 3361 physicians—involved physicians of different religious orientations and of different medical specialities. Most of the physicians involved (67%) were Catholics, and the majority were gynaecologists and paediatricians (43%) who are mainly in charge to inform future mothers about the possibility of cordonal stem cells conservation. The majority of the physicians interviewed do not have specific knowledge about stem cells (59%), most of them having only generic information (92%). The largest part of physicians prefer to use umbilical cord blood cells rather than embryonic stem cells. Nevertheless, a large percentage of physicians were in favour of embryo research, especially when embryos are supernumerary (44% versus 34%). Eighty-seven % of the physicians interviewed proved to have a general knowledge about stem cells and believe in their therapeutic potential. They prefer research on cordonal stem cells rather than on embryo stem cells. Although they are in favour of heterologous stem cells donation, they still prefer cryopreservation for personal use. PMID:24877099
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Sánchez-Argüello, Paloma; Aparicio, Natalia; Fernández, Carlos
2012-06-01
Genotoxic effects on fauna after waterborne pollutant exposure have been demonstrated by numerous research programmes. Less effort has been focused on establishing relationship between genotoxicity and long-term responses at higher levels of biological organization. Taking into account that embryos may be more sensitive indicators of reproductive impairment than alterations in fertility, we have developed two assays in multiwell plates to address correlations between embryo toxicity and genotoxicity. The potential teratogenicity was assessed by analyzing abnormal development and mortality of Physa acuta at embryonic stage. Genotoxicity was measured by the micronucleus (MN) test using embryonic cells. Our results showed that linkage between genotoxicity and embryo toxicity depends on mechanisms of action of compounds under study. Embryo toxic responses showed a clear dose-related tendency whereas no clear dose-dependent effect was observed in micronucleus induction. The higher embryo toxicity was produced by benzo(a)pyrene exposure followed by fluoxetine and bisphenol A. Vinclozolin was the lower embryo toxic compound. Binary mixtures with BaP always resulted in higher embryo toxicity than single exposures but antagonistic effects were observed for MN induction. Benzo(a)pyrene produced the higher MN induction at 0.04 mg/L, which also produced clear embryo toxic effects. Fluoxetine did not induce cytogenetic effects but 0.25mg/L altered embryonic development. Bisphenol A significantly reduced hatchability at 0.5mg/L while MN induction appeared with higher treatments than those that start causing teratogenicity. Much higher concentration of vinclozolin (5mg/L) reduced hatchability and induced maximum MN formation. In conclusion, while validating one biomarker of genotoxicity and employing one ecologically relevant effect, we have evaluated the relative sensitivity of a freshwater mollusc for a range of chemicals. The embryo toxicity test is a starting point for the development of a life cycle test with freshwater snails even for undertaking multigeneration studies focused on transgenerational effects. Copyright © 2012 Elsevier Inc. All rights reserved.
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Matsubara, Yoshiyuki; Sakai, Atsushi; Kuroiwa, Atsushi; Suzuki, Takayuki
2014-10-01
The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named "sausage-style (SS) culture", allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113-115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.
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Profound Effects of Population Density on Fitness-Related Traits in an Invasive Freshwater Snail
Zachar, Nicholas; Neiman, Maurine
2013-01-01
Population density can profoundly influence fitness-related traits and population dynamics, and density dependence plays a key role in many prominent ecological and evolutionary hypotheses. Here, we evaluated how individual-level changes in population density affect growth rate and embryo production early in reproductive maturity in two different asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail that is an important model system for ecotoxicology and the evolution of sexual reproduction as well as a potentially destructive worldwide invader. We showed that population density had a major influence on individual growth rate and early-maturity embryo production, effects that were often apparent even when comparing treatments that differed in population density by only one individual. While individual growth rate generally decreased as population density increased, we detected a hump-shaped relationship between embryo production and density, with females from intermediate-density treatments producing the most embryos and females from low- and high-density treatments producing the fewest embryos. The two lineages responded similarly to the treatments, indicating that these effects of population density might apply more broadly across P. antipodarum. These results indicate that there are profound and complex relationships between population density, growth rate, and early-maturity embryo production in at least two lineages of this important model system, with potential implications for the study of invasive populations, research on the maintenance of sex, and approaches used in ecotoxicology. PMID:24278240
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Profound effects of population density on fitness-related traits in an invasive freshwater snail.
Zachar, Nicholas; Neiman, Maurine
2013-01-01
Population density can profoundly influence fitness-related traits and population dynamics, and density dependence plays a key role in many prominent ecological and evolutionary hypotheses. Here, we evaluated how individual-level changes in population density affect growth rate and embryo production early in reproductive maturity in two different asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail that is an important model system for ecotoxicology and the evolution of sexual reproduction as well as a potentially destructive worldwide invader. We showed that population density had a major influence on individual growth rate and early-maturity embryo production, effects that were often apparent even when comparing treatments that differed in population density by only one individual. While individual growth rate generally decreased as population density increased, we detected a hump-shaped relationship between embryo production and density, with females from intermediate-density treatments producing the most embryos and females from low- and high-density treatments producing the fewest embryos. The two lineages responded similarly to the treatments, indicating that these effects of population density might apply more broadly across P. antipodarum. These results indicate that there are profound and complex relationships between population density, growth rate, and early-maturity embryo production in at least two lineages of this important model system, with potential implications for the study of invasive populations, research on the maintenance of sex, and approaches used in ecotoxicology.
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Jiang, Hao; Liang, Shuang; Yao, Xue-Rui; Jin, Yong-Xun; Shen, Xing-Hui; Yuan, Bao; Zhang, Jia-Bao; Kim, Nam-Hyung
2018-04-23
Laminarin (LMA), a β-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 μg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H 2 O 2 . Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA. Copyright © 2018 Elsevier Inc. All rights reserved.
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The cellular and molecular biology of conifer embryogenesis.
Cairney, John; Pullman, Gerald S
2007-01-01
Gymnosperms and angiosperms are thought to have evolved from a common ancestor c. 300 million yr ago. The manner in which gymnosperms and angiosperms form seeds has diverged and, although broad similarities are evident, the anatomy and cell and molecular biology of embryogenesis in gymnosperms, such as the coniferous trees pine, spruce and fir, differ significantly from those in the most widely studied model angiosperm Arabidopsis thaliana. Molecular analysis of signaling pathways and processes such as programmed cell death and embryo maturation indicates that many developmental pathways are conserved between angiosperms and gymnosperms. Recent genomics research reveals that almost 30% of mRNAs found in developing pine embryos are absent from other conifer expressed sequence tag (EST) collections. These data show that the conifer embryo differs markedly from other gymnosperm tissues studied to date in terms of the range of genes transcribed. Approximately 72% of conifer embryo-expressed genes are found in the Arabidopsis proteome and conifer embryos contain mRNAs of very similar sequence to key genes that regulate seed development in Arabidopsis. However, 1388 loblolly pine (Pinus taeda) embryo ESTs (11.4% of the collection) are novel and, to date, have been found in no other plant. The data imply that, in gymnosperm embryogenesis, differences in structure and development are achieved by subtle molecular interactions, control of spatial and temporal gene expression and the regulating agency of a few unique proteins.
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Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.
Kurth, Thomas
2003-06-01
Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.
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1987-01-01
This document summarizes Denmark's 1987 Law establishing an Ethical Council regulating health care services and biomedical research on human subjects. The work of the Council will be based on the assumption that human life begins at fertilization. The Council will recommend laws to protect fertilized human ova, living embryos, and fetuses. The Council will also make recommendations about: 1) the right to perform gene therapy on human gametes or fertilized human ova, embryos, and fetuses; 2) the right to use new diagnostic techniques to detect congenital defeats or diseases in fertilized human ova, embryos, and fetuses; and 3) the establishment of rules on deep freezing of human gametes and fertilized human ova. In addition, the Council will advise the scientific ethical committees on general ethical matters concerning experiments on human volunteers; advise health authorities on general ethical questions associated with new treatments, diagnostic techniques, and medical technology; and advise public authorities on matters relating to registration, release, and use of information on hereditary diseases. The Law prohibits experiments enabling the production of genetically identical human beings, those enabling the production of human beings by the fusion of genetically different embryos or parts of embryos prior to implantation, and those seeking to produce hybrids of human beings and other species.
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Formation and evolution of anodic TiO2 nanotube embryos
NASA Astrophysics Data System (ADS)
Jin, Rong; Liao, Maoying; Lin, Tong; Zhang, Shaoyu; Shen, Xiaoping; Song, Ye; Zhu, Xufei
2017-06-01
Anodic TiO2 nanotubes (ATNTs) have been widely investigated for decades due to their interesting nanostructures and various applications. However, the formation mechanism of ATNTs still remains unclear. To date, most of researches focus on the tubular structure but neglect the formation process of initial nanotube embryos. Herein, polyethylene glycol (PEG) is added into the traditional electrolyte to moderate the transformation process from compact layer to porous layer. Based on ‘oxygen bubble mould’ and ‘plastic flow model’ theory, the formation and evolution process of nanotube embryo is clarified firstly. Results validate the effect of ‘oxygen bubble mould’ on the formation of nanotube embryo, which has a great effect on regulating the morphology of ATNT arrays. Besides, nanotubes prepared in electrolytes with PEG show shorter tube length with larger diameter than that prepared in traditional electrolytes. The addition of PEG can also effectively avoid the breakdown phenomenon. Highlights Transformation from compact layer into porous layer is observed in PEG electrolyte. The effect of oxygen bubble mould is first demonstrated and observed. The formation process of TiO2 nanotube embryo is described systematically. TiO2 nanotubes prepared in PEG electrolyte show short length and large diameter.
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Merviel, P; Cabry, R; Lourdel, E; Brasseur, F; Devaux, A; Copin, H
2009-09-01
The revision of the bioethics law of 2004 must occur in a five year's time. For this revision, the authorities decided to organize general states of bioethics and requested the production of contributions by the companies, institutions or associations. These texts tackle various subjects, like the Assisted Reproductive Technologies, research on the embryo and the stem cells and the banks of umbilical cord blood. Certain opinions converge, others differ, but all take part in the great debate which will take place at the time of the general conference.
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van Peperstraten, Arno M; Hermens, Rosella P M G; Nelen, Willianne L D M; Stalmeier, Peep F M; Wetzels, Alex M M; Maas, Pettie H M; Kremer, Jan A M; Grol, Richard P T M
2010-01-01
When deciding how many embryos to transfer during in vitro fertilisation (IVF), clinicians and patients have to balance optimizing the chance of pregnancy against preventing multiple pregnancies and the associated complications. This paper describes the development and pilot test of a patient decision aid (DA) for this purpose. The development of the DA consisted of a literature search, establishment of the format, and a pilot test among IVF patients. The DA development was supervised by a panel of experts in the fields of subfertility, obstetrics and DA-research and it was based on the criteria of the International Patient Decision Aid Standards. One Cochrane review and 34 articles were selected for the DA content. The DA presents information in text, summaries, tables, figures and through an interactive worksheet. The DA was reviewed positively and as acceptable for use in clinical practice by patients and professionals. The DA was thoroughly developed and is likely to be helpful for the decision-making process for the number of embryos transferred after IVF. Physicians and researchers can use the DA without restriction in clinical practice or research related to decision-making.
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No effect of embryo culture media on birthweight and length of newborns.
Lin, Shengli; Li, Ming; Lian, Ying; Chen, Lixue; Liu, Ping
2013-07-01
Does the type of media used to culture embryos for IVF influence the birthweight and length of neonates? No significant differences were observed in birthweight and length among the three embryo culture media used for in vitro embryo culture. Since the establishment of IVF as an assisted reproductive technology (ART), many different culture systems have been used for the development of human embryos. Some studies have shown that the types of culture media influence the newborn birthweight; however, other studies have shown no effect. To further explore this contradictory issue, we compared the birthweight and length of neonates born after the transfer of embryos cultured in one of three commercially available media. This retrospective analysis of birthweight and length of newborns included 1201 women who delivered singletons and 445 women who delivered twins. The following three commercially available culture media were used: G5™, Global and Quinn's advantage media. Women who underwent IVF-ET cycles between 2008 and 2010 were analyzed. Patients younger than 40 years of age with a body mass index (BMI) <30 kg/m(2) were analyzed. Only data from singletons and twins born alive after the 20th week of gestation were included in the data analysis. Patients who received preimplantation genetic diagnosis (PGD) and donor oocytes were excluded. The analysis of 1201 singletons and 445 sets of twins showed no significant association between mean birthweight or mean birth length and the type of embryo culture medium. Inter-twin mean birthweight and length disparities were analyzed, but were not shown to be significantly different. Multiple linear regression analysis showed that maternal weight, maternal height, gestational age and infant gender were significantly related to birthweight, and paternal height, gestational age and newborn complications were significantly associated with birth length. The current study showed that birthweight and length of newborns were not associated with the embryo culture medium. More research needs to be performed to analyze the effects of other culture medium formulations and to evaluate the long-term effects of embryo culture medium on the health of children conceived through ART. WIDER IMPLICATIONS OF THESE FINDINGS: Our retrospective study suggests that embryo culture medium does not influence neonatal birthweight and length; however, the effects of culture medium on epigenetic variation of embryos need to be studied further.
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The Sea Urchin Embryo: A Remarkable Classroom Tool.
ERIC Educational Resources Information Center
Oppenheimer, Steven B.
1989-01-01
Discussed are the uses of sea urchins in research and their usefulness and advantages in the classroom investigation of embryology. Ideas for classroom activities and student research are presented. Lists 25 references. (CW)
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Novo, Sergi; Nogués, Carme; Penon, Oriol; Barrios, Leonardo; Santaló, Josep; Gómez-Martínez, Rodrigo; Esteve, Jaume; Errachid, Abdelhamid; Plaza, José Antonio; Pérez-García, Lluïsa; Ibáñez, Elena
2014-01-01
Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of human oocytes and embryos during assisted reproduction technologies (ARTs)? The direct tagging system based on lectin-biofunctionalized polysilicon barcodes of micrometric dimensions is simple, safe and highly efficient, allowing the identification of human oocytes and embryos during the various procedures typically conducted during an assisted reproduction cycle. Measures to prevent mismatching errors (mix-ups) of the reproductive samples are currently in place in fertility clinics, but none of them are totally effective and several mix-up cases have been reported worldwide. Using a mouse model, our group has previously developed an effective direct embryo tagging system which does not interfere with the in vitro and in vivo development of the tagged embryos. This system has now been tested in human oocytes and embryos. Fresh immature and mature fertilization-failed oocytes (n = 21) and cryopreserved day 1 embryos produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (n = 205) were donated by patients (n = 76) undergoing ARTs. In vitro development rates, embryo quality and post-vitrification survival were compared between tagged (n = 106) and non-tagged (control) embryos (n = 99). Barcode retention and identification rates were also calculated, both for embryos and for oocytes subjected to a simulated ICSI and parthenogenetic activation. Experiments were conducted from January 2012 to January 2013. Barcodes were fabricated in polysilicon and biofunctionalizated with wheat germ agglutinin lectin. Embryos were tagged with 10 barcodes and cultured in vitro until the blastocyst stage, when they were either differentially stained with propidium iodide and Hoechst or vitrified using the Cryotop method. Embryo quality was also analyzed by embryo grading and time-lapse monitoring. Injected oocytes were parthenogenetically activated using ionomycin and 6-dimethylaminopurine. Blastocyst development rates of tagged (27/58) and non-tagged embryos (24/51) were equivalent, and no significant differences in the timing of key morphokinetic parameters and the number of inner cell mass cells were detected between the two groups (tagged: 24.7 ± 2.5; non-tagged: 22.3 ± 1.9), indicating that preimplantation embryo potential and quality are not affected by the barcodes. Similarly, re-expansion rates of vitrified-warmed tagged (19/21) and non-tagged (16/19) blastocysts were similar. Global identification rates of 96.9 and 89.5% were obtained in fresh (mean barcode retention: 9.22 ± 0.13) and vitrified-warmed (mean barcode retention: 7.79 ± 0.35) tagged embryos, respectively, when simulating an automatic barcode reading process, though these rates were increased to 100% just by rotating the embryos during barcode reading. Only one of the oocytes lost one barcode during intracytoplasmic injection (100% identification rate) and all oocytes retained all the barcodes after parthenogenetic activation. Although the direct embryo tagging system developed is effective, it only allows the identification and traceability of oocytes destined for ICSI and embryos. Thus, the traceability of all reproductive samples (oocytes destined for IVF and sperm) is not yet ensured. The direct embryo tagging system developed here provides fertility clinics with a novel tool to reduce the risk of mix-ups in human ARTs. The system can also be useful in research studies that require the individual identification of oocytes or embryos and their individual tracking. This study was supported by the Sociedad Española de Fertilidad, the Spanish Ministry of Education and Science (TEC2011-29140-C03) and the Generalitat de Catalunya (2009SGR-00282 and 2009SGR-00158). The authors do not have any competing interests.
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Ethical dimensions of therapeutic human cloning.
Reiss, Michael J
2002-09-11
Therapeutic human cloning has the potential significantly to reduce human suffering and enhance human happiness. This is the main ethical argument in its favour. The main ethical arguments against it centre on questions to do with the moral status of the human embryo. A subsidiary set of arguments arises from the connections between therapeutic human cloning and reproductive cloning. Most of the ethical questions concerning the status of the human embryo have long been examined in the context of abortion, though they are being re-examined in the context of genetic screening and embryo research. A consensus on such matters seems extremely unlikely to result in the near future. The current role of ethicists may not, therefore, be so much to attempt to produce a definitive answer to the question of the status of the human embryo at the very early developmental stages at which therapeutic human cloning would take place, but more to help clarify arguments and indicate the implications of particular approaches. That is what this paper seeks to do.
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High-resolution gene expression data from blastoderm embryos of the scuttle fly Megaselia abdita
Wotton, Karl R; Jiménez-Guri, Eva; Crombach, Anton; Cicin-Sain, Damjan; Jaeger, Johannes
2015-01-01
Gap genes are involved in segment determination during early development in dipteran insects (flies, midges, and mosquitoes). We carried out a systematic quantitative comparative analysis of the gap gene network across different dipteran species. Our work provides mechanistic insights into the evolution of this pattern-forming network. As a central component of our project, we created a high-resolution quantitative spatio-temporal data set of gap and maternal co-ordinate gene expression in the blastoderm embryo of the non-drosophilid scuttle fly, Megaselia abdita. Our data include expression patterns in both wild-type and RNAi-treated embryos. The data—covering 10 genes, 10 time points, and over 1,000 individual embryos—consist of original embryo images, quantified expression profiles, extracted positions of expression boundaries, and integrated expression patterns, plus metadata and intermediate processing steps. These data provide a valuable resource for researchers interested in the comparative study of gene regulatory networks and pattern formation, an essential step towards a more quantitative and mechanistic understanding of developmental evolution. PMID:25977812
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NASA Astrophysics Data System (ADS)
Ishigaki, Mika; Hashimoto, Kosuke; Sato, Hidetoshi; Ozaki, Yukihiro
2017-03-01
Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.
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Live dynamic analysis of the developing cardiovascular system in mice
NASA Astrophysics Data System (ADS)
Lopez, Andrew L.; Wang, Shang; Larin, Kirill V.; Larina, Irina V.
2017-02-01
The study of the developing cardiovascular system in mice is important for understanding human cardiogenesis and congenital heart defects. Our research focuses on imaging early development in the mouse embryo to specifically understand cardiovascular development under the regulation of dynamic factors like contractile force and blood flow using optical coherence tomography (OCT). We have previously developed an OCT based approach that combines static embryo culture and advanced image processing with computational modeling to live-image mouse embryos and obtain 4D (3D+time) cardiodynamic datasets. Here we present live 4D dynamic blood flow imaging of the early embryonic mouse heart in correlation with heart wall movement. We are using this approach to understand how specific mutations impact heart wall dynamics, and how this influences flow patterns and cardiogenesis. We perform studies in mutant embryos with cardiac phenotypes such as myosin regulatory light chain 2, atrial isoform (Mlc2a). This work is brings us closer to understanding the connections between dynamic mechanical factors and gene programs responsible for early cardiovascular development.
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Morin, Scott J
2017-03-01
There has been much debate regarding the optimal oxygen tension in clinical embryo culture. The majority of the literature to date has compared 5% oxygen to atmospheric levels (20-21%). While the majority of modern IVF labs have accepted the superiority of 5% oxygen tension, a new debate has emerged regarding whether a further reduction after day 3 of development represents the most physiologic system. This new avenue of research is based on the premise that oxygen tension is in fact lower in the uterus than in the oviduct and that the embryo crosses the uterotubal junction sometime on day 3. While data are currently limited, recent experience with ultra-low oxygen (2%) after day 3 of development suggests that the optimal oxygen tension in embryo culture may depend on the stage of development. This review article will consider the current state of the literature and discuss ongoing efforts at studying ultra-low oxygen tension in extended culture.
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Canovas, Sebastian; Ross, Pablo J; Kelsey, Gavin; Coy, Pilar
2017-11-01
DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children. © 2017 WILEY Periodicals, Inc.
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Oxygen supply limits the heat tolerance of lizard embryos.
Smith, Colton; Telemeco, Rory S; Angilletta, Michael J; VandenBrooks, John M
2015-04-01
The mechanisms that set the thermal limits to life remain uncertain. Classically, researchers thought that heating kills by disrupting the structures of proteins or membranes, but an alternative hypothesis focuses on the demand for oxygen relative to its supply. We evaluated this alternative hypothesis by comparing the lethal temperature for lizard embryos developing at oxygen concentrations of 10-30%. Embryos exposed to normoxia and hyperoxia survived to higher temperatures than those exposed to hypoxia, suggesting that oxygen limitation sets the thermal maximum. As all animals pass through an embryonic stage where respiratory and cardiovascular systems must develop, oxygen limitation may limit the thermal niches of terrestrial animals as well as aquatic ones. © 2015 The Author(s) Published by the Royal Society. All rights reserved.
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Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián
2015-09-01
Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.
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What's Wrong with Human/Nonhuman Chimera Research?
Hyun, Insoo
2016-08-01
The National Institutes of Health (NIH) is poised to lift its funding moratorium on research involving chimeric human/nonhuman embryos, pending further consideration by an NIH steering committee. The kinds of ethical concerns that seem to underlie this research and chimera research more generally can be adequately addressed.
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Zhao, Jianguo; Ross, Jason W.; Hao, Yanhong; Spate, Lee D.; Walters, Eric M.; Samuel, Melissa S.; Rieke, August; Murphy, Clifton N.; Prather, Randall S.
2009-01-01
The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns. PMID:19386991
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Zhao, Jianguo; Ross, Jason W; Hao, Yanhong; Spate, Lee D; Walters, Eric M; Samuel, Melissa S; Rieke, August; Murphy, Clifton N; Prather, Randall S
2009-09-01
The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
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Parturition prediction and timing of canine pregnancy
Kim, YeunHee; Travis, Alexander J.; Meyers-Wallen, Vicki N.
2007-01-01
An accurate method of predicting the date of parturition in the bitch is clinically useful to minimize or prevent reproductive losses by timely intervention. Similarly, an accurate method of timing canine ovulation and gestation is critical for development of assisted reproductive technologies, e.g. estrous synchronization and embryo transfer. This review discusses present methods for accurately timing canine gestational age and outlines their use in clinical management of high-risk pregnancies and embryo transfer research. PMID:17904630
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Summary of Research Academic Departments 1992-1993
1993-10-01
States should choose where and when to future. Settling on an internationalist destiny for engage its military forces in the post-Cold War the U.S...information during the growth of fruit fly biological information during the cell cycle and viral embryos . Kinetics of The Reaction AI(’P") + H 20 Over an...those cells various cell types in a growing embryo . These are totipotent in tissue culture (i.e., all cells can should provide insight into mechanisms
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The effects of incubation temperature and experimental design on heart rates of lizard embryos.
Hulbert, Austin C; Mitchell, Timothy S; Hall, Joshua M; Guiffre, Cassia M; Douglas, Danielle C; Warner, Daniel A
2017-08-01
Many studies of phenotypic plasticity alter environmental conditions during embryonic development, yet only measure phenotypes at the neonatal stage (after embryonic development). However, measuring aspects of embryo physiology enhances our understanding of how environmental factors immediately affect embryos, which aids our understanding of developmental plasticity. While current research on reptile developmental plasticity has demonstrated that fluctuating incubation temperatures affect development differently than constant temperatures, most research on embryo physiology is still performed with constant temperature experiments. In this study, we noninvasively measured embryonic heart rates of the brown anole (Anolis sagrei), across ecologically relevant fluctuating temperatures. We incubated eggs under temperatures measured from potential nests in the field and examined how heart rates change through a diel cycle and throughout embryonic development. We also evaluated how experimental design (e.g., repeated vs. single measures designs, constant vs. fluctuating temperatures) and different protocols (e.g., removing eggs from incubators) might influence heart rate. We found that heart rates were correlated with daily temperature and increased through development. Our findings suggest that experimenters have reasonable flexibility in choosing an experimental design to address their questions; however, some aspects of design and protocol can potentially influence estimations of heart rates. Overall, we present the first ecologically relevant measures of anole embryonic heart rates and provide recommendations for experimental designs for future experiments. © 2017 Wiley Periodicals, Inc.
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Gobeli, Amanda; Crossley, Dane; Johnson, Jeff; Reyna, Kelly
2017-05-01
Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there has been considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regard to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n=390) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100, and 150mg/kg of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected, weighed, staged, and examined for any overt structural deformities after 19days of incubation. The mass of the embryonic heart, liver, lungs and kidneys was also recorded. The majority of treatments produced no discernible differences in embryo morphology; however, in some instances, embryos were subject to increased frequency of anatomical deformity and altered organ masses. Some impacts were more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are more susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid has the potential to impact bobwhite quail embryonic development and chick survival. Copyright © 2017 Elsevier Inc. All rights reserved.
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Haba, Gen; Nishigori, Hidekazu; Tezuka, Yu; Kagami, Keisuke; Sugiyama, Toru; Nishigori, Hideo
2011-11-01
Hypothyroid state during embryogenesis disturbs normal growth and brain development, influencing later life. To evaluate the harmful consequences of the state during embryogenesis using an animal model, we inhibited thyroid hormone biosynthesis in chick embryos by using methimazole (MMI). Typically, embryos were treated with MMI (20 µmol/egg) on day 14, and examined on specific days. Of the control embryos, 94% hatched on day 21, whereas 0% and 60% of MMI-treated embryos hatched on days 21 and 24, respectively. MMI retarded the rates of bodyweight gain as well as liver and heart development, and delayed hatching. However, the external differences in appearance and differences in the weights of the newly hatched control chicks on day 21 and the MMI-treated chicks on day 24 were less obvious. Embryos treated with MMI exhibited increased mass in their brain parts on day 24. Most notably, the treatment resulted in a 1.35-fold increase in cerebellum weight compared to that of the untreated animals. Acetylcholinesterase activity in the cerebellum on the day of hatching decreased significantly to 0.85-fold that of the untreated controls. Thyroid hormone receptor β mRNA was detected from day 12 and dramatically expressed from day 19 to the day of hatching. The 'fertilized hen's egg-chick embryo-chick system' is an appropriate animal model for investigating the hypothyroid state during embryogenesis. Decreased cerebellar acetylcholinesterase activity after MMI treatment was assumed to relate to a mechanism of motor and cognitive deficits in congenital hypothyroidism. © 2011 The Authors. Journal of Obstetrics and Gynaecology Research © 2011 Japan Society of Obstetrics and Gynecology.
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Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)
NASA Astrophysics Data System (ADS)
Xu, Dongli; Peng, Leilei
2016-03-01
Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.
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Fermin, Lisanne M; Pain, Sarah J; Morel, Patrick C H; Gedye, Kristene R; Kenyon, Paul R; Blair, Hugh T
2017-11-21
Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates.
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Rostamzadeh, Ayoob; Mohammadi, Mohsen; Ahmadi, Reza; Nazari, Afshin; Ghaderi, Omar; Anjomshoa, Maryam
2016-01-01
Today, the use of electromagnetic waves in medical diagnostic devices, such as magnetic resonance imaging (MRI), has increased, and many of its biological effects have been reported. The aim of the present study was to assess the biological effects of 1.5 Tesla (T) magnetic resonance imaging (MRI) on fertility and reproductive parameters. Eighty adult male and female NMRI mice (NMRI: Naval Medical Research Institute) of age 6-8 weeks were studied and randomly divided into two study and control groups. After confirmation of pregnancy, the mice in the study group were exposed to the MRI (1.5 T) machine's waves over the next three weeks, once a week for 36 minutes. One day and thirty-five days after the last radiation, the mice were killed in order to do the in vitro fertilization (IVF) by neck beads' displacement and the impact on the evolution of embryos, and its quality was studied. Data were analyzed using SPSS version 20 and the significance level of less than 0.05 was considered. Embryo morphometry showed that the total diameter and the cytoplasm diameter of the study group embryos suffered significant reduction compared to the control group, 1 day after the last irradiation (p < 0.05), but the diameter of the perivitelline space of this group's embryos had a significant increase (p < 0.05). The qualitative results during 35 days after irradiation showed that morphologically parameters of the embryos in the study group had no significant differences from the control group. Exposure to MRI irradiation can transiently disturb the development of mouse embryos and fertility, but these effects are reversible 35 days after the last irradiation.
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Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan
2012-01-01
Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952
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4D atlas of the mouse embryo for precise morphological staging.
Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark
2015-10-15
After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development. © 2015. Published by The Company of Biologists Ltd.
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Effects of heat stress on bovine preimplantation embryos produced in vitro
SAKATANI, Miki
2017-01-01
Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress. PMID:28496018
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Effects of heat stress on bovine preimplantation embryos produced in vitro.
Sakatani, Miki
2017-08-19
Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.
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Han, Hye-In; Lee, Sang-Hee; Park, Choon-Keun
2017-07-01
The aim of this study was to establish an embryo culture system using collagen gel attached with vascular endothelial growth factor (VEGF) derived from interleukin-1 beta (IL-1β)-treated endometrial tissues from pigs. Endometria were separated from the porcine uterus at the follicular phase of the estrous cycle and were cultured with IL-1β. The collagen gels coincubated with IL-1β-treated endometria (C, without endometrial tissue; CE, with endometrial tissue; and CEI, IL-1β-treated endometrial tissue) were used for embryo culture. We found that, compared with the comparable figures in the control group, prostaglandin synthase-2 (PTGS-2) mRNA was increased in IL-1β-treated endometrial tissue (p < 0.05). The VEGF protein was not observed in collagen gel coincubated without endometrial tissue (C); however, it was detected in collagen gels coincubated with endometrial tissue (CE and CEI). The embryo cleavage rates and blastocyst formation did not differ among the treatment groups. The proportion of blastocysts did not differ among the groups. However, the number of blastocyst cells was significantly (p < 0.05) higher in the CEI group than in the other groups. These results clarify the effects of the intrauterine environment on preimplantation embryos and may be useful in research on the effects of extracellular matrix- and cytokine-treated endometrial tissue on embryo development.
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Rienzi, L; Vajta, G; Ubaldi, F
2011-09-01
During the past decades, improvements in culture of preimplantation embryos have contributed substantially in the success of human assisted reproductive techniques. However, most efforts were focused on optimization of media and gas components, while the established physical conditions and applied devices have remained essentially unchanged. Very recently, however, intensive research has been started to provide a more appropriate environment for the embryos and to replace the rather primitive and inappropriate devices with more sophisticated and practical instruments. Success has been reported with simple or sophisticated tools (microwells or microchannels) that allow accumulation of autocrine factors and establishment of a proper microenvironment for embryos cultured individually or in groups. The microchannel system may also offer certain level of automation and increased standardization of culture parameters. Continuous monitoring of individual embryos by optical or biochemical methods may help to determine the optimal day of transfer, and selection of the embryo with highest developmental competence for transfer. This advancement may eventually lead to adjustment of the culture environment to each individual embryo according to its actual needs. Connection of these techniques to additional radical approaches as automated ICSI or an ultimate assisted hatching with full removal of the zona pellucida after or even before fertilization may result in devices with high reliability and consistency, to increase the overall efficiency and decrease the work-intensity, and to eliminate the existing technological gap between laboratory embryology work and most other fields of biomedical sciences. Copyright © 2011 Elsevier Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Shi, Junchao; Zhang, Xudong; Liu, Ying; Chen, Qi
2017-03-01
In their interesting article [1] Wang et al. proposed a mathematical model based on evolutionary game theory [2] to tackle the fundamental question in embryo development, that how sperm and egg interact with each other, through epigenetic processes, to form a zygote and direct successful embryo development. This work is based on the premise that epigenetic reprogramming (referring to the erasure and reconstruction of epigenetic marks, such as DNA methylation and histone modifications) after fertilization might be of paramount importance to maintain the normal development of embryos, a premise we fully agree, given the compelling experimental evidence reported [3]. Wang et al. have specifically chosen to employ the well-studied DNA methylation reprogramming process during mammalian early embryo development, as a basis to develop their mathematical model, namely epigenetic game theory (epiGame). They concluded that the DNA methylation pattern in mammalian early embryo could be formulated and quantified, and their model can be further used to quantify the interactions, such as competition and/or cooperation of expressed genes that maximize the fitness of embryos. The efforts by Wang et al. in quantitatively and systematically analyzing the beginning of life apparently hold value and represent a novel direction for future embryo development research from both theoretical and experimental biologists. On the other hand, we see their theory still at its infancy, because there are plenty more parameters to consider and there are spaces for debates, such as the cases of haploid embryo development [4]. Here, we briefly comment on the dynamic process of epigenetic reprogramming that goes beyond DNA methylation, a dynamic interplay that involves histone modifications, non-coding RNAs, transposable elements et al., as well as the potential input of the various types of 'hereditary' epigenetic information in the gametes - a game that has started before the fertilization.
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Rajhans, Rajib; Kumar, G Sai; Dubey, Pawan K; Sharma, G Taru
2010-03-29
The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.
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Assessment of aneuploidy in human oocytes and preimplantation embryos by chromosome painting
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rougier, N.; Viegas-Pequignot, E.; Plachot, M.
1994-09-01
The poor quality of chromosome preparations often observed after fixation of oocytes and embryos did not usually allow accurate identification of chromosomes involved in non-disjunctions. We, therefore, used chromosome painting to determine the incidence of abnormalities for chromosomes 1 and 7. A total of 50 oocytes inseminated for IVF and showing no signs of fertilization as well as 37 diploid embryos donated for research were fixed according to the Dyban`s technique. Fluorescence in situ hybridization was carried out using whole chromosome painting DNA probes specific for human chromosome 1 and 7. The incidence of aneuploidy was 28%, 10% and 60%more » for metaphase II, polar body and sperm chromosomes, respectively. The high incidence of aneuploidy observed in sperm prematurely condensed sperm chromosomes is due to the fact that usually far less than 23 sperm chromatids are observed, maybe as a consequence of incomplete chromosome condensation. Thirty seven embryos were analyzed with the same probes. 48% of early embryos were either monosomic 1 or 7 or mosaics comprising blastomeres with 1, 2 or 3 signals. Thus, 8 among the 11 abnormal embryos had hypodiploid cells (25 to 37 chromosomes) indicating either an artefactual loss of chromosomes or a complex anomaly of nuclear division (maltinucleated blastomeres, abnormal migration of chromosomes at anaphase). We therefore calculated a {open_quotes}corrected{close_quotes} incidence of aneuploidy for chromosomes 1 or 7 in early embryos: 18%. 86% of the blastocysts showed mosaicism 2n/3 or 4n as a consequence of the formation of the syncitiotrophoblast. To conclude, chromosome painting is an efficient method to accurately identify chromosomes involved in aneuploidy. This technique should allow us to evaluate the incidence of non-disjunction for all chromosome pairs. Our results confirm the high incidence of chromosome abnormalities occurring as a consequence of meiotic or mitotic non-disjunctions in human oocytes and embryos.« less
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Kopeika, Julia; Thornhill, Alan; Khalaf, Yacoub
2015-01-01
Cryopreservation has been extensively used in assisted reproductive technology, agriculture and conservation programmes for endangered species. The literature reports largely positive results regarding the survival of frozen-thawed cells and clinical outcomes. Nonetheless, it is unclear whether or not cryopreservation of sperm, oocytes and embryos causes any disruption in their genetic integrity. Drawing on the available published evidence, this review paper describes in detail the physical and biochemical factors of cryopreservation that could potentially affect genomic integrity. A critical review of the published literature using PubMed with particular emphasis on studies which include assessment of genetic stability after cryopreservation of oocyte, sperm and embryos. The search was performed in 2014 and covered the period from the beginning of electronic records until July 2014. No language restrictions were applied. Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Some evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation. The extent of these changes may vary between different individuals and different techniques. The addition of antioxidants to the cryopreservation media and the use of well-controlled cooling regimes could potentially improve such outcomes. Limited numbers of studies on oocytes provide controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE) and aneuploidy. The only study on human embryos suggested that vitrification affects DNA integrity to a much lesser extent than slow freezing. Animal studies show increases in mitochondrial DNA mutations in embryos after cryopreservation. The limited numbers of long-term follow-up studies in humans provide reassurance that derives mostly from retrospective studies with some methodological weaknesses. This review provides an overview of studies performed to date on the effect of cryopreservation on the oocyte, sperm and embryos. Controversy of the reported data has highlighted the gaps in our knowledge not only for clinical studies, but also for basic research in human embryos. New perspectives for future research are proposed. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Benefiting from 'evil': an incipient moral problem in human stem cell research.
Green, Ronald M
2002-11-01
When does benefiting from others' wrongdoing effectively make one a moral accomplice in their evil deeds? If stem cell research lives up to its therapeutic promise, this question (which has previously cropped up in debates over fetal tissue research or the use of Nazi research data) is likely to become a central one for opponents of embryo destruction. I argue that benefiting from wrongdoing is prima facie morally wrong under any of three conditions: (1) when the wrongdoing is one's agent; (2) when acceptance of benefit directly encourages the repetition of the wrongful deed (even though no agency relationship is involved); and (3) when acceptance of a benefit legitimates a wrongful practice. I conclude by showing that, because of the ways in which most embryonic stem cell lines come into being, people who oppose embryo destruction may use human embryonic stem cells without incurring moral blame.
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Souders, Christopher L; Liang, Xuefang; Wang, Xiaohong; Ector, Naomi; Zhao, Yuan H; Martyniuk, Christopher J
2018-06-01
Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the μM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction. Copyright © 2018 Elsevier B.V. All rights reserved.
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Segal, Nancy L
2007-12-01
In recent years, there has been growing appreciation for the complexity of gender identity. Focusing on monozygotic (MZ) twins discordant for transsexualism can offer clues to events that may trigger this behavioral difference, offering new information about critical underlying factors. An update of twin research in this area is provided, together with a preview of a compelling new film, 'Red Without Blue.' Next, twin study findings on the topics of conjoined twinning, angiographic lesions and embryo transfer are provided. This is followed by a survey of newsworthy twins and twin-related events.
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Mouse cloning and somatic cell reprogramming using electrofused blastomeres.
Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi
2011-05-01
Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.
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The exploit of cereal embryo structure for productive reasons by in vitro techniques
NASA Astrophysics Data System (ADS)
Savaskan, C.
2017-07-01
There are two main sides of our works exploiting embryo structure in durum wheat and some other cereals. First is haploid (or doubled haploid) embryo production using anther or microspore culture or intergeneric crosses, to ameliorate desirable characters genetically homozygote. Secondly, to develope convenient embryo culture technique in order to be stored and cultivated longer time of genotypes without being alien pollination etc. in field conditions. For that reason, two different auxin and also their combination with kinetin were used for mature embryos of wheat genotypes (hexaploid and tetraploid), to understand efficient dose for calli production and plant regeneration in plant tissue culture. Modified MS media were used adding a single dose of arabinogalactan protein (AGP) and without adding for regeneration. In further step of this study, most efficient auxin+kinetin combination which is determined previous research, it was used in the same modified MS medium to produce calli production and plant regeneration in three different genotypes (hexaploid and tetraploid wheat and diploid barley). Data were calculated in five different developmental stages of treatments. All statistical analysis of data were performed and means were compared with Duncan's test. Genetics and morphological effects of AGP on genotypes were discussed with the results of variance analysis. Simple correlation coefficient (r) was calculated base on the main values of replications.
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Cooper, Mark S; Szeto, Daniel P; Sommers-Herivel, Greg; Topczewski, Jacek; Solnica-Krezel, Lila; Kang, Hee-Chol; Johnson, Iain; Kimelman, David
2005-02-01
Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Copyright 2004 Wiley-Liss, Inc.
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Tan, Germaine Xin Yi; Jamil, Muhammad; Tee, Nicole Gui Zhen; Zhong, Liang; Yap, Choon Hwai
2015-11-01
Recent animal studies have provided evidence that prenatal blood flow fluid mechanics may play a role in the pathogenesis of congenital cardiovascular malformations. To further these researches, it is important to have an imaging technique for small animal embryos with sufficient resolution to support computational fluid dynamics studies, and that is also non-invasive and non-destructive to allow for subject-specific, longitudinal studies. In the current study, we developed such a technique, based on ultrasound biomicroscopy scans on chick embryos. Our technique included a motion cancelation algorithm to negate embryonic body motion, a temporal averaging algorithm to differentiate blood spaces from tissue spaces, and 3D reconstruction of blood volumes in the embryo. The accuracy of the reconstructed models was validated with direct stereoscopic measurements. A computational fluid dynamics simulation was performed to model fluid flow in the generated construct of a Hamburger-Hamilton (HH) stage 27 embryo. Simulation results showed that there were divergent streamlines and a low shear region at the carotid duct, which may be linked to the carotid duct's eventual regression and disappearance by HH stage 34. We show that our technique has sufficient resolution to produce accurate geometries for computational fluid dynamics simulations to quantify embryonic cardiovascular fluid mechanics.
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Effects of Multimodal Analgesia on the Success of Mouse Embryo Transfer Surgery
Parker, John M.; Austin, Jamie; Wilkerson, James; Carbone, Larry
2011-01-01
Multimodal analgesia is promoted as the best practice pain management for invasive animal research procedures. Universal acceptance and incorporation of multimodal analgesia requires assessing potential effects on study outcome. The focus of this study was to assess effects on embryo survival after multimodal analgesia comprising an opioid and nonsteroidal antiinflammatory drug (NSAID) compared with opioid-only analgesia during embryo transfer procedures in transgenic mouse production. Mice were assigned to receive either carprofen (5 mg/kg) with buprenorphine (0.1 mg/kg; CB) or vehicle with buprenorphine (0.1 mg/kg; VB) in a prospective, double-blinded placebo controlled clinical trial. Data were analyzed in surgical sets of 1 to 3 female mice receiving embryos chimeric for a shared targeted embryonic stem-cell clone and host blastocyst cells. A total of 99 surgical sets were analyzed, comprising 199 Crl:CD1 female mice and their 996 offspring. Neither yield (pups weaned per embryo implanted in the surgical set) nor birth rate (average number of pups weaned per dam in the set) differed significantly between the CB and VB conditions. Multimodal opioid–NSAID analgesia appears to have no significant positive or negative effect on the success of producing novel lines of transgenic mice by blastocyst transfer. PMID:21838973
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The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)
Veres, Monika; Duselis, Amanda R.; Graft, Audrey; Pryor, William; Crossland, Janet; Vrana, Paul B.; Szalai, Gabor
2011-01-01
Although laboratory-reared species of the genus Peromyscus - including deer mice - are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte/embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of PMSG was given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in KSOM media in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus pre-implantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g. chimeras, transgenics), and preservation of genetic lines by cryopreservation. PMID:21924468
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Loren, Pía; Sánchez, Raúl; Arias, María-Elena; Felmer, Ricardo; Risopatrón, Jennie; Cheuquemán, Carolina
2017-01-01
Oxidative and nitrosative stress are common problems when handling gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because their absence or overproduction causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during in vitro fertilization (IVF) has deleterious or positive effects, depending on the concentration used in the culture medium, demonstrating the delicate balance between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in humans and other mammals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology. PMID:28613231
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Komori, M; Miura, T; Shiota, K; Minato, K; Takahashi, T
1995-01-01
The volumetric shape of a human embryo and its development is hard to comprehend as they have been viewed as a 2D schemes in a textbook or microscopic sectional image. In this paper, a CAI and research support system for human embryology using multimedia presentation techniques is described. In this system, 3D data is acquired from a series of sliced specimens. Its 3D structure can be viewed interactively by rotating, extracting, and truncating its whole body or organ. Moreover, the development process of embryos can be animated using a morphing technique applied to the specimen in several stages. The system is intended to be used interactively, like a virtual reality system. Hence, the system is called Virtual Embryology.
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Camporesi, Silvia; Cavaliere, Giulia
2016-01-01
This paper provides an overview of the ethical issues in the international clustered regularly interspaced short palindromic repeats (CRISPR) genome editing debate from March 2015 to September 2016. We present the regulatory framework for embryo research in the UK, and explain why CRISPR is not a significant break with the past. We discuss the ethical issues arising from CRISPR applications beyond human embryos, namely the use of gene drive-engineered mosquitoes to eradicate diseases, engineering nonhuman animals to harvest organs for human transplant and engineering crops. We discuss the experiments that have demonstrated the technical feasibility of cultivating embryos in vitro for up to 14 days, and possibly beyond this limit, and the ethical issues arising from the proposal to extend the limit beyond 14 days. PMID:28757883
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Camporesi, Silvia; Cavaliere, Giulia
2016-11-01
This paper provides an overview of the ethical issues in the international clustered regularly interspaced short palindromic repeats (CRISPR) genome editing debate from March 2015 to September 2016. We present the regulatory framework for embryo research in the UK, and explain why CRISPR is not a significant break with the past. We discuss the ethical issues arising from CRISPR applications beyond human embryos, namely the use of gene drive-engineered mosquitoes to eradicate diseases, engineering nonhuman animals to harvest organs for human transplant and engineering crops. We discuss the experiments that have demonstrated the technical feasibility of cultivating embryos in vitro for up to 14 days, and possibly beyond this limit, and the ethical issues arising from the proposal to extend the limit beyond 14 days.
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CRISPR mediated somatic cell genome engineering in the chicken.
Véron, Nadège; Qu, Zhengdong; Kipen, Phoebe A S; Hirst, Claire E; Marcelle, Christophe
2015-11-01
Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model. Copyright © 2015 Elsevier Inc. All rights reserved.
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Ribatti, Domenico
2018-06-07
Sir Mac Farlane Burnet was the most honored of all Australian scientists. In 1960, Burnet shared the Nobel Prize for Medicine with Peter Medawar of Britain for the discovery of acquired immunological tolerance. He developed techniques for growing influenza viruses in the chorioallantoic membrane of the chick embryo. This became a standard laboratory practice. He continued to work with chick embryos long after the use of cell cultures had become general. His virology research resulted in significant discoveries concerning the nature and replication of viruses and their interaction with the immune system. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
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Genetic transformation protocols using zygotic embryos as explants: an overview.
Tahir, Muhammad; Waraich, Ejaz A; Stasolla, Claudio
2011-01-01
Genetic transformation of plants is an innovative research tool which has practical significance for the development of new and improved genotypes or cultivars. However, stable introduction of genes of interest into nuclear genomes depends on several factors such as the choice of target tissue, the method of DNA delivery in the target tissue, and the appropriate method to select the transformed plants. Mature or immature zygotic embryos have been a popular choice as explant or target tissue for genetic transformation in both angiosperms and gymnosperms. As a result, considerable protocols have emerged in the literature which have been optimized for various plant species in terms of transformation methods and selection procedures for transformed plants. This article summarizes the recent advances in plant transformation using zygotic embryos as explants.
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Du, Tong; Wang, Yun; Fan, Yong; Zhang, Shiyi; Yan, Zhiguang; Yu, Weina; Xi, Qianwen; Chen, Qiuju; Mol, Ben W; Lyu, Qifeng; Kuang, Yanping
2018-06-01
Is transferring embryos that achieve blastulation on Day 7 effective and safe? Embryos that achieve blastulation on Day 7 resulted in clinically relevant rates of clinical pregnancy (32.5%) and live birth (25.2%), and newborns have a similar risk of low birth weight, congenital malformations or early neonatal death compared with those derived from Days 5 and 6 blastocysts. Potential advantages of blastocyst transfer over cleavage embryo transfer have led to a shift toward the former in IVF practice. However, published data about the fertility outcomes of transferring embryos with a delayed blastulation on Day 7 are scarce and controversial. Moreover, there are few data available on the neonatal outcomes of Day 7 blastocysts. As a result, the clinical value of Day 7 blastocysts is uncertain. This was a retrospective cohort study that included 2908 women undergoing frozen-thawed embryo transfer cycles of IVF/ICSI from January 2006 to May 2015, and reported on the 1518 live born infants from those cycles. We used propensity score matching to compare the fertility outcomes of women undergoing Day-5, Day-6 and Day-7 vitrified embryo transfers in three matched comparisons (Day 5 vs Day 6, Day 5 vs Day 7 and Day 6 vs Day 7). We also compared neonatal outcomes among babies derived from Day-5, Day-6 and Day-7 vitrified embryo transfers. We studied 922 Day-5, 1752 Day-6 and 234 Day-7 vitrified embryo transfers. Day-7 vitrified embryo transfers had significantly lower implantation, clinical pregnancy and live birth rates than both Day-5 (23.9 vs 49.9%, 31.7 vs 58.1% and 25.1 vs 46.5%, all P < 0.001, respectively) and Day-6 (24.7 vs 42.3%, 33.0 vs 53.2% and 25.6 vs 41.4%, all P < 0.001, respectively) vitrified embryo transfers. Assessment of babies showed no statistically significant difference in the rates of low birth weight, congenital malformations and early neonatal death among the 585, 869 and 64 babies born from Day-5, Day-6 and Day-7 vitrified embryo transfer groups, respectively. This was a single center retrospective study, and most of the neonatal data were extracted from parental questionnaires. Besides, the number of Day-7 vitrified embryo transfer cycles and babies born from these cycles was still limited, thus reducing the power of our study in assessing neonatal outcomes. In addition, only the morphologically poorer Day 3 embryos were extendedly cultured, and poorer blastocysts were qualified for vitrification on Day 7 than on Day 5 or 6, both of which might bias clinical pregnancy rates. Transfer of embryos that reach the blastocyst stage on Day 7 results in lower but still acceptable live birth rate, and seems to be safe for the offspring. Extension of the culture time in embryos that do not reach blastocyst stage by Day 6 should be assessed in randomized clinical trials. This work was supported by the National Nature Science Foundation of China (Grant nos. 81771533, 81571397, 81571486, 31770989 and 81501319), the Nature Science Foundation of Shanghai (Grant nos. 15ZR1424900 and 1441196300), and the Foundation of Health and Family Planning Commission of Shanghai (Grant no. 201540237). B.W.M is supported by the National Health and Medical Research Council (NHMRC) Practitioner Fellowship (GNT1082548), B.W.M reports consultancy for ObsEva, Merck and Guerbet. Not applicable.
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Frith, Lucy; Blyth, Eric; Lui, Steve
2017-05-01
What contact arrangements are established between providers and recipients of embryos using Snowflakes® Embryo Adoption Program? Contact arrangements varied considerably and were generally positively described, although some challenges were acknowledged. Reproductive technologies create new and diverse family forms, and the ways in which families created by embryo adoption are negotiated in practice have not been extensively investigated. This exploratory, mixed-methods study had two phases: (i) an online survey (open May-September 2013) and (ii) qualitative semi-structured interviews by email (conducted between 2014 and 2015), exploring participants' experiences of contact with their embryo provider or recipient. Phase I included 17 providers (14 women and 3 men) and 28 recipients (27 women and 1 man). Phase II included 8 providers (5 women and 3 men) and 12 recipients (10 women and 2 men). All participants, except one, were located in the US. This study illustrates how embryo adoption in the US, as a form of conditional donation, can operate and how the participants define and negotiate these emerging relationships. All families were open with their children about how they were conceived and early contact between recipients and providers (frequently before birth) was valued. On the whole, participants were happy with the amount and type of contact they had, and where the current contact did not involve the children, it was seen as a way of keeping the channels open for future contact when the children were older. Participants often portrayed the opportunities for contact as being in the best interests of the child. The study participants are a particular group who had chosen to either receive or give their embryos via a conditional embryo adoption agency in the US and had established contact. Therefore, this is not a representative sample of those who provide or receive embryos for family building. This embryo adoption model clearly fulfils a need; some people want to use a conditional embryo donation programme such as Snowflakes®. Some form of 'ongoing support mechanism' such as counselling could be useful for those negotiating the complex sets of new kinship patterns and balancing these relationships with their children's welfare. The authors have no conflict of interest to declare. Snowflakes is a commercial adoption agency and the authors have no relation to the organization, other than requesting that they participate in this research project. N/A. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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ERIC Educational Resources Information Center
Oppenheimer, Steven B.
1989-01-01
To integrate a research/discovery component into one course, an experiment using the sea urchin embryo system is introduced because the teacher manages only one organism and students provide the variables. The materials, handling methods, and experimental methods of the experiment are described. (YP)
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Behavorial assessments of larval zebrafish neurotoxicology
Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...
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A descriptive study of culture media in Brazilian assisted reproduction clinics.
Bartmann, Ana; Amaral, Amanda Turato Barbosa do; Gonçalves, Letícia
2016-08-01
The present study aimed to draw a profile of the most commonly used media and protocol characteristics from assisted reproduction technology (ART) facilities in Brazil. To obtain an overview of ART methods and culture media, a questionnaire was given to embryologists from ART clinics in Brazil. Further research in scientific papers and journals was carried out for describing the processes around Brazil, USA and Europe. From the questionnaire, we found that the embryo medium mostly used is CSCMTM from Irvine Scientific, represented 37.04% in Brazilian ART clinics; interestingly, 70.37% of clinics exchange the embryo media bath; however, 70.37% do not change the media type. Transfers in Brazilian clinics were variable, but day 3 transfer was a procedure seen in 37.04%. The remaining embryos are habitually maintained in prolonged cultivation in 51.85% of the clinics interviewed. Although there are numerous studies trying to better understand embryo culture media influences, there is a lack of evidence for choosing one as the most appropriate. In short, it is a random decision for such an essential stage of In Vitro Fertilization.
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A descriptive study of culture media in Brazilian assisted reproduction clinics
Bartmann, Ana; do Amaral, Amanda Turato Barbosa; Gonçalves, Letícia
2016-01-01
Objective The present study aimed to draw a profile of the most commonly used media and protocol characteristics from assisted reproduction technology (ART) facilities in Brazil. Methods To obtain an overview of ART methods and culture media, a questionnaire was given to embryologists from ART clinics in Brazil. Further research in scientific papers and journals was carried out for describing the processes around Brazil, USA and Europe. Results From the questionnaire, we found that the embryo medium mostly used is CSCMTM from Irvine Scientific, represented 37.04% in Brazilian ART clinics; interestingly, 70.37% of clinics exchange the embryo media bath; however, 70.37% do not change the media type. Transfers in Brazilian clinics were variable, but day 3 transfer was a procedure seen in 37.04%. The remaining embryos are habitually maintained in prolonged cultivation in 51.85% of the clinics interviewed. Conclusion Although there are numerous studies trying to better understand embryo culture media influences, there is a lack of evidence for choosing one as the most appropriate. In short, it is a random decision for such an essential stage of In Vitro Fertilization. PMID:27584601
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To QC or not to QC: the key to a consistent laboratory?
Lane, Michelle; Mitchell, Megan; Cashman, Kara S; Feil, Deanne; Wakefield, Sarah; Zander-Fox, Deirdre L
2008-01-01
A limiting factor in every embryology laboratory is its capacity to grow 'normal' embryos. In human in vitro fertilisation (IVF), there is considerable awareness that the environment of the laboratory itself can alter the quality of the embryos produced and the industry as a whole has moved towards the implementation of auditable quality management systems. Furthermore, in some countries, such as Australia, an established quality management system is mandatory for clinical IVF practice, but such systems are less frequently found in other embryology laboratories. Although the same challenges of supporting consistent and repeatable embryo development are paramount to success in all embryology laboratories, it could be argued that they are more important in a research setting where often the measured outcomes are at an intracellular or molecular level. In the present review, we have outlined the role and importance of quality control and quality assurance systems in any embryo laboratory and have highlighted examples of how simple monitoring can provide consistency and avoid the induction of artefacts, irrespective of the laboratory's purpose, function or species involved.
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Gurkov, Anton; Sadovoy, Anton; Shchapova, Ekaterina; Teh, Cathleen; Meglinski, Igor; Timofeyev, Maxim
2017-01-01
In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the changes in physiological parameters in the same individual. In this study, we introduce microencapsulated fluorescent probes as a promising tool for in vivo monitoring of internal pH variation in zebrafish embryos. The pH alteration identified under stress conditions demonstrates the applicability of the microencapsulated fluorescent probes for the repeated analysis of the embryo's physiological state. The proposed approach has strong potential to simultaneously measure a range of physiological characteristics using a set of specific fluorescent probes and to finally bring toxicological bioassays and related research fields to a new level of effectiveness and sensitivity.
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Mapping a multiplexed zoo of mRNA expression.
Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A
2016-10-01
In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.
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Mapping a multiplexed zoo of mRNA expression
Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.
2016-01-01
In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788
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NASA Astrophysics Data System (ADS)
Nadort, Annemarie; Liang, Liuen; Grebenik, Ekaterina; Guller, Anna; Lu, Yiqing; Qian, Yi; Goldys, Ewa; Zvyagin, Andrei
2015-12-01
Nanoparticle-based delivery of drugs and contrast agents holds great promise in cancer research, because of the increased delivery efficiency compared to `free' drugs and dyes. A versatile platform to investigate nanotechnology is the chick embryo chorioallantoic membrane tumour model, due to its availability (easy, cheap) and accessibility (interventions, imaging). In our group, we developed this model using several tumour cell lines (e.g. breast cancer, colon cancer). In addition, we have synthesized in-house silica coated photoluminescent upconversion nanoparticles with several functional groups (COOH, NH2, PEG). In this work we will present the systematic assessment of their in vivo blood circulation times. To this end, we injected chick embryos grown ex ovo with the functionalized UCNPs and obtained a small amount of blood at several time points after injection to create blood smears The UCNP signal from the blood smears was quantified using a modified inverted microscope imaging set-up. The results of this systematic study are valuable to optimize biochemistry protocols and guide nanomedicine advancement in the versatile chick embryo tumour model.
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Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda
2016-01-01
Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or "frosties," show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50-85%). We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol.
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Nazari Tavakkoli, Saeid
2017-10-01
One of the most controversial issues related to the human embryo is the determination of the moment when an embryo is considered a human being and acquires a moral status. Although personhood and moral status are frequently mentioned in medical ethics, they are considered interdisciplinary as concepts that shape the debate in medical law (fiqh) since their consequences are influential in the way which the parents and other individuals behave towards the embryo. This analytical-descriptive research gathered relevant data in a literature search. After a description of the fundamentals and definitions, we subsequently analyzed juridical texts and selected one of the viewpoints that regarded the surrogacy contract revocation. The surrogacy contract is a contract based upon which two sides (infertile couple and surrogate mother) involved in making the contract are obligated to fulfill its terms. Therefore, contract revocation can be surveyed from three perspectives: mutual revocation (iqala), legal unilateral wills (khiar al-majlis, khiar al-ayb), and contractual wills (khiar al-shart). Revocation of a surrogacy contract either by the genetic parents, surrogate or the fertility clinic is allowed by Muslim jurists only when the embryo lacks personhood. Based on Islamic teachings, the termination of a surrogacy contract in and after the sixteenth week of pregnancy, when the embryo acquires a human soul (ensoulment), is not allowed. However religious thought emphasizes the moral status of the fetus before the sixteenth week and states that optional termination of the surrogacy contract is not permitted while the fetus becomes a human being. Copyright© by Royan Institute. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shirakashi, Ryo, E-mail: aa21150@iis.u-tokyo.ac.jp; Mischke, Miriam; Fischer, Peter
2012-11-09
Highlights: Black-Right-Pointing-Pointer Electrorotation offers a non-invasive tool for dielectric analysis of fish embryos. Black-Right-Pointing-Pointer The three-shell dielectric model matches the rotation spectra of medaka eggs. Black-Right-Pointing-Pointer The capacitance value suggests a double-membrane structure of yolk envelope. -- Abstract: The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to earlymore » somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.« less
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Sreetharan, S; Thome, C; Mitz, C; Eme, J; Mueller, C A; Hulley, E N; Manzon, R G; Somers, C M; Boreham, D R; Wilson, J Y
2015-09-01
A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development. © 2015 The Fisheries Society of the British Isles.
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[Performance of in vitro fertilization in Germany].
van der Ven, Hans; Montag, Markus; van der Ven, Katrin
2002-07-01
In Germany the application of assisted reproductive techniques (ART) is regulated by federal legislation. Compared with the international situation the "German Embryo Protection Law" is very "restrictive" and various methods of ART are prohibited, e.g. oocyte/embryo donation, embryo cryopreservation and Preimplantation Genetic Diagnosis (PGD). Furthermore, in Germany only 1 to 3 fertilized oocytes may be cultured to embryo. All these embryos then have to be transferred into the uterus of a particular patient. Additional fertilized oocytes can only be cryopreserved in a pronuclear state. The success rate of ART has increased significantly over the past few years owing to the introduction of blastocyst cultures and the selection of 1 to 2 good quality blastocysts for embryo transfer. Furthermore, the transfer of only 1 to 2 blastocysts effectively reduces the risk of high rank multiple pregnancies. In Germany, however, the selection of only a few good quality blastocysts for transfer is prohibited by law. New laboratory techniques, e.g. pronuclear scoring and polar body biopsy screening for aneuploidy are in accordance with German law. The application of these methods provides a selection of "good quality oocytes" and seems to increase the overall success rate. Further studies are required, however. The success rate, quality and cost effectiveness of ART in Germany appears compromised when compared with many other countries. What is more, in contrast to the international situation research and development in ART in Germany has been decreasing constantly over the past few years, due to the inappropriate regulations of the German health care system and the insufficient support given to university-based centers.
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An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.
Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H
1986-04-01
The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.
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Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy
NASA Astrophysics Data System (ADS)
Gareau, Daniel
2014-03-01
The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy
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Souza, Iara Maria de Almeida; Caitité, Amanda Muniz Logeto
2010-06-01
Based on news reports from Brazilian papers, the article examines the case of scientific fraud involving cloned embryos, committed by South Korean scientist Hwang. The media generally focus on the intellectual process of science, its discoveries, and the new possibilities it promises. In this case, however, science is shown the other way around, revealing a web that interweaves elements of a radically disparate nature, like the Korean government, researchers, tools, research funds, human eggs and funguses, scientific journals, among others. These ties are what make up science in practice, yet they only become visible in the media when there is tension between them and, in this case, when something illicit happens.
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Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe
2016-09-01
The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.
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The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus).
Veres, Monika; Duselis, Amanda R; Graft, Audrey; Pryor, William; Crossland, Janet; Vrana, Paul B; Szalai, Gabor
2012-01-15
Although laboratory-reared species of the genus Peromyscus-including deer mice-are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.
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Zebrafish Embryo Disinfection with Povidone–Iodine: Evaluating an Alternative to Chlorine Bleach
Amack, Jeffrey D.; Whipps, Christopher M.
2016-01-01
Abstract Mycobacteriosis is a common bacterial infection in laboratory zebrafish caused by several different species and strains of Mycobacterium, including both rapid and slow growers. One control measure used to prevent mycobacterial spread within and between facilities is surface disinfection of eggs. Recent studies have highlighted the effectiveness of povidone–iodine (PVPI) on preventing propagation of Mycobacterium spp. found in zebrafish colonies. We evaluated the effect of disinfection using 12.5–50 ppm PVPI (unbuffered and buffered) on zebrafish exposed at 6 or 24 h postfertilization (hpf) to determine if this treatment is suitable for use in research zebrafish. Our results show that 6 hpf embryos are less sensitive to treatment as fewer effects on mortality, developmental delay, and deformity were observed. We also found that buffered PVPI treatment results in a greater knockdown of Mycobacterium chelonae and Mycobacterium marinum, as well as results in decreased harmful effects on embryos. Treatments of shorter (2 min vs. 5 min) duration were also more effective at killing mycobacteria in addition to resulting in fewer effects on embryo health. In addition, we compared the efficacy of a rinsing regimen to rinsing and disinfecting. Based on the findings of this study, we recommend disinfecting embryos for 2 min with buffered PVPI at 12.5–25 ppm. PMID:27351620
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Ethics of PGD: thoughts on the consequences of typing HLA in embryos.
Edwards, R G
2004-08-01
As with so many fields of study associated with assisted human reproduction, many ethical issues are raised by the practice of preimplantation diagnosis of inherited disease (PGD). Some are part and parcel of assisted conception, e.g.the rights of human embryos in vitro and of embryologists to establish them, carry out research and discard them. Others unique to clinical PGD were discussed at an earlier meeting on PGD (Edwards et al., 2003). Recent developments in PGD are discussed briefly in this Commentary, especially the ethics of designer babies.
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Wolpert, Lewis
2015-01-01
I am a developmental biologist, but I started off as a civil engineer. I did some research on soil mechanics but decided to change to biology. A friend changed my life when he told me about the mechanics of cell division, on which I did my PhD at Kings College. I then worked on the morphogenesis of the sea urchin embryo and became interested in how embryos are patterned, and I proposed positional information as a basic mechanism. I was a professor at the Middlesex Hospital Medical School, where we concentrated on how the chick limb developed.
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Considerations Regarding Embryo Culture Conditions: From Media to Epigenetics.
Simopoulou, Mara; Sfakianoudis, Konstantinos; Rapani, Anna; Giannelou, Polina; Anifandis, George; Bolaris, Stamatis; Pantou, Agni; Lambropoulou, Maria; Pappas, Athanasios; Deligeoroglou, Efthimios; Pantos, Konstantinos; Koutsilieris, Michael
2018-01-01
There are numerous reports on embryo culture media and conditions in the laboratory, as the subject is multifaceted and complex, reflecting the variation in practice. In this scoping review, we attempt to approach the topic of culture media and conditions from the practitioners' perspective aiming to highlight, in a comprehensive fashion, important aspects regarding the options available, introduce points of debate and controversy, while maintaining the viewpoint of the practicing embryologist's concerns. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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Is the hypothesis of preimplantation genetic screening (PGS) still supportable? A review.
Gleicher, Norbert; Orvieto, Raoul
2017-03-27
The hypothesis of preimplantation genetic diagnosis (PGS) was first proposed 20 years ago, suggesting that elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos during in vitro fertilization (IVF), increase pregnancy and live birth rates and reduce miscarriages. The aforementioned improved outcome was based on 5 essential assumptions: (i) Most IVF cycles fail because of aneuploid embryos. (ii) Their elimination prior to embryo transfer will improve IVF outcomes. (iii) A single trophectoderm biopsy (TEB) at blastocyst stage is representative of the whole TE. (iv) TE ploidy reliably represents the inner cell mass (ICM). (v) Ploidy does not change (i.e., self-correct) downstream from blastocyst stage. We aim to offer a review of the aforementioned assumptions and challenge the general hypothesis of PGS. We reviewed 455 publications, which as of January 20, 2017 were listed in PubMed under the search phrase < preimplantation genetic screening (PGS) for aneuploidy>. The literature review was performed by both authors who agreed on the final 55 references. Various reports over the last 18 months have raised significant questions not only about the basic clinical utility of PGS but the biological underpinnings of the hypothesis, the technical ability of a single trophectoderm (TE) biopsy to accurately assess an embryo's ploidy, and suggested that PGS actually negatively affects IVF outcomes while not affecting miscarriage rates. Moreover, due to high rates of false positive diagnoses as a consequence of high mosaicism rates in TE, PGS leads to the discarding of large numbers of normal embryos with potential for normal euploid pregnancies if transferred rather than disposed of. We found all 5 basic assumptions underlying the hypothesis of PGS to be unsupported: (i) The association of embryo aneuploidy with IVF failure has to be reevaluated in view how much more common TE mosaicism is than has until recently been appreciated. (ii) Reliable elimination of presumed aneuploid embryos prior to embryo transfer appears unrealistic. (iii) Mathematical models demonstrate that a single TEB cannot provide reliable information about the whole TE. (iv) TE does not reliably reflect the ICM. (v) Embryos, likely, still have strong innate ability to self-correct downstream from blastocyst stage, with ICM doing so better than TE. The hypothesis of PGS, therefore, no longer appears supportable. With all 5 basic assumptions underlying the hypothesis of PGS demonstrated to have been mistaken, the hypothesis of PGS, itself, appears to be discredited. Clinical use of PGS for the purpose of IVF outcome improvements should, therefore, going forward be restricted to research studies.
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A life in Science with the avian embryo.
Le Douarin, Nicole M
2018-01-01
My career in research was a second thought. I first (during 8 years) worked as a secondary school teacher and after 4-5 years, during which my two daughters were born, I found a way to escape from what was to be a lifetime job. For two years, my initiation to research was limited to the free time left by my teaching duties. This period of time was a bit "complicated" but not enough to prevent me to realize that research was really what I wanted to do for the rest of my life… And this was when I became acquainted with the chick embryo. This companionship later became extended to another representative of the avian world: the quail (Coturnix coturnix japonica). I recall in the following lines a survey of scientific stories that came out from my association with these precious animals, ... not without a feeling of gratitude.
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Donating embryos to stem cell research.
Scully, Jackie Leach; Haimes, Erica; Mitzkat, Anika; Porz, Rouven; Rehmann-Sutter, Christoph
2012-03-01
This paper is based on linked qualitative studies of the donation of human embryos to stem cell research carried out in the United Kingdom, Switzerland, and China. All three studies used semi-structured interview protocols to allow an in-depth examination of donors' and non-donors' rationales for their donation decisions, with the aim of gaining information on contextual and other factors that play a role in donor decisions and identifying how these relate to factors that are more usually included in evaluations made by theoretical ethics. Our findings have implications for one factor that has previously been suggested as being of ethical concern: the role of gratitude. Our empirical work shows no evidence that interpersonal gratitude is an important factor, but it does support the existence of a solidarity-based desire to "give something back" to medical research. Thus, we use empirical data to expand and refine the conceptual basis of bioethically theorizing the IVF-stem cell interface.
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Human embryonic stem cell research: implications from an ethical and legal standpoint.
Trepagnier, D M
2000-12-01
The purpose of this paper is to discuss the ethical and legal implications of one of the newest and most controversial medical breakthroughs. Stem cell research has been performed on mice for many years, but human embryonic stem cells are believed by scientists to be the basis for possible treatments and/or cures to many diseases affecting millions of people around the world. In order to perform research on human embryonic stem cells, numerous ethical issues must be addressed. Guidelines and protocols can be established in order to allow scientists to pursue new medical advances while maintaining the highest ethical standards in the use of human embryos. An alternative to using embryos is adult stem cells which have recently proven to be more versatile than previously believed. Opposing views will always be encountered when facing new science technologies. Where should the ethical line be drawn?
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Gately, Pauline
2011-01-01
In 2006 the Government issued a white paper in which it proposed a ban on human-animal embryo research pending greater clarity on its potential. The Commons Select Committee on Science and Technology initiated an Inquiry and concluded that such research was necessary and should be permitted immediately. The Government agreed and this is reflected in revised legislation. The Government has issued guidelines on the gathering and use of scientific advice and evidence, designed to ensure that these are "credible, reliable and objective." This article tests the Committee's approach in the light of its remit and these, and other, relevant guidelines and concludes that it failed to meet these standards. Rather it effectively ceded to an interest group the regulation of its own activities. The article ends by suggesting alterations to the Committee's remit and composition designed to ensure that the public interest is better protected in future.
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Embryo density and medium volume effects on early murine embryo development.
Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C
1992-10-01
One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.
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Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L
2012-06-15
MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.
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Elevated nitrate alters the metabolic activity of embryonic zebrafish.
Conlin, Sarah M; Tudor, M Scarlett; Shim, Juyoung; Gosse, Julie A; Neilson, Andrew; Hamlin, Heather J
2018-04-01
Nitrate accumulation in aquatic reservoirs from agricultural pollution has often been overlooked as a water quality hazard, yet a growing body of literature suggests negative effects on human and wildlife health following nitrate exposure. This research seeks to understand differences in oxygen consumption rates between different routes of laboratory nitrate exposure, whether via immersion or injection, in zebrafish (Danio rerio) embryos. Embryos were exposed within 1 h post fertilization (hpf) to 0, 10, and 100 mg/L NO 3 -N with sodium nitrate, or to counter ion control (CIC) treatments using sodium chloride. Embryos in the immersion treatments received an injection of 4 nL of appropriate treatment solution into the perivitelline space. At 24 hpf, Oxygen Consumption Rates (OCR) were measured and recorded in vivo using the Agilent Technologies XF e 96 Extracellular Flux Analyzer and Spheroid Microplate. Immersion exposures did not induce significant changes in OCR, yet nitrate induced significant changes when injected through the embryo chorion. Injection of 10 and 100 mg/L NO 3 -N down-regulated OCR compared to the control treatment group. Injection of the 100 mg/L CIC also significantly down-regulated OCR compared to the control treatment group. Interestingly, the 100 mg/L NO 3 -N treatment further down-regulated OCR compared to the 100 mg/L CIC treatment, suggesting the potential for additive effects between the counter ion and the ion of interest. These data support that elevated nitrate exposure can alter normal metabolic activity by changing OCR in 24 hpf embryos. These results highlight the need for regularly examining the counter ion of laboratory nitrate compounds while conducting research with developing zebrafish, and justify examining different routes of laboratory nitrate exposure, as the chorion may act as an effective barrier to nitrate penetration in zebrafish, which may lead to conservative estimates of significant effects in other species for which nitrate more readily penetrates the chorion. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Newkirk, Catherine E; Gagnon, Zofia E; Pavel Sizemore, Ioana E
2014-01-01
Research was conducted to examine the hematological effects of heavy metals (platinum (Pt ((IV))), palladium (Pd ((II))), rhodium (Rh ((III))), antimony (Sb ((III)) and Sb ((V))), and silver nanoparticles (AgNPs)) on white blood cells in mammalian (rat) and avian (chick embryo) models. These metals are used in many everyday products and are accumulating in our environment. Six-week old Sprague-Dawley female rats were treated daily by gavage and six-day old, fertile, specific pathogen-free white leghorn strain chick embryos' eggs were injected on days 7 and 14 of incubation with 0.0, 1.0, 5.0 or 10.0 ppm concentrations of Pt ((IV)) and a platinum group metal (PGM) mix of Pt ((IV)), Pd ((II)) and Rh ((III)). Chick embryos were also tested with 1.0 or 5.0 ppm of antimony compounds (Sb ((III)) and Sb ((V))) and 0.0, 15.0, 30.0, 60.0, or 100.0 ppm of silver nanoparticles (AgNPs). After 8 weeks of treatment, blood was obtained from the rats by jugular cut down and from chick embryos on day 20 of incubation by heart puncture. Blood smears were made and stained and a differential white cell count was performed on each. Examination of the smears revealed unconventional dose responses, stimulation of the immune response, and decreases in leukocyte production with various metals and concentrations. Chick embryos responded differently than rats to Pt and the PGM mix; suggesting that species differences and/or stage of development are important components of response to heavy metals. Route of administration of the metals might also influence the response. All of the heavy metals tested affected the immune responses of the tested animals as demonstrated by changes in the types and numbers of leukocytes. Our findings warrant further research to determine the mechanism of these effects and to understand and prevent toxicological effects in humans and other living organisms.
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How do laboratory embryo transfer techniques affect IVF outcomes? A review of current literature.
Sigalos, George; Triantafyllidou, Olga; Vlahos, Nikos
2017-04-01
Over the last few years, many studies have focused on embryo selection methods, whereas little attention has been given to the standardization of the procedure of embryo transfer. In this review, several parameters of the embryo transfer procedure are examined, such as the: (i) culture medium volume and loading technique; (ii) syringe and catheters used for embryo transfer; (iii) viscosity and composition of the embryo transfer medium; (iv) environment of embryo culture; (v) timing of embryo transfer; (vi) and standardization of the embryo transfer techniques. The aim of this manuscript is to review these factors and compare the existing embryo transfer techniques and highlight the need for better embryo transfer standardization.
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Ozáez, Irene; Morcillo, Gloria; Martínez-Guitarte, José-Luis
2016-07-01
Several organic UV filters have hormonal activity in vertebrates, as demonstrated in fishes, rodents and human cells. Despite the accumulation of filter contaminants in aquatic systems, research on their effects on the endocrine systems of freshwaters invertebrates is scarce. In this work, the effects of five frequently used UV filters were investigated in embryos and larvae of Chironomus riparius, which is a reference organism in ecotoxicology. LC50 values for larvae as well as the percentage of eclosion of eggs were determined following exposures to: octyl-p-methoxycinnamate (OMC) also known as 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4MBC); 4-hydroxybenzophenone (4HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). To assess sublethal effects, expression levels of the genes coding for the ecdysone receptor (EcR) and heat shock protein HSP70 were investigated as biomarkers for endocrine and stress effects at the cellular level. Life-stage-dependent sensitivity was found. In embryos, all of the UV filters provoked a significant overexpression of EcR at 24h after exposure. OC, 4MBC and OD-PABA also triggered transcriptional activation of the hsp70 stress gene in embryos. In contrast, in larvae, only 4MBC and OMC/EHMC increased EcR and hsp70 mRNA levels and OD-PABA upregulated only the EcR gene. These results revealed that embryos are particularly sensitive to UV filters, which affect endocrine regulation during development. Most UV filters also triggered the cellular stress response, and thus exhibit proteotoxic effects. The differences observed between embryos and larvae and the higher sensitivity of embryos highlight the importance of considering different life stages when evaluating the environmental risks of pollutants, particularly when analyzing endocrine effects. Copyright © 2016 Elsevier B.V. All rights reserved.
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Rios, G L; Mucci, N C; Kaiser, G G; Alberio, R H
2010-03-01
The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P<0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1 microL, 61.3% 0.5 microL and 80.5% control, P<0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P<0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification-warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.
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González Altamiranda, Erika; Manrique, Julieta M; Pérez, Sandra E; Ríos, Glenda L; Odeón, Anselmo C; Leunda, María R; Jones, Leandro R; Verna, Andrea
2015-01-01
Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.
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Editing the human genome: where ART and science intersect.
Hershlag, Avner; Bristow, Sara L
2018-06-07
The rapid development of gene-editing technologies has led to an exponential rise in both basic and translational research initiatives studying molecular processes and investigating possible clinical applications. Early experiments using genome editing to study human embryo development have contradicted findings in studies on model organisms. Additionally, a series of four experiments over the past 2 years set out to investigate the possibilities of introducing genetic modifications to human embryos, each with varying levels of success. Here, we discuss the key findings of these studies, including the efficiency, the safety, the potential untoward effects, major flaws of the studies, and emerging alternative genome editing methods that may allow overcoming the hurdles encountered so far. Given these results, we also raise several questions about the clinical utilization of germline gene editing: For which indications is gene editing appropriate? How do gene-editing technologies compare with genetic testing methods currently used for screening embryos? What are the ethical considerations we should be concerned about? While further research is underway, and our understanding of how to implement this technology continues to evolve, it is critical to contemplate if and how it should be translated from the bench to clinical practice.
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Research on tomato seed vigor based on X-ray digital image
NASA Astrophysics Data System (ADS)
Zhao, Xueguan; Gao, Yuanyuan; Wang, Xiu; Li, Cuiling; Wang, Songlin; Feng, Qinghun
2016-10-01
Seed size, interior abnormal and damage of the tomato seeds will affect the germination. The purpose of this paper was to study the relationship between the internal morphology, seed size and seed germination of tomato. The preprocessing algorithm of X-ray image of tomato seeds was studied, and the internal structure characteristics of tomato seeds were extracted by image processing algorithm. By developing the image processing software, the cavity area between embryo and endosperm and the whole seed zone were determined. According to the difference of area of embryo and endosperm and Internal structural condition, seeds were divided into six categories, Respectively for three kinds of tomato seed germination test, the relationship between seed vigor and seed size , internal free cavity was explored through germination experiment. Through seedling evaluation test found that X-ray image analysis provide a perfect view of the inside part of the seed and seed morphology research methods. The larger the area of the endosperm and the embryo, the greater the probability of healthy seedlings sprout from the same size seeds. Mechanical damage adversely effects on seed germination, deterioration of tissue prone to produce week seedlings and abnormal seedlings.
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Williams, Clare; Kitzinger, Jenny; Henderson, Lesley
2003-11-01
How is the embryo defined, envisaged, imagined? Who speaks on its behalf, and how? Based on a study of UK press and TV news reporting, this paper identifies the rhetorical strategies used to assert competing ethical positions around embryonic stem cell research. We show how both sides in the dispute mobilise metaphors and use personification to recruit support; and how they promote different ideas about the embryo's significance, size, and social embeddedness and present competing narratives about its origins, destiny and 'death'. The role of visual representation is key here. It does not follow the usual pattern whereby, in the abortion debate, those 'on the side' of the fetus display its image while those who are 'pro-choice' shy away from this. In the stem cell debate the pattern is inverted, highlighting the role of technologies of visualisation in defining what counts as human. Our analysis also shows how the media coverage marginalises women's perspectives, disregards more fundamental challenges to science, side-lines concerns about effectiveness or safety and curtails discussion of broader issues. We reflect on the media processes restricting debate in this way and conclude by identifying opportunities for a more inclusive discussion of science ethics.
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Informed consent and federal funding for stem cell research.
Streiffer, Robert
2008-01-01
A review of the consent forms signed by those who donated embryos for the NIH-approved embryonic stem cell lines reveals several problems, providing ethical as well as scientific reasons to overturn the Bush administration's restrictions on federal funding for stem cell research.
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Brüstle v. Greenpeace: Implications for Commercialisation of Translational Stem Cell Research.
Mansnérus, Juli
2015-04-01
The lack of consensus on a common definition of the term 'embryo' has resulted in legal uncertainty affecting the permissibility of human embryonic stem cell (hESC) research and the commercialisation prospects and patenting of inventions of hESC origin in the EU. The Brüstle v. Greenpeace case, which by providing a very broad definition of a human embryo restricts the patentability of hESC-based inventions, aims at harmonising the patenting practices regarding interpretation of Article 6.2.c of Directive 98/44/ EC. It fills the gaps in national laws by providing binding interpretation guidelines for national courts. As currently no marketing authorisations have been granted to hESC-based products, implications of this judgment for translational hESC research together with other barriers to commercialisation of such research need to be analysed. In addition, whether the main obstacles relate to patenting restrictions or whether something else in the innovation system is impeding the market entry of these innovative products is discussed.
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Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif
2015-01-01
Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881
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Qi, Shu-Tao; Liang, Li-Feng; Xian, Ye-Xing; Liu, Jian-Qiao; Wang, Weihua
2014-01-01
Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.
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Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.
Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter
2017-08-01
Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p < 0.001; D3: 19.7 vs. 27.1 vs. 21.2%; p = 0.029; Group 1. vs. Group 2. vs. Group 3). Cell number on Day 3 differed between Groups 1 and 2 (6.8 ± 2.2; 7.3 ± 2.1; p = 0.004) and Groups 2 and 3 (7.3 ± 2.1 vs. 7.0 ± 2.0; p = 0.014). Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.
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Zhang, Xinyu; Ma, Caihong; Wu, Zhangxin; Tao, Liyuan; Li, Rong; Liu, Ping; Qiao, Jie
2017-01-01
To evaluate the risk of ectopic pregnancy of embryo transfer. A retrospective cohort study on the incidence of ectopic pregnancy in fresh and frozen-thawed embryo transfer cycles from January 1 st , 2010, to January 1 st , 2015. Infertile women undergoing frozen-thawed transfer cycles or fresh transfer cycles. In-vitro fertilization, fresh embryo transfer, frozen-thawed embryo transfer, ectopic pregnancy. Ectopic pregnancy rate and clinical pregnancy rate. A total of 69 756 in vitro fertilization-embryo transfer cycles from 2010 to 2015 were analyzed, including 45 960 (65.9%) fresh and 23 796 (34.1%) frozen-thawed embryo transfer cycles. The clinical pregnancy rate per embryo transfer was slightly lower in fresh embryo transfer cycles compared with frozen-thawed embryo transfer cycles (40.8% vs 43.1%, P < .001). Frozen-thawed embryo transfer is associated with a lower incidence of ectopic pregnancy per clinical pregnancy, compared with fresh embryo transfers (odds ratio = 0.31; 95% confidence interval = 0.24-0.39). Female age and body mass index have no influence on ectopic pregnancy. In the frozen-thawed embryo transfer cycles, blastocyst transfer shows a significantly lower incidence of ectopic pregnancy (0.8% vs 1.8%, P = .002) in comparison with day 3 cleavage embryo transfer. The risk of ectopic pregnancy is lower in frozen-thawed embryo transfer cycles than fresh embryo transfer cycles, and blastocyst transfer could further decrease the ectopic pregnancy rate in frozen-thawed embryo transfer cycles.
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Ushijima, Hitoshi; Akiyama, Kiyoshi; Tajima, Toshio
2008-08-01
The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.
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Cánepa, Maria Jesús; Ortega, Nicolás Matías; Monteleone, Melisa Carolina; Mucci, Nicolas; Kaiser, German Gustavo; Brocco, Marcela; Mutto, Adrián
2014-01-01
Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART).
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Shorter Exposures to Harder X-Rays Trigger Early Apoptotic Events in Xenopus laevis Embryos
Dong, JiaJia; Mury, Sean P.; Drahos, Karen E.; Moscovitch, Marko
2010-01-01
Background A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. Methodology/Principal Findings We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. Conclusions/Significance Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic purposes. PMID:20126466
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Tavakkoli, Saeid Nazari
2017-01-01
Background One of the most controversial issues related to the human embryo is the determination of the moment when an embryo is considered a human being and acquires a moral status. Although personhood and moral status are frequently mentioned in medical ethics, they are considered interdisciplinary as concepts that shape the debate in medical law (fiqh) since their consequences are influential in the way which the parents and other individuals behave towards the embryo. Materials and Methods This analytical-descriptive research gathered relevant data in a literature search. After a description of the fundamentals and definitions, we subsequently analyzed juridical texts and selected one of the viewpoints that regarded the surrogacy contract revocation. Results The surrogacy contract is a contract based upon which two sides (infertile couple and surrogate mother) involved in making the contract are obligated to fulfill its terms. Therefore, contract revocation can be surveyed from three perspectives: mutual revocation (iqala), legal unilateral wills (khiar al-majlis, khiar al-ayb), and contractual wills (khiar al-shart). Conclusion Revocation of a surrogacy contract either by the genetic parents, surrogate or the fertility clinic is allowed by Muslim jurists only when the embryo lacks personhood. Based on Islamic teachings, the termination of a surrogacy contract in and after the sixteenth week of pregnancy, when the embryo acquires a human soul (ensoulment), is not allowed. However religious thought emphasizes the moral status of the fetus before the sixteenth week and states that optional termination of the surrogacy contract is not permitted while the fetus becomes a human being. PMID:28868846
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Brent, R.L.
Many professionals are unfamiliar with radiation biology or the quantitative nature of the risks. Frequently, microwave, ultrasound, and ionizing radiation risks are confused. Although it is impossible to prove no risk for any environmental hazard, it appears that exposure to microwave radiation below the maximal permissible levels present no measurable risk to the embryo. Ultrasound exposure from diagnostic ultrasonographic imaging equipment also is quite innocuous. It is true that continued surveillance and research into potential risks of these low-level exposures should continue, but at present ultrasound not only improves obstetric care but also reduces the necessity of diagnostic x-ray procedures.more » In the field of ionizing radiation, we have as good a comprehension of the biologic effects and the quantitative maximum risks as of any other environmental hazard. Although the animal and human data support the conclusion that no increases in the incidence of gross congenital malformations, intrauterine growth retardation, or abortion will occur with exposures less than 5 rad, that does not mean that there are definitely no risks to the embryo exposed to lower doses of radiation. Whether there exists a linear or exponential dose-response relationship or a threshold exposure for genetic, carcinogenic, cell-depleting, and life-shortening effects has not been determined. In establishing maximum permissible levels for the embryo at low exposures, refer to Tables 4, 5, 6, 8, and 9. It is obvious that the risks of 1-rad or 5-rad acute exposure are far below the spontaneous risks of the developing embryo because 15 per cent of human embryos abort, 2.7 to 3.0 per cent of human embryos have major malformations, 4 per cent have intrauterine growth retardation, and 8 to 10 per cent have early- or late-onset genetic disease. 98 references.« less
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Pre-implantation Development of Domestic Animals.
Piliszek, Anna; Madeja, Zofia E
2018-01-01
During the first days following fertilization, cells of mammalian embryo gradually lose totipotency, acquiring distinct identity. The first three lineages specified in the mammalian embryo are pluripotent epiblast, which later gives rise to the embryo proper, and two extraembryonic lineages, hypoblast (also known as primitive endoderm) and trophectoderm, which form tissues supporting development of the fetus in utero. Most of our knowledge regarding the mechanisms of early lineage specification in mammals comes from studies in the mouse. However, the growing body of evidence points to both similarities and species-specific differences. Understanding molecular and cellular mechanisms of early embryonic development in nonrodent mammals expands our understanding of basic mechanisms of differentiation and is essential for the development of effective protocols for assisted reproduction in agriculture, veterinary medicine, and for biomedical research. This review summarizes the current state of knowledge on key events in epiblast, hypoblast, and trophoblast differentiation in domestic mammals. © 2018 Elsevier Inc. All rights reserved.
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Spencer, Ricky-John; Janzen, Fredric J
2011-07-01
Incubation temperature plays a prominent role in shaping the phenotypes and fitness of embryos, including affecting developmental rates. In many taxa, including turtles, eggs are deposited in layers such that thermal gradients alter developmental rates within a nest. Despite this thermal effect, a nascent body of experimental work on environmentally cued hatching in turtles has revealed unexpected synchronicity in hatching behavior. This review discusses environmental cues for hatching, physiological mechanisms behind synchronous hatching, proximate and ultimate causes for this behavior, and future directions for research. Four freshwater turtle species have been investigated experimentally, with hatching in each species elicited by different environmental cues and responding via various physiological mechanisms. Hatching of groups of eggs in turtles apparently involves some level of embryo-embryo communication and thus is not a purely passive activity. Although turtles are not icons of complex social behavior, life-history theory predicts that the group environment of the nest can drive the evolution of environmentally cued hatching.
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MicroRNA Signaling in Embryo Development
Gross, Nicole; Khatib, Hasan
2017-01-01
Expression of microRNAs (miRNAs) is essential for embryonic development and serves important roles in gametogenesis. miRNAs are secreted into the extracellular environment by the embryo during the preimplantation stage of development. Several cell types secrete miRNAs into biological fluids in the extracellular environment. These fluid-derived miRNAs have been shown to circulate the body. Stable transport is dependent on proper packaging of the miRNAs into extracellular vesicles (EVs), including exosomes. These vesicles, which also contain RNA, DNA and proteins, are on the forefront of research on cell-to-cell communication. Interestingly, EVs have been identified in many reproductive fluids, such as uterine fluid, where their miRNA content is proposed to serve as a mechanism of crosstalk between the mother and conceptus. Here, we review the role of miRNAs in molecular signaling and discuss their transport during early embryo development and implantation. PMID:28906477
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Epigenetic disorders and male subfertility.
Boissonnas, Céline Chalas; Jouannet, Pierre; Jammes, Hélène
2013-03-01
To provide a link between epigenetics and male subfertility at the DNA, histone-protamine, and RNA levels and its consequences on fertilization and embryo development. Review of the relevant literature. University-based clinical and research laboratories. Fertile and infertile men. None. Critical review of the literature. Epigenetic markers can be modified in infertile patients. Epigenetic modifications include methylation loss or gain on the global level and on imprinted genes, high levels of histone retention in spermatozoa, and deficiencies in some transcripts involved in spermatogenesis. Interestingly, these abnormalities are all linked together, because DNA methylation maintenance depends on DNA histone-protamine configuration which itself is stabilized by spermatozoal RNAs. The paternal genome has long been considered to be silent and passive in embryo formation. The epigenetic processes associated with the paternal DNA genome highlights its importance in male fertility as well as for embryo development. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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9 CFR 98.9 - Embryos refused entry.
Code of Federal Regulations, 2012 CFR
2012-01-01
... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...
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9 CFR 98.9 - Embryos refused entry.
Code of Federal Regulations, 2014 CFR
2014-01-01
... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...
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9 CFR 98.9 - Embryos refused entry.
Code of Federal Regulations, 2013 CFR
2013-01-01
... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...
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9 CFR 98.9 - Embryos refused entry.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...
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9 CFR 98.9 - Embryos refused entry.
Code of Federal Regulations, 2011 CFR
2011-01-01
... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...
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Nagy, R A; van Montfoort, A P A; Dikkers, A; van Echten-Arends, J; Homminga, I; Land, J A; Hoek, A; Tietge, U J F
2015-05-01
Are bile acids (BA) and their respective subspecies present in human follicular fluid (FF) and do they relate to embryo quality in modified natural cycle IVF (MNC-IVF)? BA concentrations are 2-fold higher in follicular fluid than in serum and ursodeoxycholic acid (UDCA) derivatives were associated with development of top quality embryos on Day 3 after fertilization. Granulosa cells are capable of synthesizing BA, but a potential correlation with oocyte and embryo quality as well as information on the presence and role of BA subspecies in follicular fluid have yet to be investigated. Between January 2001 and June 2004, follicular fluid and serum samples were collected from 303 patients treated in a single academic centre that was involved in a multicentre cohort study on the effectiveness of MNC-IVF. Material from patients who underwent a first cycle of MNC-IVF was used. Serum was not stored from all patients, and the available material comprised 156 follicular fluid and 116 matching serum samples. Total BA and BA subspecies were measured in follicular fluid and in matching serum by enzymatic fluorimetric assay and liquid chromatography-mass spectrometry, respectively. The association of BA in follicular fluid with oocyte and embryo quality parameters, such as fertilization rate and cell number, presence of multinucleated blastomeres and percentage of fragmentation on Day 3, was analysed. Embryos with eight cells on Day 3 after oocyte retrieval were more likely to originate from follicles with a higher level of UDCA derivatives than those with fewer than eight cells (P < 0.05). Furthermore, follicular fluid levels of chenodeoxycholic derivatives were higher and deoxycholic derivatives were lower in the group of embryos with fragmentation compared with those without (each P < 0.05). Levels of total BA were 2-fold higher in follicular fluid compared with serum (P < 0.001), but had no predictive value for oocyte and embryo quality. Only samples originating from first cycle MNC-IVF were used, which resulted in 14 samples only from women with an ongoing pregnancy, therefore further prospective studies are required to confirm the association of UDCA with IVF pregnancy outcomes. The inter-cycle variability of BA levels in follicular fluid within individuals has yet to be investigated. We checked for macroscopic signs of contamination of follicular fluid by blood but the possibility that small traces of blood were present within the follicular fluid remains. Finally, although BA are considered stable when stored at -20°C, there was a time lag of 10 years between the collection and analysis of follicular fluid and serum samples. The favourable relation between UDCA derivatives in follicular fluid and good embryo development and quality deserves further prospective research, with live birth rates as the end-point. This work was supported by a grant from the Netherlands Organisation for Scientific Research (VIDI Grant 917-56-358 to U.J.F.T.). No competing interests are reported. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda
2016-01-01
Background: Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. Materials and Methods: We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. Results: The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or “frosties,” show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50–85%). Conclusions: We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol. PMID:27512686
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de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José
2014-04-01
To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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Development, differentiation and manipulation of chicken germ cells.
Nakamura, Yoshiaki; Kagami, Hiroshi; Tagami, Takahiro
2013-01-01
Germ cells are the only cell type capable of transmitting genetic information to the next generation. During development, they are set aside from all somatic cells of the embryo. In many species, germ cells form at the fringe of the embryo proper and then traverse through several developing somatic tissues on their migration to the emerging gonads. Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. Unlike other species, in avian embryos, PGCs use blood circulation for transport to the future gonadal region. This unique accessibility of avian PGCs during early development provides an opportunity to collect and transplant PGCs. The recent development of methods for production of germline chimeras by transfer of PGCs, and long-term cultivation methods of chicken PGCs without losing their germline transmission ability have provided important breakthroughs for the preservation of germplasm , for the production of transgenic birds and study the germ cell system. This review will describe the development, migration, differentiation and manipulation of germ cells, and discuss the prospects that germ cell technologies offer for agriculture, biotechnology and academic research. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.
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NASA Astrophysics Data System (ADS)
Selvaggi, L.; Pasakarnis, L.; Brunner, D.; Aegerter, C. M.
2018-04-01
Magnetic tweezers are mainly divided into two classes depending on the ability of applying torque or forces to the magnetic probe. We focused on the second category and designed a device composed by a single electromagnet equipped with a core having a special asymmetric profile to exert forces as large as 230 pN-2.8 μm Dynabeads at distances in excess of 100 μm from the magnetic tip. Compared to existing solutions our magnetic tweezers overcome important limitations, opening new experimental paths for the study of a wide range of materials in a variety of biophysical research settings. We discuss the benefits and drawbacks of different magnet core characteristics, which led us to design the current core profile. To demonstrate the usefulness of our magnetic tweezers, we determined the microrheological properties inside embryos of Drosophila melanogaster during the syncytial stage. Measurements in different locations along the dorsal-ventral axis of the embryos showed little variation, with a slight increase in cytoplasm viscosity at the periphery of the embryos. The mean cytoplasm viscosity we obtain by active force exertion inside the embryos is comparable to that determined passively using high-speed video microrheology.
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Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition
NASA Astrophysics Data System (ADS)
Wang, Qian; Gosik, Kirk; Xing, Sujuan; Jiang, Libo; Sun, Lidan; Chinchilli, Vernon M.; Wu, Rongling
2017-03-01
Epigenetic reprogramming is thought to play a critical role in maintaining the normal development of embryos. How the methylation state of paternal and maternal genomes regulates embryogenesis depends on the interaction and coordination of the gametes of two sexes. While there is abundant research in exploring the epigenetic interactions of sperms and oocytes, a knowledge gap exists in the mechanistic quantitation of these interactions and their impact on embryo development. This review aims at formulating a modeling framework to address this gap through the integration and synthesis of evolutionary game theory and the latest discoveries of the epigenetic control of embryo development by next-generation sequencing. This framework, named epigenetic game theory or epiGame, views embryogenesis as an ecological system in which two highly distinct and specialized gametes coordinate through either cooperation or competition, or both, to maximize the fitness of embryos under Darwinian selection. By implementing a system of ordinary differential equations, epiGame quantifies the pattern and relative magnitude of the methylation effects on embryogenesis by the mechanisms of cooperation and competition. epiGame may gain new insight into reproductive biology and can be potentially applied to design personalized medicines for genetic disorder intervention.
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Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej
2017-06-23
Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.
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Kufner, K; Tonne, M; Barth, J
2009-01-01
Improved pregnancy rates in IVF have led to increasing numbers of surplus embryos which can potentially be used for purposes like donation to another infertile couple or further research. Individuals report high levels of ambivalence concerning the donation of surplus embryos. This study examined which strategies infertile patients use to deal with this ambivalence when asked to evaluate potential dispositions of surplus embryos created during IVF. Guideline-based interviews with fertility patients were audio-recorded and transcribed verbatim. Following the principle of theoretical sampling, eight interviews were analysed by use of Grounded Theory. Analyses focused on processes of individual attitude formation. Strategies for handling ambivalence during attitude formation were identified: the six strategies comprise cognitive and communicative strategies, and were integrated into a model of attitude formation under ambivalence. As ambivalence is a relevant phenomenon in attitude formation within IVF treatment, assessment of ambivalence is strongly recommended in social science studies investigating ethical problems in patient care. In the context of informed consent, there is a need for individual counselling which draws attention to the conflicting values during attitude formation. Counsellors should be aware of the signs of and the strategies to deal with ambivalence.
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Isom, S Clay; Li, Rong Feng; Whitworth, Kristin M; Prather, Randall S
2012-03-01
Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality. Copyright © 2011 Wiley Periodicals, Inc.
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Transfer of bovine demi-embryos with and without the zona pellucida.
Warfield, S J; Seidel, G E; Elsden, R P
1987-09-01
Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.
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The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.
Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat
2015-04-01
The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.
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Zhao, H; Teng, X M; Li, Y F
2017-11-25
Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.
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Surgical manipulation of mammalian embryos in vitro.
Naruse, I; Keino, H; Taniguchi, M
1997-04-01
Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.
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Equine cloning: in vitro and in vivo development of aggregated embryos.
Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F
2012-07-01
The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.
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[Stem cells and therapeutic cloning, medical perspectives under discussion].
Manuel, Catherine; Lafon, Claude; Hairion, Dominique; Antoniotti, Stéphanie
2004-03-13
Innovative biotechnical progress over the past few years regards stem cells and therapeutic cloning, which open promising medical horizons for many presently incurable diseases. THE CURRENT DEBATE: The research work in France has been stalled because of the prohibitions listed in the so-called "bioethical" laws of 1994. The ongoing revision of these laws is based on a certain number of ethical questions and launches a disputable parlementary debate. Other than reproductive cloning and research on the embryo, the possibilities provided by stem cells and therapeutic cloning should be emphasized and the different positions advanced specified, showing an evolution in the laws in France. ABUSIVE LEGISLATIVE PROHIBITIONS: The proposed law, which maintains the prohibition for research on the embryo, with a 5-Year dispensation, and which explicitly prohibits therapeutic cloning, is not in keeping with the widening of in this field expected by research teams. Many scientists and physicians, supported by patients' associations, are aware of the importance of therapeutic progress attached to such research. They should not be stalled in their studies by the prohibitions maintained in the new law.
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Kaser, Daniel J
2017-03-01
Fellows in Reproductive Endocrinology and Infertility training are expected to complete 18 months of clinical, basic, or epidemiological research. The goal of this research is not only to provide the basis for the thesis section of the oral board exam but also to spark interest in reproductive medicine research and to provide the next generation of physician-scientists with a foundational experience in research design and implementation. Incoming fellows often have varying degrees of training in research methodology and, likewise, different career goals. Ideally, selection of a thesis topic and mentor should be geared toward defining an "answerable" question and building a practical skill set for future investigation. This contribution to the JARG Young Investigator's Forum revisits the steps of the scientific method through the lens of one recently graduated fellow and his project aimed to test the hypothesis that "sequential oxygen exposure (5% from days 1 to 3, then 2% from days 3 to 5) improves blastocyst yield and quality compared to continuous exposure to 5% oxygen among human preimplantation embryos."
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The importance of Zebrafish in biomedical research.
Tavares, Bárbara; Santos Lopes, Susana
2013-01-01
Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.
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Building extended families through embryo donation: the experiences of donors and recipients.
Goedeke, S; Daniels, K; Thorpe, M; Du Preez, E
2015-10-01
How do embryo donors and recipients understand and experience embryo donation (ED)? Donors and recipients regard the genetic link between donors and donor-conceived child as significant, and draw on metaphors of ED as adoption and as building extended family networks in order to manage the complex interplay of genetic, gestational and social aspects of reproduction and family-building. The uptake of embryo donation internationally is relatively low and there has been little research into the outcomes and experiences of ED for donor and recipient families. Embryo donors and recipients in New Zealand were interviewed between March and October 2012 as to their understandings and experiences of embryo donation. Participants were recruited from across New Zealand and interviews were conducted with 22 donors (10 couples, 2 individuals) and 15 recipients (5 couples, 5 individuals). Interview data were analysed thematically to identify factors that influenced their perspectives and affected their experiences. The genetic link between donors and offspring is regarded as bestowing immutable social ties by both donors and recipients. This makes ED a difficult choice for both donors, who experience a sense of ongoing responsibility for offspring welfare, and for recipients, who may have concerns about the donor-offspring bond. Disclosure of genetic background is regarded as critical for the welfare of both the donor-conceived child as well as siblings in the donor family. Both donors and recipients draw parallels between ED and open adoption practices, and draw on extended family constructs in order to describe their relationship to each other. Donors and recipients were interviewed at various stages of the ED process, and experiences may change over time. The comparison of ED with open adoption practice and the metaphor of extended family-building both help make sense of and complicate the ED experience. These concepts help to structure expectations for the assessment for ED, the disclosure of donor conception, and the nature of relationships, information-exchange and contact. This study was funded by an AUT University Faculty of Health Environmental Sciences research grant. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.
Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe
2016-01-01
In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.
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Comparison of effects of albendazole sulfoxide on in vitro produced bovine embryos and rat embryos.
Piscopo, S E; Smoak, I W
1997-09-01
To evaluate and compare effects of albendazole sulfoxide (ABZSO) on rat embryos and bovine embryos produced in vitro. In vitro produced bovine embryos. Rat embryos recovered from naturally bred Sprague-Dawley rats. 4- and 8-cell bovine embryos were randomly allocated to ABZSO or vehicle control groups. After 48 hours, embryos were evaluated for cell number and blastomere morphology. Rat embryos of similar stages, flushed from the uterine tube on gestational day 2-5, were randomly allocated to treatment or control groups. After 24 hours, embryos were evaluated as described previously. 44% of control bovine embryos divided in culture (> or = 16-cell stage). Fifteen percent of the controls had morphologic abnormalities, including disparity in blastomere size and cytoplasmic vacuoles and stippling. Treated (> or = 1 microgram of ABZSO/ml) bovine embryos differed (P < 0.0001) from controls, with 4% development and 93% abnormal morphology. Forty-five percent of control rat embryos divided in culture. Treated (> or = 500 ng of ABZSO/ml) rat embryos differed (P < 0.0003) from controls with regard to ability to divide. There were no consistent morphologic abnormalities in rat embryos. In vitro produced bovine embryos were susceptible to ABZSO at a concentration > or = 1 microgram/ ml, resulting in decreased ability to divide and presence of gross morphologic abnormalities. Rat embryos produced in vivo and exposed in vitro to ABZSO at a concentration > or = 500 ng/ml had decreased ability to divide in culture. Despite severe effects of ABZSO (> or = 1 microgram/ml) on bovine embryo development in vitro, it is beyond the scope of this study to speculate whether a therapeutic dosage of albendazole (10 mg/kg of body weight) would result in necessary concentrations of ABZSO in vivo to disrupt embryogenesis.
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SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei
2012-01-01
Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384
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Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.
Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito
2016-05-01
The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. © 2015 Japanese Teratology Society.
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Yu, Kaimin; Li, Guochao; Feng, Weimin; Liu, Lili; Zhang, Jiayu; Wu, Wei; Xu, Lei; Yan, Yanchun
2015-09-05
The potential interference of endocrine disrupting chemicals (EDCs) on aquatic animals and humans has drawn wide attention in recent years. Reports have shown that some organophosphorus pesticides were a kind of EDCs, but their effects on fish species are still under research. In present study, flow cytometry data of HEC-1B cell line showed that chlorpyrifos (CPF) could increase cell proliferation index like 17β-estradiol (E2), but the effect of CPF was weaker than of E2 in the same concentration. Moreover, CPF altered the expression pattern of estrogen-responsive gene VTG and ERα in zebrafish embryos. When exposed to CPF at various concentrations (0, 0.10, 0.25, 0.50, 0.75 and 1.00mg/L) for 48h during the embryo stage, compared with controls, the hatching rate of treated groups significantly increased at the same time and the hatching rate of embryos was proportional to CPF concentration. The mRNA expression levels of c-myc, cyclin D1, Bax and Bcl-2, which are closely related to cell proliferation and cell apoptosis, were disturbed by CPF in zebrafish embryos after exposure treated for 48h. In addition, acridine orange (AO) staining of zebrafish embryos showed that cell apoptosis was appeared in the 0.75, 1.00mg/L CPF treated groups. Taken together, the results obtained in the present study indicated that chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish. Copyright © 2015. Published by Elsevier Ireland Ltd.
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van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer
2017-11-25
We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.
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Helicobacter Infection Significantly Alters Pregnancy Success in Laboratory Mice.
Bracken, Tara C; Cooper, Caitlin A; Ali, Zil; Truong, Ha; Moore, Julie M
2017-05-01
Helicobacter spp. are gram-negative, helically shaped bacteria that cause gastric and enterohepatic infections in mammalian species. Although Helicobacter infection frequently is implicated to interfere with reproductive success, few experimental data support these claims. We therefore retrospectively investigated the effect of Helicobacter infection on murine pregnancy outcome after the identification of endemic Helicobacter infection in an animal research facility. Multiplex conventional PCR analysis was used to characterize Helicobacter infection status in one inbred and 2 transgenic strains of mice in 2 self-contained rooms assigned to the same investigator. Outcomes of timed-mating experiments were compared among Helicobacter spp.-infected and uninfected mice of the same strain; Helicobacter infection was eradicated from the colony through fostering with uninfected dams. Although Helicobacter infection affected fecundity in only one strain of transgenic mouse, the total number of embryos per gravid uterus was significantly reduced in C57BL/6J mice that were infected with a single Helicobacter species, H. typhlonius. Helicobacter infection was also associated with a significant increase in the number of resorbing embryos per uterus and significant decreases in pregnancy-associated weight gain relative to uninfected mice in C57BL6/J mice and one transgenic strain. Helicobacter spp.-infected mice of all tested strains exhibited higher frequency of intrauterine hemorrhaging relative to uninfected mice. These results indicate that naturally-acquired Helicobacter infection not only reduces the productivity of a research animal breeding colony, but also negatively impacts embryo health. Despite these deleterious effects, these data suggest that colonies can be rederived to be Helicobacter-free by Cesarean section and fostering with uninfected dams. This paper provides the first evidence that H. typhlonius infection is sufficient to interfere with reproductive success and embryo health of C57BL/6J mice. Animal research facilities should therefore implement Helicobacter spp. surveillance and control practices to avoid confounding experimental results and to improve breeding colony efficiency.
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ERIC Educational Resources Information Center
Keller, Dolores Elaine
1972-01-01
Summarizes evidence that mammals are basically female, with masculine characteristics being imposed by hormonal changes in embryos or post-natally. Advocates the removal of male-dominant terminology in biological research and teaching. (AL)
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Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu
2007-09-01
To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.
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Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).
Correia, Sandra M; Canhoto, Jorge M
2010-06-01
The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.
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Wu, Yan-Guang; Lazzaroni-Tealdi, Emanuela; Wang, Qi; Zhang, Lin; Barad, David H; Kushnir, Vitaly A; Darmon, Sarah K; Albertini, David F; Gleicher, Norbert
2016-08-24
Previously manual human embryology in many in vitro fertilization (IVF) centers is rapidly being replaced by closed embryo incubation systems with time-lapse imaging. Whether such systems perform comparably to manual embryology in different IVF patient populations has, however, never before been investigated. We, therefore, prospectively compared embryo quality following closed system culture with time-lapse photography (EmbryoScope™) and standard embryology. We performed a two-part prospectively randomized study in IVF (clinical trial # NCT92256309). Part A involved 31 infertile poor prognosis patients prospectively randomized to EmbryoScope™ and standard embryology. Part B involved embryos from 17 egg donor-recipient cycles resulting in large egg/embryo numbers, thus permitting prospectively alternative embryo assignments to EmbryoScope™ and standard embryology. We then compared pregnancy rates and embryo quality on day-3 after fertilization and embryologist time utilized per processed embryo. Part A revealed in poor prognosis patients no differences in day-3 embryo scores, implantation and clinical pregnancy rates between EmbryoScope™ and standard embryology. The EmbryoScope™, however, more than doubled embryology staff time (P < 0.0001). In Part B, embryos grown in the EmbyoScope™ demonstrated significantly poorer day-3 quality (depending on embryo parameter between P = 0.005 and P = 0.01). Suspicion that conical culture dishes of the EmbryoScope™ (EmbryoSlide™) may be the cause was disproven when standard culture dishes demonstrated no outcome difference in standard incubation. Though due to small patient numbers preliminary, this study raises concerns about the mostly uncontrolled introduction of closed incubation systems with time lapse imaging into routine clinical embryology. Appropriately designed and powered prospectively randomized studies appear urgently needed in well-defined patient populations before the uncontrolled utilization of these instruments further expands. NCT02246309 Registered September 18, 2014.
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Rho, G J; Johnson, W H; Betteridge, K J
1998-10-15
The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.
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Roberts, Stephen A; Hann, Mark; Brison, Daniel R
2016-02-01
Many studies have identified prognostic factors for IVF treatment outcome; however, little information is available on the mechanism of their action. Embryo-uterus models have the potential to distinguish between factors acting on the embryo directly and those acting through the uterine environment. Here we apply embryo-uterus models to comprehensive UK registry data from two periods, 2000-2005 and 2007-2011, containing 139,444 and 226,542 embryo transfer cycles, respectively. Given this large dataset, the embryo-uterus model is capable of distinguishing between uterine and embryo effects. Maternal age is the predominant predictor of live birth and acts on both the embryo and uterine components, but with larger effects on the embryo. Prolonged embryo culture is associated with greater embryo viability, reflecting the greater degree of selection, but is also associated with greater uterine receptivity. Cryopreserved embryos are less viable and were associated with poorer uterine receptivity. This work suggests that, in addition to the direct effects of in-vitro culture on the embryonic environment during the first few days of the embryo's life, the delay in transfer after extended culture or cryopreservation can lead to an altered uterine environment for the embryo after transfer. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Clinical evaluation of frozen/thawed embryo transfer following transport of oocytes and embryos
2004-01-01
Background and Aims: We evaluated the efficacy of the transport oocyte/embryo frozen/thawed embryo transfer method, in which oocytes or embryos were transported from satellite clinics to the main assisted reproductive technology (ART) center, and surplus embryos were placed in cryopreservation. Methods: We evaluated 41 cycles in 34 patients in the transport oocyte group (TO group). In the TO group the oocytes were collected at the satellite clinics, transported to the main ART center and underwent in vitro fertilization or intracytoplasmic sperm injection. Surplus embryos were used for frozen/thawed embryo transfer. We also evaluated 17 cycles in 10 patients in the transport embryo group (TE group), where surplus embryos were transported to the main ART center and used for frozen/thawed embryo transfer; and 189 cycles in 134 patients in the center group (C group), where surplus embryos collected at the same time at the main ART center were used for frozen/thawed embryo transfer. Oocytes were transported from satellite clinics in HEPES buffered human tubal fluid (HTF) culture medium, and embryos in 30% synthetic serum substitute + HEPES buffered HTF, using a portable incubator we devised. Results: The proportions of undamaged embryos after freeze/thawing were 47% for the C group, 46% for the TO group, and 46% for the TE group. The numbers of embryos transferred were 2.0 ± 0.7 for the C group, 2.0 ± 0.6 for the TO group, and 2.2 ± 0.4 for the TE group. The rate of embryo transfer was 63% for the C group, 68% for the TO group, and 76% for the TE group. Pregnancy rates per patient were 16% for the C group, 24% for the TO group, and 40% for the TE group. The embryo survival rates (number of embryos with ≥50% viable blastomeres/total number of embryos) were 55% for the C group, 60% for the TO group, and 54% for the TE group. No significant differences were seen between the C group and either the TO or TE groups in any of these parameters. Conclusions: Favorable results were achieved with the transport oocyte/embryo frozen/thawed embryo transfer method, and it is suitable for widespread clinical application. (Reprod Med Biol 2004; 3: 1–8) PMID:29662379
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Novo, Sergi; Penon, Oriol; Barrios, Leonardo; Nogués, Carme; Santaló, Josep; Durán, Sara; Gómez-Matínez, Rodrigo; Samitier, Josep; Plaza, José Antonio; Pérez-García, Luisa; Ibáñez, Elena
2013-06-01
Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process. The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested. The direct embryo tagging system developed here, once tested in human embryos, could provide fertility clinics with a novel tool to reduce the risk of mix-ups in human assisted reproduction technologies.
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Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D
2010-10-01
To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.
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Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.
Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J
2018-05-01
The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.
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Comparison of different cryopreservation methods for horse and donkey embryos.
Pérez-Marín, C C; Vizuete, G; Vazquez-Martinez, R; Galisteo, J J
2018-05-01
Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. Randomised controlled experiment. Horse (n=19) and donkey (n=16) embryos (≤300 μm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L glycerol, 1.4 mol/L glycerol + 3.6 mol/L EG and 3.4 mol/L glycerol + 4.6 mol/L EG, using Fibreplug or a 0.25 mL straw; donkey embryos were vitrified using Fibreplug with similar EG-glycerol solutions to above or 7.0 mol/L EG. Dead cells, apoptotic and fragmented nuclei, and cytoskeleton quality were assessed on thawed/warmed embryos. A significant decrease in embryo quality was observed after cryopreservation (P<0.05). Although the percentage of dead cells was lower (P<0.05) in control than in cryopreserved embryos, no differences were observed between freezing protocols used for horse or donkey embryos. While no differences were detected in the number of apoptotic cells in warmed horse embryos, in donkey embryos a higher incidence of apoptosis was measured after vitrification with EG-glycerol in Fibreplug (P<0.05). Vitrified horse embryos had a significantly (P<0.05) higher percentage of nonviable cells than donkey embryo. Actin cytoskeleton quality did not differ between treatments. Difficulties in obtaining a large number of embryos meant that the number of embryos per group was low. Vitrified horse and donkey embryos did not show higher susceptibility to cell damage than those preserved by slow freezing, whether using straws or Fibreplug. However, Fibreplug with EG 7 mol/L resulted in fewer nonviable and apoptotic cells in donkey embryos. Donkey embryos showed lower susceptibility to vitrification than horse embryos. THE SUMMARY IS AVAILABLE IN SPANISH - SEE SUPPORTING INFORMATION. © 2017 EVJ Ltd.
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Stojkovic, M; Büttner, M; Zakhartchenko, V; Riedl, J; Reichenbach, H D; Wenigerkind, H; Brem, G; Wolf, E
1999-04-30
Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.
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9 CFR 98.20 - Embryos refused entry.
Code of Federal Regulations, 2013 CFR
2013-01-01
... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...
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9 CFR 98.16 - The embryo collection unit.
Code of Federal Regulations, 2013 CFR
2013-01-01
... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...
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9 CFR 98.16 - The embryo collection unit.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...
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9 CFR 98.20 - Embryos refused entry.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...
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9 CFR 98.20 - Embryos refused entry.
Code of Federal Regulations, 2012 CFR
2012-01-01
... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...
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9 CFR 98.16 - The embryo collection unit.
Code of Federal Regulations, 2011 CFR
2011-01-01
... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...
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9 CFR 98.20 - Embryos refused entry.
Code of Federal Regulations, 2014 CFR
2014-01-01
... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...
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9 CFR 98.16 - The embryo collection unit.
Code of Federal Regulations, 2014 CFR
2014-01-01
... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...
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9 CFR 98.20 - Embryos refused entry.
Code of Federal Regulations, 2011 CFR
2011-01-01
... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...
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9 CFR 98.16 - The embryo collection unit.
Code of Federal Regulations, 2012 CFR
2012-01-01
... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...
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Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige
2015-07-01
The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. © 2014 Japanese Society of Animal Science.
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Ideta, Atsushi; Urakawa, Manami; Aoyagi, Yoshito; Saeki, Kazuhiro
2005-04-01
We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.
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Fugel, Hans-Joerg; Connolly, Mark; Nuijten, Mark
2014-10-09
New techniques in assessing oocytes and embryo quality are currently explored to improve pregnancy and delivery rates per embryo transfer. While a better understanding of embryo quality could help optimize the existing "in vitro fertilization" (IVF) therapy schemes, it is essential to address the economic viability of such technologies in the healthcare setting. An Embryo-Dx economic model was constructed to assess the cost-effectiveness of 3 different IVF strategies from a payer's perspective; it compares Embryo-Dx with single embryo transfer (SET) to elective single embryo transfer (eSET) and to double embryo transfer (DET) treatment practices. The introduction of a new non-invasive embryo technology (Embryo-Dx) associated with a cost up to €460 is cost-effective compared to eSET and DET based on the cost per live birth. The model assumed that Embryo-Dx will improve ongoing pregnancy rate/realize an absolute improvement in live births of 9% in this case. This study shows that improved embryo diagnosis combined with SET may have the potential to reduce the cost per live birth per couple treated in IVF treatment practices. The results of this study are likely more sensitive to changes in the ongoing pregnancy rate and consequently the live birth rate than the diagnosis costs. The introduction of a validated Embryo-Dx technology will further support a move towards increased eSET procedures in IVF clinical practice and vice versa.
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The pros and cons of human therapeutic cloning in the public debate.
Nippert, Irmgard
2002-09-11
Few issues linked to genetic research have raised as much controversial debate as the use of somatic cell nuclear transfer technology to create embryos specifically for stem cell research. Whereas European countries unanimously agree that reproductive cloning should be prohibited there is no agreement to be found on whether or not research into therapeutic cloning should be permitted. Since the UK took the lead and voted in favour of regulations allowing therapeutic cloning the public debate has intensified on the Continent. This debate reflects the wide spectrum of diverse religious and secular moralities that are prevalent in modern multicultural European democratic societies. Arguments range from putting forward strictly utilitarian views that weight the moral issues involved against the potential benefits that embryonic stem cell research may harbour to considering the embryo as a human being, endowed with human dignity and human rights from the moment of its creation, concluding that its use for research is unethical and should be strictly prohibited. Given the current state of dissension among the various European states, it is difficult to predict whether 'non-harmonisation' will prevail or whether in the long run 'harmonisation' of legislation that will allow stem cell research will evolve in the EU.
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Isasi, Rosario M; Knoppers, Bartha M
2006-10-01
This report provides a comparative analysis of the regulation of embryonic stem cells and cloning research in 50 countries. The development of international stem cell consortia involving the exchange of materials, data and knowledge presumes 'policy know-how' on the varying positions and governing regulations of the various partners; knowledge is essential for the feasibility of such international collaborative projects. Across the spectrum of restrictive-to-liberal policies, requirements regarding the justification for or the setting of substantive limits on (i) embryo use and/or (ii) destruction in research are often present. These goals justify the regulation, the control and even the prohibition of embryonic stem cell and cloning research. Moreover, irrespective of whether a country adopts a restrictive or a liberal approach, there is significant symmetry in both the substantive and the procedural requirements. Procedural safeguards provide another layer of protection and control over the research. In reality, such safeguards may have a greater systemic impact than the substantive requirements. They can be subdivided into three broad categories: (i) safeguards relating to the stage of embryonic development, (ii) safeguards relating to the donors of blastocysts, gametes, embryos and somatic cells and (iii) requirements for research governance.
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Applying embryo cryopreservation technologies to the production of domestic and black-footed cats.
Pope, C E; Gómez, M C; Galiguis, J; Dresser, B l
2012-12-01
Our objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra- and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies--three of six (50%) and one of two (50%) that received embryos from in vivo- and in vitro-matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out--two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra- and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black-footed cat. © 2012 Blackwell Verlag GmbH.
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Embryo loss in cattle between Days 7 and 16 of pregnancy.
Berg, D K; van Leeuwen, J; Beaumont, S; Berg, M; Pfeffer, P L
2010-01-15
Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).
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Nontransmission of porcine circovirus 2 (PCV2) by embryo transfer.
Bielanski, A; Algire, J; Lalonde, A; Garceac, A; Pollard, J W; Plante, C
2013-07-15
Two experiments were conducted to determine the association of porcine circovirus type 2 (PCV2) with embryos and the risk of viral transmission by embryo transfer. In the first experiment, 240 embryos from uninfected donors were exposed to PCV2a 10(4)TCID50/mL in vitro before transfer to seronegative recipients; in the second experiment, 384 embryos recovered from infected donors, 10 days after donor inoculation with PCV2, were transferred to seronegative recipients. In total, 1120 embryos and/or ova were collected from 37 viral-free donors (experiment 1) and 1019 from 59 PCV2-infected donors (experiment 2) (P < 0.01). The washing and/or disinfection procedure recommended by the International Embryo Transfer Society was applied to embryos in both experiments. Transfer of embryos experimentally exposed in vitro to high titers of virus caused seroconversion of recipients (58%; N = 7/12) and their piglets (81%; N = 13/16). Postmortem, PCV2 DNA was detected in various organs of embryo transfer recipients and their embryo transfer-derived piglets. In contrast, the transfer of embryos recovered from infectious PCV2 donors did not result in the seroconversion of embryo recipients (N = 24) or their embryo transfer-derived piglets (N = 76). Neither PCV2 DNA nor infectious virus was detected in the tissues of either recipients or embryo transfer-derived piglets collected postmortem in the second experiment. The results obtained in this study indicate that the transmission of PCV2 from infected donors by embryo transfer is unlikely if the sanitary recommendations of the International Embryo Transfer Society are followed. In practical terms, this means that embryo transfer can be successfully used for the intentional elimination of PCV2 and to create virus-free offspring for the safe exchange of swine genetic materials. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.
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The dangers of disease transmission by artificial insemination and embryo transfer.
Philpott, M
1993-01-01
This review summarizes the major infectious diseases of the three major agricultural species (cattle, sheep and pigs) and horses, and presents the evidence for and against the possibility of infectious agents being transmitted between animals via the venereal route or by the use of semen or early embryos in commercial artificial insemination (AI) or embryo transfer (ET). Cattle feature most prominently in the widespread distribution of frozen semen, and national and international organizations have set out guidelines to work towards disease-free bull studs with semen free from potential pathogens. With the control of major epizootic diseases, attention has been focused on such diseases as IBR, BVD and blue tongue, where clinical signs are rarely evident but the detection of virus in semen is of great importance. New information on the relevance of bacterial disease such as Mycobacterium paratuberculosis, campylobacteriosis and leptospirosis is reviewed, along with details of the mycoplasma and ureaplasma species of the bull's genital tract. Bovine spongiform encephalopathy (BSE) has attracted much research and semen is not regarded as a source of infection. New work on the pathogenesis of a number of diseases and the use of new biotechnology in diagnosis is included. The International Embryo Transfer Society (IETS) has encouraged a great deal of experimental work--much originating in Canada--on the risk of transmission of disease from donors to recipients via a 7-day-old blastocyst. There has been much success in demonstrating that with an approved protocol of handling the embryos, to date there is very little danger in disease transmission with both viruses and bacteria. The mycoplasma group appear more intractable and the role of BSE is still being evaluated. In sheep, scrapie, Brucella ovis infection and blue tongue feature in current work. In the pig there is a surge in international movement of pig semen, and Aujeszky's disease and the new so-called Blue Ear disease feature prominently. Much work is in progress on infectious agents likely to be found in the semen of stallions, with an expanding trade in the international movement of chilled and frozen semen. Equine embryo transfer experiments are hampered by the very limited number of embryos available. Reference is also made to the further risk of disease transmission by in vitro manipulated embryos.
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2010-01-01
Background Pregnancies induced by in vitro fertilisation (IVF) often result in twin gestations, which are associated with both maternal and perinatal complications. An effective way to reduce the number of IVF twin pregnancies is to decrease the number of embryos transferred from two to one. The interpretation of current studies is limited because they used live birth as outcome measure and because they applied limited time horizons. So far, research on long-term outcomes of IVF twins and singletons is scarce and inconclusive. The objective of this study is to investigate the short (1-year) and long-term (5 and 18-year) costs and health outcomes of IVF singleton and twin children and to consider these in estimating the cost-effectiveness of single embryo transfer compared with double embryo transfer, from a societal and a healthcare perspective. Methods/Design A multi-centre cohort study will be performed, in which IVF singletons and IVF twin children born between 2003 and 2005 of whom parents received IVF treatment in one of the five participating Dutch IVF centres, will be compared. Data collection will focus on children at risk of health problems and children in whom health problems actually occurred. First year of life data will be collected in approximately 1,278 children (619 singletons and 659 twin children). Data up to the fifth year of life will be collected in approximately 488 children (200 singletons and 288 twin children). Outcome measures are health status, health-related quality of life and costs. Data will be obtained from hospital information systems, a parent questionnaire and existing registries. Furthermore, a prognostic model will be developed that reflects the short and long-term costs and health outcomes of IVF singleton and twin children. This model will be linked to a Markov model of the short-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies to enable the calculation of the long-term cost-effectiveness. Discussion This is, to our knowledge, the first study that investigates the long-term costs and health outcomes of IVF singleton and twin children and the long-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies. PMID:20961411
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van Heesch, Mirjam M J; Bonsel, Gouke J; Dumoulin, John C M; Evers, Johannes L H; van der Hoeven, Mark Ahbm; Severens, Johan L; Dykgraaf, Ramon H M; van der Veen, Fulco; Tonch, Nino; Nelen, Willianne L D M; van Zonneveld, Piet; van Goudoever, Johannes B; Tamminga, Pieter; Steiner, Katerina; Koopman-Esseboom, Corine; van Beijsterveldt, Catharina E M; Boomsma, Dorret I; Snellen, Diana; Dirksen, Carmen D
2010-10-20
Pregnancies induced by in vitro fertilisation (IVF) often result in twin gestations, which are associated with both maternal and perinatal complications. An effective way to reduce the number of IVF twin pregnancies is to decrease the number of embryos transferred from two to one. The interpretation of current studies is limited because they used live birth as outcome measure and because they applied limited time horizons. So far, research on long-term outcomes of IVF twins and singletons is scarce and inconclusive. The objective of this study is to investigate the short (1-year) and long-term (5 and 18-year) costs and health outcomes of IVF singleton and twin children and to consider these in estimating the cost-effectiveness of single embryo transfer compared with double embryo transfer, from a societal and a healthcare perspective. A multi-centre cohort study will be performed, in which IVF singletons and IVF twin children born between 2003 and 2005 of whom parents received IVF treatment in one of the five participating Dutch IVF centres, will be compared. Data collection will focus on children at risk of health problems and children in whom health problems actually occurred. First year of life data will be collected in approximately 1,278 children (619 singletons and 659 twin children). Data up to the fifth year of life will be collected in approximately 488 children (200 singletons and 288 twin children). Outcome measures are health status, health-related quality of life and costs. Data will be obtained from hospital information systems, a parent questionnaire and existing registries. Furthermore, a prognostic model will be developed that reflects the short and long-term costs and health outcomes of IVF singleton and twin children. This model will be linked to a Markov model of the short-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies to enable the calculation of the long-term cost-effectiveness. This is, to our knowledge, the first study that investigates the long-term costs and health outcomes of IVF singleton and twin children and the long-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies.
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Virtual Tissue Models in Developmental Toxicity Research
Prenatal exposure to drugs and chemicals may perturb, directly or indirectly, core developmental processes in the embryo (patterning, morphogenesis, proliferation and apoptosis, and cell differentiation), leading to adverse developmental outcomes. Because embryogenesis entails a...
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Segal, Nancy L
2014-02-01
The story of her allegedly stolen twin brother in Armenia is recounted by a 'singleton twin' living in the United States. The behavioral consequences and societal implications of this loss are considered. This case is followed by twin research reports on the evolution of sleep length, dental treatment of craniopagus conjoined twins, cryopreserved double embryo transfer (DET), and gender options in multiple pregnancy. Current events include the diagnosis of appendectomy in one identical twin, the accomplishments of autistic twin marathon runners, the power of three-dimensional (3D) facial recognition, and the goals of twin biathletes heading to the 2014 Sochi Olympics in Russia.
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Cryopreservation of day 2-3 embryos by vitrification yields better outcome than slow freezing.
Levron, Jacob; Leibovitz, Oshrit; Brengauz, Masha; Gitman, Hila; Yerushalmi, Gil M; Katorza, Eldad; Gat, Itai; Elizur, Shai E
2014-03-01
To compare the outcome of vitrification versus slow freezing cryopreservation for cleavage stage day 2-3 embryos. A retrospective observational study. All thawed embryos assisted reproduction cycles between January 2010 and December 2012 at a single IVF laboratory of a Tertiary Medical Center. Five hundred and thirty-nine cycles of day 2-3 thawed embryos. In 327 of the thawed cycles, the embryos were vitrified and in 212 of the cycles the embryos were derived from slow freezing embryos. Embryo survival rate, blastomere surviving rate and pregnancy rate. Embryo survival rate was significantly higher after vitrification compared with slow freezing (81.6%, 647/793 versus 70.0%, 393/562 embryos, p < 0.0001). The clinical pregnancy rate per ET was significantly higher following vitrification compared to slow freezing, 20.0%, 63/314 versus 11.9%, 23/193, respectively (p = 0.02). Vitrification of day 2-3 cleavage stage embryos yields better cycle outcome in all the parameters compared to slow freezing.
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Timing embryo biopsy for PGD - before or after cryopreservation?
Shinar, S; Kornecki, N; Schwartz, T; Mey-Raz, N; Amir, H; Almog, B; Shavit, T; Hasson, J
2016-09-01
Pre-implantation genetic diagnosis (PGD) is required in order to screen and diagnose embryos of patients at risk of having a genetically affected offspring. A biopsy to diagnose the genetic profile of the embryo may be performed either before or after cryopreservation. The aim of this study was to determine which biopsy timing yields higher embryo survival rates. Retrospective cohort study of all PGD patients in a public IVF unit between 2010 and 2013. Inclusion criteria were patients with good-quality embryos available for cryopreservation by the slow freezing method. Embryos were divided into two groups: biopsy before and biopsy after cryopreservation. The primary outcome was embryo survival rates post thawing. Sixty-five patients met inclusion criteria. 145 embryos were biopsied before cryopreservation and 228 embryos were cryopreserved and biopsied after thawing. Embryo survival was significantly greater in the latter group (77% vs. 68%, p < 0.0001). Cryopreservation preceding biopsy results in better embryo survival compared to biopsy before cryopreservation.
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Goose embryonic development from oviposition through 16 hours of incubation.
Lukaszewicz, E; Lason, M; Rosenberger, J; Kowalczyk, A; Bakst, M
2017-06-01
Normal tables provide an objective step-wise description of the morphological development of an embryo. Such tables have been described for the chicken, turkey, quail, and duck embryos, but there is no such staging table for goose embryos. As the goose has one of the longest incubation periods of all the poultry species and embryo mortality during incubation is relatively high, a normal table of goose embryo development would be useful in assessing the morpho-genetic status of the goose embryo before and during incubation. In this study, embryos were isolated from commercial White Koluda goose eggs stored no longer than four days in a cool room (18°C) prior to incubation and after 4, 8, 12, and 16 h of incubation. Embryo staging was based on the normal tables described for the chicken by Eyal-Giladi and Kochav (EGK) and Hamburger and Hamilton (HH). Goose embryos from unincubated eggs were at Stage X and XI EGK and after 16 h of incubation the majority of embryos were between Stages 2 and 4 HH. Our results suggest that while the stage of development of the embryo in the unincubated goose egg is similar to that reported for the chicken, although the diameter of goose embryo is slighter larger. Following incubation, a goose embryo advances more slowly than a chicken embryo up to 16 h of incubation. © 2016 Poultry Science Association Inc.
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Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.
2014-01-01
Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230
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Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder
2016-10-01
Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.
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Culture media for human pre-implantation embryos in assisted reproductive technology cycles.
Youssef, Mohamed M A; Mantikou, Eleni; van Wely, Madelon; Van der Veen, Fulco; Al-Inany, Hesham G; Repping, Sjoerd; Mastenbroek, Sebastiaan
2015-11-20
Many media are commercially available for culturing pre-implantation human embryos in assisted reproductive technology (ART) cycles. It is unknown which culture medium leads to the best success rates after ART. To evaluate the safety and effectiveness of different human pre-implantation embryo culture media in used for in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles. We searched the Cochrane Menstrual Disorders and Subfertility Group's Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the National Research Register, the Medical Research Council's Clinical Trials Register and the NHS Center for Reviews and Dissemination databases from January 1985 to March 2015. We also examined the reference lists of all known primary studies, review articles, citation lists of relevant publications and abstracts of major scientific meetings. We included all randomised controlled trials which randomised women, oocytes or embryos and compared any two commercially available culture media for human pre-implantation embryos in an IVF or ICSI programme. Two review authors independently selected the studies, assessed their risk of bias and extracted data. We sought additional information from the authors if necessary. We assessed the quality of the evidence using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methods. The primary review outcome was live birth or ongoing pregnancy. We included 32 studies in this review. Seventeen studies randomised women (total 3666), three randomised cycles (total 1018) and twelve randomised oocytes (over 15,230). It was not possible to pool any of the data because each study compared different culture media.Only seven studies reported live birth or ongoing pregnancy. Four of these studies found no evidence of a difference between the media compared, for either day three or day five embryo transfer. The data from the fifth study did not appear reliable.Six studies reported clinical pregnancy rate. One of these found a difference between the media compared, suggesting that for cleavage-stage embryo transfer, Quinn's Advantage was associated with higher clinical pregnancy rates than G5 (odds ratio (OR) 1.56; 95% confidence interval (CI) 1.12 to 2.16; 692 women). This study was available only as an abstract and the quality of the evidence was low.With regards to adverse effects, three studies reported multiple pregnancies and six studies reported miscarriage. None of them found any evidence of a difference between the culture media used. None of the studies reported on the health of offspring.Most studies (22/32) failed to report their source of funding and none described their methodology in adequate detail. The overall quality of the evidence was rated as very low for nearly all comparisons, the main limitations being imprecision and poor reporting of study methods. An optimal embryo culture medium is important for embryonic development and subsequently the success of IVF or ICSI treatment. There has been much controversy about the most appropriate embryo culture medium. Numerous studies have been performed, but no two studies compared the same culture media and none of them found any evidence of a difference between the culture media used. We conclude that there is insufficient evidence to support or refute the use of any specific culture medium. Properly designed and executed randomised trials are necessary.
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Van Landuyt, L; Van de Velde, H; De Vos, A; Haentjens, P; Blockeel, C; Tournaye, H; Verheyen, G
2013-11-01
Is the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos? Vitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight. After slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage. Survival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos. Embryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed. Survival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification. This analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos. Although it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed. none.
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Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J; Dickinson, Mary; Greene, Nicholas D E; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David
2013-05-01
Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.
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Michalik, O; Dobosz, S; Zalewski, T; Sapota, M; Ocalewicz, K
2015-04-01
Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation-induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes. © 2015 Blackwell Verlag GmbH.
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Acute embryo toxicity and teratogenicity of three potential biofuels also used as flavor or solvent.
Bluhm, Kerstin; Seiler, Thomas-Benjamin; Anders, Nico; Klankermayer, Jürgen; Schaeffer, Andreas; Hollert, Henner
2016-10-01
The demand for biofuels increases due to concerns regarding greenhouse gas emissions and depletion of fossil oil reserves. Many substances identified as potential biofuels are solvents or already used as flavors or fragrances. Although humans and the environment may be readily exposed little is known regarding their (eco)toxicological effects. In this study, the three potential biofuels ethyl levulinate (EL), 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) were investigated for their acute embryo toxicity and teratogenicity using the fish embryo toxicity (FET) test to identify unknown hazard potentials and to allow focusing further research on substances with low toxic potentials. In addition, two fossil fuels (diesel and gasoline) and an established biofuel (rapeseed oil methyl ester) were investigated as references. The FET test is widely accepted and used in (eco)toxicology. It was performed using the zebrafish Danio rerio, a model organism useful for the prediction of human teratogenicity. Testing revealed a higher acute toxicity for EL (LC50: 83mg/L) compared to 2-MTHF (LC50: 2980mg/L), 2-MF (LC50: 405mg/L) and water accommodated fractions of the reference fuels including gasoline (LC50: 244mg DOC/L). In addition, EL caused a statistically significant effect on head development resulting in elevated head lengths in zebrafish embryos. Results for EL reduce its likelihood of use as a biofuel since other substances with a lower toxic potential are available. The FET test applied at an early stage of development might be a useful tool to avoid further time and money requiring steps regarding research on unfavorable biofuels. Copyright © 2016 Elsevier B.V. All rights reserved.
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Tereso, Susana; Zoglauer, Kurt; Milhinhos, Ana; Miguel, Célia; Oliveira, M Margarida
2007-05-01
We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.
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Morton, Katherine M; Rowe, Anthony M; Chis Maxwell, W M; Evans, Gareth
2006-04-15
Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.
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Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K
2008-03-15
The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.
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Effects of laser polar-body biopsy on embryo quality.
Levin, Ishai; Almog, Benny; Shwartz, Tamar; Gold, Veronica; Ben-Yosef, Dalit; Shaubi, Michal; Amit, Ami; Malcov, Mira
2012-05-01
To evaluate the effect of laser polar-body biopsy (PBB) for preimplantation genetic diagnosis on embryo quality. Retrospective case-control analysis. The quality of 145 embryos after PBB was compared to 276 embryos of the same group of women without biopsy. University-based tertiary-care medical center. Women with inherited genetics disease. Laser PBB of IVF embryos for genetic diagnosis. The study and control embryos were compared for fertilization rate, pronuclear grading, and cleavage-stage parameters on days 1, 2, and 3 after oocyte retrieval. The study embryos demonstrated higher rates of cleavage arrest (3.6% vs. 0.7%), higher rate of significant fragmentation on day 2 (9.5% vs. 3.0%), and lower rate of good cleavage embryos on day 2 (69.1% vs. 78.4%) compared with control embryos. On day 3, the study embryos had lower cleavage rates (six or more blastomeres; 56.5% vs. 74.5%), higher fragmentation (11.7% vs. 3.9%), higher rate of embryos presenting inferior cleavage pattern (57.2% vs. 38.5%), and lower mean blastomere number (5.8 ± 2.1 vs. 6.6 ± 1.9) compared with control embryos. Polar-body biopsy may have a negative effect on embryo quality. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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Zhang, Fengjiao; Wang, Zhiquan; Dong, Wen; Sun, Chunqing; Wang, Haibin; Song, Aiping; He, Lizhong; Fang, Weimin; Chen, Fadi; Teng, Nianjun
2014-10-07
Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.
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Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.
Balbach, Sebastian T; Boiani, Michele
2015-01-01
Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.
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Why the moratorium on human-animal chimera research should not be lifted.
Moy, Alan
2017-08-01
The National Institutes of Health (NIH) announced its plans to lift its moratorium on funding research that involves injecting human embryonic stem cells into animal embryos, which would allow for the creation of part-human and part-animal organisms known as chimeras. The NIH allowed only one month to receive public comments in the midst of a presidential election campaign. Lifting the moratorium means that, for the first time, the federal government will begin spending taxpayer dollars on the creation and manipulation of new organisms that would blur the line between humans and animals. Interestingly, this government effort is creating an uncommon coalition between pro-life groups and animal rights activists that oppose this medical research on ethical grounds; the former seeking to ensure the welfare of human embryos and the latter seeking to protect the well-being of animals. Unlike the issue of abortion, this research is complex. Yet, it is important that the pro-life laity and clergy be adequately informed on some of the basic science and ethics that surround this research. To fully understand why this research is unethical and why the NIH is pursuing this particular research, it is important to understand the ethical tenets governing human-subject research and why secular scientists are pursuing this scientific field.
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Skene, Loane
2008-03-01
Committees appointed by governments to inquire into specific policy issues often have no further role when the Committee's report is delivered to government, but that is not always so. This paper describes the activities of members of the Australian Committee on human cloning and embryo research (the Lockhart Committee) to inform Parliament and the community about the Committee's recommendations after its report was tabled in Parliament. It explains their participation in the political process as their recommendations were debated and amending legislation was passed by Parliament. It illustrates a method of communication about scientific and policy issues that explores people's concerns and what they 'need to know' to make a judgment; and then responds to questions they raise, with the aim of facilitating discussion, not arguing for one view. The paper considers whether this type of engagement and communication is appropriate and could be used in other policy discussions.
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González Altamiranda, Erika; Manrique, Julieta M.; Pérez, Sandra E.; Ríos, Glenda L.; Odeón, Anselmo C.; Leunda, María R.; Jones, Leandro R.; Verna, Andrea
2015-01-01
Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as “starting material” for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds. PMID:26177382
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40 CFR 26.116 - General requirements for informed consent.
Code of Federal Regulations, 2011 CFR
2011-07-01
... PROTECTION OF HUMAN SUBJECTS Basic EPA Policy for Protection of Subjects in Human Research Conducted or... policy, no investigator may involve a human being as a subject in research covered by this policy unless... subject (or to the embryo or fetus, if the subject is or may become pregnant) which are currently...
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Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song
2014-06-01
To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.
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NASA Astrophysics Data System (ADS)
Itoh, Harumi; Arai, Tsunenori; Kikuchi, Makoto
1997-06-01
We measured angular distribution of the light scattering from live mouse embryo with 632.8nm in wavelength to evaluate the embryo viability. We aim to measure the mitochondrial density in human embryo which have relation to the embryo viability. We have constructed the light scattering measurement system to detect the mitochondrial density non-invasively. We have employed two optical fibers for the illumination and sensing to change the angle between these fibers. There were two dips on the scattering angular distribution from the embryo. These dips existed on 30 and 85 deg. We calculated the scattering angular pattern by Mie theory to fit the measured scattering estimated scattering size and density. The best fitting was obtained when the particle size and density were 0.9 micrometers and 1010 particles per ml, respectively. These values coincided with the approximated values of mitochondrial in the embryo. The measured light scattering may mainly originated from mitochondria in spite of the existence of the various scattering particles in the embryo. Since our simple scattering measurement may offer the mitochondrial density in the embryo, it might become the practical method of human embryo on in vitro fertilization-embryo transfer.
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Pollen source effects on growth of kernel structures and embryo chemical compounds in maize.
Tanaka, W; Mantese, A I; Maddonni, G A
2009-08-01
Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos. Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos. Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0.01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0.98, P < 0.01) and soluble sugars (r = 0.95, P < 0.05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0.05-0.10) scutellum cell area occupied by starch granules and fewer oil bodies. The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the embryos.
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Onimaru, Koh; Motone, Fumio; Kiyatake, Itsuki; Nishida, Kiyonori
2018-01-01
Background: Studying cartilaginous fishes (chondrichthyans) has helped us understand vertebrate evolution and diversity. However, resources such as genome sequences, embryos, and detailed staging tables are limited for species within this clade. To overcome these limitations, we have focused on a species, the brownbanded bamboo shark (Chiloscyllium punctatum), which is a relatively common aquarium species that lays eggs continuously throughout the year. In addition, because of its relatively small genome size, this species is promising for molecular studies. Results: To enhance biological studies of cartilaginous fishes, we establish a normal staging table for the embryonic development of the brownbanded bamboo shark. Bamboo shark embryos take around 118 days to reach the hatching period at 25°C, which is approximately 1.5 times as fast as the small‐spotted catshark (Scyliorhinus canicula) takes. Our staging table divides the embryonic period into 38 stages. Furthermore, we found culture conditions that allow early embryos to grow in partially opened egg cases. Conclusions: In addition to the embryonic staging table, we show that bamboo shark embryos exhibit relatively fast embryonic growth and are amenable to culture, key characteristics that enhance their experimental utility. Therefore, the present study is a foundation for cartilaginous fish research. Developmental Dynamics 247:712–723, 2018. © 2017 Wiley Periodicals, Inc. PMID:29396887
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Fleischmann, Thea; Arras, Margarete; Sauer, Mareike; Saleh, Lanja; Rülicke, Thomas; Jirkof, Paulin
2017-04-01
Embryo transfer (ET) in mice is a key technique in biomedical research, and is carried out mostly via surgery by transferring founder embryos into pseudo-pregnant recipient females. To cover post-operative analgesic requirements in surrogate mothers, oral self-administration of painkillers has several advantages, but its effectiveness has also been criticized as voluntary ingestion of the drug can be uncertain. Additionally, concerns about potential negative side effects of analgesics on embryo viability and development have been raised. In this regard, we investigated the impact of orally administered analgesia by comparing the outcome of ET with and without paracetamol in the drinking water (3.5mg/ml) of surrogate mothers. Water intake increased significantly when paracetamol, as a sweet-tasting formulation (children's syrup), was added to the drinking water. Measurements of paracetamol concentrations in blood serum confirmed reasonable drug uptake. Success rate of ETs and the body weight of newborn offspring were not different whether paracetamol was administered for two days after surgery or not. In conclusion, paracetamol in drinking water was consumed voluntarily in substantial doses, without detectable side-effects, by freshly operated surrogate mothers, and can therefore be recommended as a feasible method for providing analgesic treatment for surgical ET in mice. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Demakovsky, Leah; Tesla, Blanka; Goodfellow, Forrest T.; Stice, Steven L.; Murdock, Courtney C.
2017-01-01
Zika virus (ZIKV) has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in Aedes aegypti mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes. PMID:29258204
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Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.
Verdi, Camila M; Jesus, Francielli P K; Kommers, Glaucia; Ledur, Pauline C; Azevedo, Maria I; Loreto, Erico S; Tondolo, Juliana S M; Andrade, Eduardo N C; Schlemmer, Karine B; Alves, Sydney H; Santurio, Janio M
2018-02-01
Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis. © 2017 Blackwell Verlag GmbH.
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Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology.
Amin, Nirav M; Womble, Mandy; Ledon-Rettig, Cristina; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette
2015-09-15
The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. Copyright © 2015 Elsevier Inc. All rights reserved.
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Budgett’s frog (Lepidobatrachus laevis): a new amphibian embryo for developmental biology
Amin, Nirav M.; Womble, Mandy; Ledon-Rettig, Cris; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette
2015-01-01
The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett’s frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett’s frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett’s tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett’s frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. PMID:26169245
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Construction and application of 3D model sequence to illustrate the development of the human embryo
NASA Astrophysics Data System (ADS)
Mizuta, Shinobu; Kakusho, Koh; Minekura, Yutaka; Minoh, Michihiko; Nakatsu, Tomoko; Shiota, Kohei
2002-05-01
Embryology is one of the basic subjects in medical education, to learn the process of human development especially from fertilization to birth. The shape deformation in the development of human embryo is one of the most important points to be comprehended, but it is difficult to illustrate the deformation by texts, 2D drawings, photographs and so on, because it is extremely complicated. The purpose of our research is to construct a 3D model sequence to illustrate the deformation of human embryo, and to make the model sequence into the teaching materials for medical education. Firstly, 3D images of the specimens of human embryo were acquired using MR microscopy. Next, an initial 3D model sequence was manually modified by comparing with the features of the acquired images under the supervision of medical doctors, because the images were influenced not only by the noise or limitation of resolution in MR image acquisition, but also by the variation of shape depending on the difference of subject. Using the constructed 3D model sequence, CG animations and an interactive VRML system were composed as the teaching materials for embryology. These materials were quite helpful to understand the shape deformation compared with the conventional materials.
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Pasparakis, Christina; Sweet, Lauren E; Stieglitz, John D; Benetti, Daniel; Casente, Conrad T; Roberts, Aaron P; Grosell, Martin
2017-10-01
The Deepwater Horizon oil spill occurred in the summer of 2010 and coincided with the spawning window of the ecologically and economically important pelagic fish mahi-mahi (Coryphaena hippurus). During summer months, early life stage mahi-mahi were likely also exposed to other naturally occurring stressors such as increased temperature and ultraviolet radiation (UV). Previous research has shown that co-exposure to oil and additional natural stressors can affect the timing and duration of negative buoyancy in mahi-mahi embryos. The current study aimed to elucidate the factors affecting the onset of negative buoyancy and to also explore possible mechanisms behind buoyancy change. Embryos co-exposed to oil and/or increased temperature and UV radiation displayed early onset of negative buoyancy with concurrent increases in oxygen consumption and sinking rates, which are normally only seen during the period directly preceding hatch. Results also suggest a behavioral response in which embryos avoid UV radiation by sinking down the water column but reestablish positive buoyancy once the UV radiation is removed. These findings imply that embryos can dynamically change their position in the water column in response to external cues and thus may have much greater control over buoyancy than previously thought. Copyright © 2017 Elsevier B.V. All rights reserved.
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Williams, Ruth M; Senanayake, Upeka; Artibani, Mara; Taylor, Gunes; Wells, Daniel; Ahmed, Ahmed Ashour; Sauka-Spengler, Tatjana
2018-02-23
CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo . © 2018. Published by The Company of Biologists Ltd.
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Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition.
Wang, Qian; Gosik, Kirk; Xing, Sujuan; Jiang, Libo; Sun, Lidan; Chinchilli, Vernon M; Wu, Rongling
2017-03-01
Epigenetic reprogramming is thought to play a critical role in maintaining the normal development of embryos. How the methylation state of paternal and maternal genomes regulates embryogenesis depends on the interaction and coordination of the gametes of two sexes. While there is abundant research in exploring the epigenetic interactions of sperms and oocytes, a knowledge gap exists in the mechanistic quantitation of these interactions and their impact on embryo development. This review aims at formulating a modeling framework to address this gap through the integration and synthesis of evolutionary game theory and the latest discoveries of the epigenetic control of embryo development by next-generation sequencing. This framework, named epigenetic game theory or epiGame, views embryogenesis as an ecological system in which two highly distinct and specialized gametes coordinate through either cooperation or competition, or both, to maximize the fitness of embryos under Darwinian selection. By implementing a system of ordinary differential equations, epiGame quantifies the pattern and relative magnitude of the methylation effects on embryogenesis by the mechanisms of cooperation and competition. epiGame may gain new insight into reproductive biology and can be potentially applied to design personalized medicines for genetic disorder intervention. Copyright © 2016 Elsevier B.V. All rights reserved.
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Ata, Baris; Zeng, Xing; Son, Weon Y; Holzer, Hananel; Tan, Seang L
2011-11-01
The aim of this retrospective study was to compare the oocyte yield with the luteal estradiol patch (LPA) - GnRH antagonist and microdose (MD) flare-up protocols in anticipated poor responders. Fifty-seven women who underwent IVF treatment following stimulation with LPA or MD protocols at McGill Reproductive Centre were matched for age and markers of ovarian reserve. Numbers of oocytes collected (6 vs 7), mature oocytes collected (5 vs 5), and oocyte maturation rates (72% vs 74%) were similar. The numbers of good quality embryos available (2 vs 1) and embryos transferred (3 vs 3) were likewise similar. Embryo implantation rate of 16.7% and clinical pregnancy rate of 38.9% achieved in the LPA group were almost 50% higher than the corresponding figures at 10.3% and 22.2% in the MD group; however, the differences were not statistically significant (p > 0.05 for all comparisons). Although the results do not suggest an increased oocyte yield or follicular synchronization with the LPA protocol, the observed trend toward higher embryo implantation and clinical pregnancy rates requires further research.
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[A new method for quantitative measurement of the cadmium absorbed by chick embryos].
Gottofrey, J
1984-01-01
We attempted to determine the quantity of cadmium incorporated in hens eggs after immersion in cadmium solutions, and the cadmium concentration measured in embryos. We discussed equipment allowing simultaneous treatment of up to 42 samples, and called it " digestor ". It consisted of two gas-heated sand baths, two stands for cooling down solutions and an evacuation system for toxic vapours. Our method was based on wet mineralisation. It consisted of desintegrating experimental chick embryos in a HNO3/H2O2 mixed solution. After heating and evaporating, the quantity of cadmium in the remnant was determined by atomic absorption spectrophotometry. The reliability of such a technique was tested by studying as controls controls 17 days-old chick embryos injected with a known quantity of Cd(NO3). It showed no loss of cadmium. We also compared our procedure with a dry ashing method. The latter showed unacceptable losses and insufficient precision for the problems we wanted to investigate. Our method gave us much more precise results. The equipment we developed has functioned wholly satisfactorily and allowed us to investigate for instance cadmium distribution and concentration in embryonic organs of 17 days-old chicks. It could also be useful for researches concerning other biological samples analyzed for different heavy metals.
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Development of whole and demi-embryos of mice in culture and in vivo after supercooled storage.
Fuku, E; Fiser, P S; Marcus, G J; Sasada, H; Downey, B R
1993-12-01
Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at -5 degrees C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant (P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.
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Research on the Reproductive Effects of Endocrine Disrupting Chemicals
Reproductive hormones exert their effects at many life stages. During embryo and fetus development in the womb, these hormones direct formation of sex organs and the cells that give rise to sperm and eggs.
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Safari, Somayyeh; Faramarzi, Azita; Agha-Rahimi, Azam; Khalili, Mohammad Ali
2016-09-01
The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B-C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF.
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Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd.
Arave, C W; Bunch, T D; Mickelsen, C H; Warnick, K
1987-09-01
Sixty Holstein donor cows were superovulated and embryos were collected during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Forty-three donor cows yielded embryos. Ninety-one embryos graded 1 or 2 were split and transferred to 181 recipient Holsteins. Demi-embryos were graded 2, 2-, 3 and 3- prior to transfer. Pregnancy and calving percentages were similar for all demi-embryo grades, averaging 59 and 53% from the two donor groups, respectively. Twin demi-embryo pregnancies averaged 36 and 19% for embryos split at the compacted morula and blastocyst stages, respectively. Twin demi-embryo calvings averaged 30 and 15% for these same groups. Progesterone levels of recipients (of either whole or demi-embryos) of second period donors were measured. Pregnancy rate increased generally with level of progesterone; however, calving percentage was slightly greater for recipients with intermediate levels of progesterone (2-6 ng/ml). Multiparous cow (20) recipients of demi-embryos had 45% pregnancy and 40% calving, while nulliparous heifer (161) recipients averaged 59 and 53% pregnancy and calving, respectively.
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Tan, Xiu-Wen; Ji, Chang-Li; Zheng, Liang-Liang; Zhang, Jie; Yuan, Hong-Jie; Gong, Shuai; Zhu, Jiang; Tan, Jing-He
2017-08-01
What are the mechanisms by which corticotrophin-releasing hormone (CRH) and corticosterone impair the development of preimplantation embryos in the oviduct. CRH and corticosterone do not affect preimplantation embryos directly, but impair their development indirectly by triggering apoptosis of oviductal epithelial cells (OECs) through activation of the Fas system. Studies report that stress impairs embryo development with facilitated secretion of CRH and glucocorticoids. Although an in vivo study demonstrated that preimplantation stress impaired embryo development in conjunction with oviductal apoptosis and activation of the Fas system, whether CRH or glucocorticoids damage embryos directly or indirectly by way of oviductal cells remains to be clarified. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in Fas ligand in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female mice were used 8-10 weeks after birth. While some female mice were killed 48 h after being injected with equine CG to collect oviducts and prepare OECs, others were killed to recover zygotes after mating with males following superovulation with eCG and hCG. The zygotes obtained were cultured with or without CRH or corticosterone (CRH/Cort) either in Chatot-Ziomek-Bavister (CZB) medium with or without OECs or in conditioned medium (CM) conditioned with OECs pretreated or not with CRH/Cort. Preimplantation development, levels of redox potential and apoptosis, and expression of CRH receptor 1 (CRHR1), glucocorticoid receptor (GR), Fas and 11β-hydroxysteroid dehydrogenase (HSD) were observed in embryos recovered at different times of in vitro culture. After culture of OECs with or without CRH/Cort, levels of redox potential and apoptosis, mRNA and protein expression of growth factors, and protein expression of CRHR1, GR and Fas were examined in OECs and the level of FasL was measured in CM. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. This study showed that blastocyst development was unaffected when mouse zygotes were cultured in CZB medium containing various concentrations of CRH/Cort but was impaired when embryos were cultured with CRH/Cort plus OECs or in CM conditioned with OECs pretreated with CRH/Cort (treatment CM). Culture in treatment-CM induced oxidative stress and apoptosis in embryos. Preimplantation embryos expressed GR and Fas at all stages and CRHR1 at the blastocyst stage only. Mouse 4-cell embryos and blastocysts expressed HSD2 but not HSD1. Culture of OECs with CRH/Cort increased their oxidative stress, apoptosis, CRHR1, Fas and FasL while decreasing their GR and growth factors. Blastocyst development in treatment-CM conditioned with OECs from gld mice harboring FasL mutations was superior to treatment-CM conditioned with wild-type mouse OECs. The results suggest that CRH/Cort impairs embryo development indirectly by inducing oviductal apoptosis via activating the Fas system. The insensitivity of preimplantation embryos to CRH and corticosterone is due to, respectively, a lack of CRHR and the exclusive expression of HSD2 that inactivate corticosterone. Not applicable. Although significant, the conclusions were drawn from limited results obtained using mice and thus they need further verification in other species. For example, bovine embryos express both HSD1 and HSD2 at all the preimplantation stages whereas mouse preimplantation embryos express HSD2 exclusively without HSD1. The data are important for our understanding of the mechanisms by which stress affects female reproduction in both human and animals, as early stages of pregnancy are considered more vulnerable to stress than the late stages. This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
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Kurome, Mayuko; Leuchs, Simon; Kessler, Barbara; Kemter, Elisabeth; Jemiller, Eva-Maria; Foerster, Beatrix; Klymiuk, Nikolai; Zakhartchenko, Valeri; Wolf, Eckhard
2017-04-01
Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.
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Effect of road deicing salt on the susceptibility of amphibian embryos to infection by water molds.
Karraker, Nancy E; Ruthig, Gregory R
2009-01-01
Some causative agents of amphibian declines act synergistically to impact individual amphibians and their populations. In particular, pathogenic water molds (aquatic oomycetes) interact with environmental stressors and increase mortality in amphibian embryos. We documented colonization of eggs of three amphibian species, the wood frog (Rana sylvatica), the green frog (Rana clamitans), and the spotted salamander (Ambystoma maculatum), by water molds in the field and examined the interactive effects of road deicing salt and water molds, two known sources of mortality for amphibian embryos, on two species, R. clamitans and A. maculatum in the laboratory. We found that exposure to water molds did not affect embryonic survivorship in either A. maculatum or R. clamitans, regardless of the concentration of road salt to which their eggs were exposed. Road salt decreased survivorship of A. maculatum, but not R. clamitans, and frequency of malformations increased significantly in both species at the highest salinity concentration. The lack of an effect of water molds on survival of embryos and no interaction between road salt and water molds indicates that observations of colonization of these eggs by water molds in the field probably represent a secondary invasion of unfertilized eggs or of embryos that had died of other causes. Given increasing salinization of freshwater habitats on several continents and the global distribution of water molds, our results suggest that some amphibian species may not be susceptible to the combined effects of these factors, permitting amphibian decline researchers to devote their attention to other potential causes.
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Vertical Transmission of H9N2 Avian Influenza Virus in Goose.
Yu, Guanliu; Wang, Aihua; Tang, Yi; Diao, Youxiang
2017-01-01
During a study on high mortality cases of goose embryo in Shandong Province, China (2014-2015), we isolated an H9N2 avian influenza virus (AIV) strain (A/goose/Shandong/DP01/2014, DP01), which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups (A, B, C, D, and E) for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible.
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Opiela, J; Samiec, M; Romanek, J
2017-07-15
Artificial epigenomic modulation of in vitro cultured mesenchymal stem cells (MSCs) by applying a non-selective HDAC inhibitor, termed TSA, can facilitate more epigenetic reprogramming of transcriptional activity of the somatic cell-descended nuclear genome in NT pig embryos. The results of the present investigation showed that TSA-dependent epigenomic modulation of nuclear donor MSCs highly affects both the in vitro developmental capability and the cytological quality of inter-species (porcine→bovine) cloned embryos. The developmental competences to reach the blastocyst stage among hybrid (porcine→bovine) nuclear-transferred embryos that had been reconstructed with bovine ooplasts and epigenetically modulated porcine MSCs were maintained at a relatively high level. These competences were higher than those noted in studies by other authors, but they were still decreased compared to those of intra-species (porcine) cloned embryos that had been reconstituted with porcine ooplasts and either the cell nuclei of epigenetically transformed MSCs or the cell nuclei of epigenetically non-transformed MSCs. In conclusion, MSCs undergoing TSA-dependent epigenetic transformation were used for the first time as a source of nuclear donor cells not only for inter-species somatic cell cloning in pigs but also for inter-species somatic cell cloning in other livestock species. Moreover, as a result of the current research, efficient sequential physicochemical activation of inter-species nuclear-transferred clonal cybrids derived from bovine ooplasm and porcine MSC nuclei was developed. Copyright © 2017 Elsevier Inc. All rights reserved.
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[A European discussion about stem cells for therapeutic use].
Boer, G J
2002-06-29
Stem cells as a source material for growing cellular transplants to repair dysfunctional organs appear to be a new challenge for medical science. Though stem cells are also present in foetal and adult organs, embryonic stem cells from the pre-implantation embryo in particular have the potency to proliferate easily in vitro and the capacity to differentiate into all the body's organ-specific cells. Therefore, these are the ideal cells for developing new cell transplantation therapies for diseases such as Parkinson's disease, diabetes mellitus and heart failure. The use of spare in vitro fertilization (IVF) embryos or pre-implantation embryos specially created to harvest human embryonic stem cells is, however, controversial and an ethical problem. In a European discussion platform organised by the European Commission Research Directorate-General, the status quo of the progress was presented and subsequently commented upon and discussed in terms of medical-ethical, social, industrial and patient interests. The expectations of this new medical technology were high, but clinical trials seem only acceptable once the in vitro differentiation of stem cells can be adequately controlled and once it is known how in vitro prepared stem cells behave after implantation. The ethical justification of the use of in vitro pre-implantation embryos remains controversial. The prevailing view is that the interests of severely ill patients for whom no adequate therapy exists, surmounts the interest of protection of a human in vitro pre-implantation embryo, regardless of whether it was the result of IVF or of transplantation of a somatic cell nucleus of the patient in an enucleated donor egg cell (therapeutic cloning).
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Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos
Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan
2009-01-01
Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066
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Blackburn, Daniel G; Weaber, Kera K; Stewart, James R; Thompson, Michael B
2003-05-01
Although pregnant viviparous squamates are sometimes claimed to be able to resorb inviable eggs and embryos from the uterus, definitive evidence for such resorption is not available. After placing pregnant female Pseudemoia pagenstecheri into conditions under which embryonic development is terminated, we periodically harvested the gravid oviducts and examined them histologically. Females contained abnormal and degenerating eggs and embryos that had died in various stages of development. Dead embryos had undergone extensive cytolysis, dissolution, and aseptic necrosis and vitelline masses showed signs of deterioration and passage down the oviduct. The uterine mucosa lay in direct contact with the vitelline material, with no intact shell membrane intervening between them. Yolk was sometimes displaced into the exocoelom and allantoic cavity due to rupture of the extraembryonic membranes. Histological examination revealed no evidence of the uptake of yolk by the uterine epithelium or its accumulation in the subepithelial connective tissue. In many specimens, the uterine epithelium showed minuscule, apical granules. The position, appearance, and staining properties of the granules suggests them to be secretory, a manifestation of placentotrophy. Our observations indicate that P. pagenstecheri females retain dead eggs and embryos for several weeks or longer, yet do not resorb them during that period. This lizard is the second placentotrophic skink species in which resorption has been suspected, but in which abortive eggs appear to be retained or extruded instead of being resorbed by the oviducts. Researchers should not assume that squamates can digest and resorb oviductal eggs without definitive morphological evidence. Copyright 2003 Wiley-Liss, Inc.
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Vergouw, Carlijn G; Kostelijk, E Hanna; Doejaaren, Els; Hompes, Peter G A; Lambalk, Cornelis B; Schats, Roel
2012-09-01
Does the type of medium used to culture fresh and frozen-thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen-thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight. Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook(®) Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage(®)), not previously analysed, and it includes data on frozen-thawed SETs. This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen-thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011. Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage(®), Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage(®)) and singletons born after a frozen-thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage(®) and 86 in Sage(®) only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets, babies with congenital or chromosomal abnormalities and babies born before 22 weeks of gestation. Analysis of 358 singletons born after a fresh SET and 159 singletons born after a frozen-thawed SET showed no significant difference between the HTF and Sage(®) groups in terms of birthweight. Gestational age, parity and gender of the baby were significantly related to birthweight in multiple linear regression analyses, and other possible confounding factors included maternal age, BMI and smoking, the number of blastomeres in the transferred embryo and the type of culture medium. Maternal age, BMI and smoking, gestational age at birth, gender of the baby and the percentage of firstborns did not differ significantly between the HTF and Sage(®) groups; however, among the fresh embryos, those cultured in Sage(®) had significantly more blastomeres at the time of embryo transfer compared with the embryos cultured in HTF. Birthweights adjusted for gestational age and gender or gestational age and parity (z-scores) were not significantly different between the HTF and Sage(®) groups for fresh or frozen-thawed SETs. Mean birthweight, as well as the mean birthweight among firstborns and the mean birthweights adjusted for gestational age and gender or parity (z-scores) were significantly higher in the cryopreservation group compared with the fresh embryo transfer group. Our study is limited by its retrospective design and only two commercially available types of culture media were tested. More research is necessary to investigate the potential influence of culture media on gene expression. Although our data do not indicate the major influences of the HTF and Sage(®) culture media on birthweight, our results cannot be extrapolated to other culture media types. Furthermore, there remains a potential influence of embryo culture environment on epigenetic variation not represented by birthweight differences but by more subtle features.
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2011-01-01
Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner. PMID:21569635
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Novel embryo selection techniques to increase embryo implantation in IVF attempts.
Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F
2016-11-01
The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.
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Sundvall, Linda; Kirkegaard, Kirstine; Ingerslev, Hans Jakob; Knudsen, Ulla Breth
2015-07-01
Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Factors other than anovulation, such as low embryo quality have been suggested to contribute to the infertility in these women. This 2-year retrospective study used timelapse technology to investigate the PCOS-influence on timing of development in the pre-implantation embryo (primary endpoint). The secondary outcome measure was live birth rates after elective single-embryo transfer. In total, 313 embryos from 43 PCOS women, and 1075 embryos from 174 non-PCOS women undergoing assisted reproduction were included. All embryos were monitored until day 6. Differences in embryo kinetics were tested in a covariance regression model to account for potential confounding variables: female age, BMI, fertilization method and male infertility. Time to initiate compaction and reach the morula stage as well as the duration of the 4th cleavage division was significantly shorter in PCOS embryos compared with non-PCOS embryos. No other kinetic differences were found at any time-points annotated. The proportion of multi-nucleated cells at the 2-cell stage was significantly higher in PCOS embryos compared with non-PCOS embryos. The live birth rates were comparable between the two groups. The findings suggest that the causative factor for subfertility in PCOS is not related to timing of development in the pre-implantation embryo.
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Full allogeneic fusion of embryos in a holothuroid echinoderm.
Gianasi, Bruno L; Hamel, Jean-François; Mercier, Annie
2018-05-30
Whole-body chimaeras (organisms composed of genetically distinct cells) have been directly observed in modular/colonial organisms (e.g. corals, sponges, ascidians); whereas in unitary deuterostosmes (including mammals) they have only been detected indirectly through molecular analysis. Here, we document for the first time the step-by-step development of whole-body chimaeras in the holothuroid Cucumaria frondosa , a unitary deuterostome belonging to the phylum Echinodermata. To the best of our knowledge, this is the most derived unitary metazoan in which direct investigation of zygote fusibility has been undertaken. Fusion occurred among hatched blastulae, never during earlier (unhatched) or later (larval) stages. The fully fused chimaeric propagules were two to five times larger than non-chimaeric embryos. Fusion was positively correlated with propagule density and facilitated by the natural tendency of early embryos to agglomerate. The discovery of natural chimaerism in a unitary deuterostome that possesses large externally fertilized eggs provides a framework to explore key aspects of evolutionary biology, histocompatibility and cell transplantation in biomedical research. © 2018 The Author(s).
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Evans, B R
1999-01-01
Historically, international regulatory interventions in the area of animal reproductive technologies have focused on the need for mitigation against the dissemination of diseases with the movement of genetics and germplasm across international borders. The continued globalization of agriculture under the Sanitary/Phytosanitary (SPS) Agreement of the World Trade Organization (WTO) ensures that disease considerations arising from third and fourth generation reproductive technologies such as in vitro fertilized embryos, transgenics and xenotransplantation will continue to give rise to animal health regulatory measures. Furthermore, in the aftermath of the raising of the public consciousness and the ensuing consumer confidence crisis concerning animal husbandry and livestock production practices following the Bovine Spongiform Encephalopathy outbreak, evolving societal values are expected to expand regulatory considerations to address veterinary public health and ethical concerns. Consequently, it is expected that the role of the International Embryo Transfer Society in fostering meaningful dialogue and profiling of the research necessary to provide for appropriate science based regulation development will increase in importance.
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NASA Astrophysics Data System (ADS)
Lopez, Andrew L.; Wang, Shang; Garcia, Monica; Valladolid, Christian; Larin, Kirill V.; Larina, Irina V.
2015-03-01
Understanding mouse embryonic development is an invaluable resource for our interpretation of normal human embryology and congenital defects. Our research focuses on developing methods for live imaging and dynamic characterization of early embryonic development in mouse models of human diseases. Using multidisciplinary methods: optical coherence tomography (OCT), live mouse embryo manipulations and static embryo culture, molecular biology, advanced image processing and computational modeling we aim to understand developmental processes. We have developed an OCT based approach to image live early mouse embryos (E8.5 - E9.5) cultured on an imaging stage and visualize developmental events with a spatial resolution of a few micrometers (less than the size of an individual cell) and a frame rate of up to hundreds of frames per second and reconstruct cardiodynamics in 4D (3D+time). We are now using these methods to study how specific embryonic lethal mutations affect cardiac morphology and function during early development.
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Legal considerations for cryopreservation of sperm and embryos.
Schuster, Timothy G; Hickner-Cruz, Kathryn; Ohl, Dana A; Goldman, Edward; Smith, Gary D
2003-07-01
To summarize the case law for cryopreservation of sperm and embryos and make recommendations for desirable characteristics that should be included in a cryopreservation clinic's disposition agreements. A literature review of case law and legal review articles was performed. Academic research center. None. None. None. Two court cases involving cryopreserved semen and five cases involving cryopreserved embryos are reviewed. The state of the law surrounding cryopreservation is recent and unsettled. Disposition agreements can provide cryopreservation clinics some degree of certainty in this unsettled area of law. Those drafting cryopreservation disposition agreements are assisted by lessons learned from cryopreservation cases and insight gained from law review articles. It is evident that cryopreservation agreements will be most successful and most likely to be enforced if they are unambiguous, consistent with public policy, and include: a duration provision, each individual's contact information, the individual's provisions for use of their gametes in case of death, and responsibilities of cryopreservation clinic and individuals. Individuals must enter into agreements with a true understanding of the contained provisions.
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Automated phenotype pattern recognition of zebrafish for high-throughput screening.
Schutera, Mark; Dickmeis, Thomas; Mione, Marina; Peravali, Ravindra; Marcato, Daniel; Reischl, Markus; Mikut, Ralf; Pylatiuk, Christian
2016-07-03
Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.
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Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi
2015-11-01
Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P < 0.01). To predict implantation outcomes, we examined the area under the ROC curve (AUCROC), which was significantly higher for the viability than for the morphology score (0.94 vs. 0.55; P < 0.01); however, the AUCROCs for the composite and viability scores did not differ significantly (0.92 vs. 0.94; P > 0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.
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Mognetti, B; Leppens, G; Sakkas, D
1996-04-01
Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.
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AFREEN, F.; ZOBAYED, S. M. A.; KOZAI, T.
2002-01-01
Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 µmol m–2 s–1) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90–130 µg g–1 fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300–500 µg g–1 fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar‐free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50 %, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100–150 µmol m–2 s–1) and increased CO2 concentration (1100 µmol mol–1) were found to be necessary for the development of plantlets from cotyledonary stage embryos. PMID:12125763
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Cost-effectiveness of single versus double embryo transfer in IVF in relation to female age.
van Loendersloot, Laura L; Moolenaar, Lobke M; van Wely, Madelon; Repping, Sjoerd; Bossuyt, Patrick M; Hompes, Peter G A; van der Veen, Fulco; Mol, Ben Willem J
2017-07-01
To evaluate the cost-effectiveness of single embryo transfer followed by an additional frozen-thawed single embryo transfer, if more embryos are available, as compared to double embryo transfer in relation to female age. We used a decision tree model to evaluate the costs from a healthcare provider perspective and the pregnancy rates of two embryo transfer policies: one fresh single embryo transfer followed by an additional frozen-thawed single embryo transfer, if more embryos are available (strategy I), and double embryo transfer (strategy II). The analysis was performed on an intention-to-treat basis. Sensitivity analyses were carried out to evaluate the robustness of our model and to identify which model parameters had the strongest impact on the results. SET followed by an additional frozen-thawed single embryo transfer if available was dominant, less costly and more effective, over DET in women under 32 years. In women aged 32 or older DET was more effective than SET followed by an additional frozen-thawed single embryo transfer if available but also more costly. SET followed by an additional frozen-thawed single embryo transfer should be the preferred strategy in women under 32 undergoing IVF. The choice for SET followed by an additional frozen-thawed single embryo transfer or DET in women aged 32 or older depends on individual patient preferences and on how much society is willing to pay for an extra child. There is a strong need for a randomized clinical trial comparing the cost and effects of SET followed by an additional frozen-thawed single embryo transfer and DET in the latter category of women. Copyright © 2017 Elsevier B.V. All rights reserved.
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Dyrlund, Thomas F; Kirkegaard, Kirstine; Poulsen, Ebbe Toftgaard; Sanggaard, Kristian W; Hindkjær, Johnny J; Kjems, Jørgen; Enghild, Jan J; Ingerslev, Hans Jakob
2014-11-01
Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Geber, Selmo; Bossi, Renata; Lisboa, Cintia B; Valle, Marcelo; Sampaio, Marcos
2011-04-28
We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.
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Inbreeding effects on in vitro embryo production traits in Guzerá cattle.
Perez, B C; Balieiro, J C C; Ventura, R V; Bruneli, F A T; Peixoto, M G C D
2017-11-01
Inbreeding has been associated with the impairment of reproductive performance in many cattle breeds. Although the usage of reproductive biotechnologies has been increasing in bovine populations, not much attention has been given to the impact of inbreeding over cow's performance on artificial reproduction. The objective of this study was to estimate the impact of inbreeding on in vitro embryo production in a Guzerá breed population. The inbreeding coefficient (F), calculated as half of the co-ancestry of the individual's parents, was used as an estimate of inbreeding. The inbreeding coefficients of the donor, sire (used on in vitro fertilization) and of the embryos were included, separately, in the proposed models either as classificatory or continuous variables (linear and quadratic effects). The percentage of non-inbred individuals (or embryos) and mean F of donors, embryos and sires were 29.38%; 35.76%; 42.86% and 1.98±2.68; 1.32±3.13; 2.08±2.79, respectively. Two different models were considered, one for oocyte production traits and other for embryo production traits. The increase of F of the donor significantly (P<0.05) impaired the number of viable oocytes (N OV), number of grade I oocytes (N GI) and number of cleaved embryos (N CLV). Moreover, the donor's F influenced the percentage of grade I oocytes (P GI), percentage of viable embryos (P EMB) and percentage of cleaved embryos that turned into embryos (P CXE). No significant (P>0.05) effects were observed for the sire (father of the embryos) inbreeding coefficient over the traits analysed. Embryo's F influenced (P<0.05) the number of viable embryos (N EMB), percentage of viable embryos (P EMB) and percentage of cleaved embryos that turn into embryos (P CXE). Results suggested that an increase in the inbreeding coefficient might impair the embryos ability to survive through challenges imposed by the in vitro environment. Submitting highly inbred Guzerá female donors to in vitro embryo production may, in the long-term, have negative implications on the number of embryos obtained per cow and increase the relative costs of the improvement programmes based on this technology. High levels of inbreeding should be avoided when selecting Guzerá female donors and planning in vitro fertilization mating.
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A mathematical model of in vivo bovine blastocyst developmental to gestational Day 15.
Shorten, P R; Donnison, M; McDonald, R M; Meier, S; Ledgard, A M; Berg, D
2018-06-20
Bovine embryo growth involves a complex interaction between the developing embryo and the growth-promoting potential of the uterine environment. We have previously established links between embryonic factors (embryo stage, embryo gene expression), maternal factors (progesterone, body condition score), and embryonic growth to 8 d after bulk transfer of Day 7 in vitro-produced blastocysts. In this study we recovered blastocysts on Days 7 and 15 after artificial insemination to test the hypothesis that in vivo and in vitro embryos follow a similar growth program. We conducted our study using 4 commercial farms and repeated our study over 2 yr (2014, 2015), with data available from 2 of the 4 farms in the second year. Morphological and gene expression measurements (196 candidate genes) of the Day 7 embryos were measured and the progesterone concentration of the cows were measured throughout the reproductive cycle as a reflection of the state of the uterine environment. These data were also used to assess the interaction between the uterine environment and the developing embryo and to examine how well Day 7 embryo stage can be predicted from the Day 7 gene expression profile. Progesterone was not a strong predictor of in vivo embryo growth to Day 15. This contrasts with a range of Day 7 embryo transfer studies which demonstrated that progesterone is a very good predictor of embryo growth to Day 15. Our analysis demonstrates that in vivo embryos are 3 times less sensitive to progesterone than in vitro-transferred embryos (up to Day 15). This highlights that caution must be applied when extrapolating the results of in vitro embryo transfer studies to the in vivo situation. The similar variance in measured and predicted (based on Day 15 length) Day 7 embryo stage indicate low stochastic perturbations for in vivo embryo growth (large stochastic growth effects would generate a significantly larger standard deviation in measured embryo length on Day 15). We also identified that Day 7 embryo stage could be predicted based on the Day 7 gene expression profile (58% overall success rate for classification of 5 embryo stages). Our analysis also associated genes with each developmental stage and demonstrates the high level of temporal regulation of genes that occurs during early embryonic development. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Hendriks, W K; Roelen, B A J; Colenbrander, B; Stout, T A E
2015-11-01
Equine embryos are cryopreserved by slow-freezing or vitrification. While small embryos (<300 μm) survive cryopreservation reasonably well, larger embryos do not. It is not clear if slow-freezing or vitrification is less damaging to horse embryos. To compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-freezing vs. vitrification. Sixty-three Day 6.5-7 embryos were subdivided by size and assigned to one of 5 treatments: control, exposure to slow-freezing or vitrification cryoprotectants (CPs), and cryopreservation by either technique. After thawing/CP removal, embryos were stained with fluorescent stains for various parameters of cellular integrity, and assessed by multiphoton microscopy. Exposing large embryos to vitrification CPs resulted in more dead cells (6.8 ± 1.3%: 95% confidence interval [CI], 3.1-10.4%) than exposure to slow-freezing media (0.3 ± 0.1%; 95% CI 0.0-0.6%: P = 0.001). Cryopreservation by either technique induced cell death and cytoskeleton disruption. Vitrification of small embryos resulted in a higher proportion of cells with fragmented or condensed (apoptotic) nuclei (P = 0.002) than slow-freezing (6.7 ± 1.5%, 95% CI 3.0-10.4% vs. 5.0 ± 2.1%, 95% CI 4.0-14.0%). Slow-freezing resulted in a higher incidence of disintegrated embryos (P = 0.01) than vitrification. Mitochondrial activity was low in control embryos, and was not differentially affected by cryopreservation technique, whereas vitrification changed mitochondrial distribution from a homogenous crystalline pattern in control embryos to a heterogeneous granulated distribution in vitrified embryos (P = 0.05). Cryopreservation caused more cellular damage to large embryos than smaller ones. While vitrification is more practical, it is not advisable for large embryos due to a higher incidence of dead cells. The choice is less obvious for small embryos, as vitrification led to occasionally very high percentages of dead or damaged cells, but a lower incidence of embryo disintegration. Modifications that reduce the level of cellular damage induced by vitrification are required before it can be considered the method of choice for cryopreserving equine embryos. © 2014 EVJ Ltd.
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Code of Federal Regulations, 2013 CFR
2013-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...
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Code of Federal Regulations, 2012 CFR
2012-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...
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Code of Federal Regulations, 2014 CFR
2014-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...
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Transcriptome analysis of PCOS arrested 2-cell embryos.
Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie
2018-06-18
In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.
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The European embryonic stem-cell debate and the difficulties of embryological Kantianism.
Mauron, Alexandre; Baertschi, Bernard
2004-10-01
As elsewhere, the ethical debate on embryonic stem cell research in Central Europe, especially Germany and Switzerland, involves controversy over the status of the human embryo. There is a distinctive Kantian flavor to the standard arguments however, and we show how they often embody a set of misunderstandings and argumentative shortcuts we term"embryological Kantianism."We also undertake a broader analysis of three arguments typically presented in this debate, especially in official position papers, namely the identity, continuity, and potentiality arguments. It turns out that these arguments do not support the strong, quasi-personal status accorded to the embryos in these official opinions.
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Italy enacts new law on medically assisted reproduction.
Boggio, Andrea
2005-05-01
In 2004, the Italian Parliament enacted a law regulating medically assisted reproduction. Although the law recognizes as legal certain assisted reproduction techniques, several other procedures are implicitly or expressly banned: oocyte and sperm donation, using embryos for the scientific research purposes and reproductive cloning. In this article, I outline the new legal framework, pointing out some of the shortcomings of its provisions, such as the failure to define what an 'embryo' is, the contradictions between this law and the law on abortion, the opportunity for Italian couples to circumvent some of the prohibitions by resorting to 'reproductive tourism', and the central role that physicians play in the new legal framework.
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The argument from potentiality in the embryo protection debate: finally "depotentialized"?
Stier, Marco; Schoene-Seifert, Bettina
2013-01-01
Debates on the moral status of human embryos have been highly and continuously controversial. For many, these controversies have turned into a fruitless scholastical endeavor. However, recent developments and insights in cellular biology have cast further doubt on one of the core points of dissent: the argument from potentiality. In this article we want to show in a nonscholastical way why this argument cannot possibly survive. Getting once more into the intricacies of status debates is a must in our eyes. Not merely intellectual coherence but the standing and self-understanding of current stem cell research might profit from finally taking leave of the argument from potentiality.
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NASA Astrophysics Data System (ADS)
Wu, Chen; Ran, Shihao; Le, Henry; Singh, Manmohan; Larina, Irina V.; Mayerich, David; Dickinson, Mary E.; Larin, Kirill V.
2017-02-01
Both optical coherence tomography (OCT) and selective plane illumination microscopy (SPIM) are frequently used in mouse embryonic research for high-resolution three-dimensional imaging. However, each of these imaging methods provide a unique and independent advantage: SPIM provides morpho-functional information through immunofluorescence and OCT provides a method for whole-embryo 3D imaging. In this study, we have combined rotational imaging OCT and SPIM into a single, dual-modality device to image E9.5 mouse embryos. The results demonstrate that the dual-modality setup is able to provide both anatomical and functional information simultaneously for more comprehensive tissue characterization.
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Construction vs. development: polarizing models of human gestation.
Stith, Richard
2014-12-01
This essay argues that the polarization of our public debate over embryo-destructive research may be due, to a large extent, not to different valuations of individual human life but to different conceptions of the process of gestation, with one group treating the process as a making or construction and the other treating it as a development. These two incompatible models of reproduction are shown to explain the various positions commonly encountered in this debate over the treatment of embryos, and to a significant degree those encountered in the debate over abortion as well. Finally, the historical, theoretical, and intuitive strengths of each model are examined.
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Impact of PCOS on early embryo cleavage kinetics.
Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L
2014-04-01
This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Yoder, Nicole; Tal, Reshef; Martin, J Ryan
2016-10-19
Ectopic pregnancy is the leading cause of maternal morbidity and mortality during the first trimester and the incidence increases dramatically with assisted-reproductive technology (ART), occurring in approximately 1.5-2.1 % of patients undergoing in-vitro fertilization (IVF). Abdominal ectopic pregnancy is a rare yet clinically significant form of ectopic pregnancy due to potentially high maternal morbidity. While risk factors for ectopic pregnancy after IVF have been studied, very little is known about risk factors specific for abdominal ectopic pregnancy. We present a case of a 30 year-old woman who had an abdominal ectopic pregnancy following IVF and elective single embryo transfer, which was diagnosed and managed by laparoscopy. We performed a systematic literature search to identify case reports of abdominal or heterotopic abdominal ectopic pregnancies after IVF. A total of 28 cases were identified. Patients' ages ranged from 23 to 38 (Mean 33.2, S.D. = 3.2). Infertility causes included tubal factor (46 %), endometriosis (14 %), male factor (14 %), pelvic adhesive disease (7 %), structural/DES exposure (7 %), and unexplained infertility (14 %). A history of ectopic pregnancy was identified in 39 % of cases. A history of tubal surgery was identified in 50 % of cases, 32 % cases having had bilateral salpingectomy. Transfer of two embryos or more (79 %) and fresh embryo transfer (71 %) were reported in the majority of cases. Heterotopic abdominal pregnancy occurred in 46 % of cases while 54 % were abdominal ectopic pregnancies. Our systematic review has revealed several trends in reported cases of abdominal ectopic pregnancy after IVF including tubal factor infertility, history of tubal ectopic and tubal surgery, higher number of embryos transferred, and fresh embryo transfers. These are consistent with known risk factors for ectopic pregnancy following IVF. Further research focusing on more homogenous population may help in better characterizing this rare IVF complication and its risks.
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Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos
Nakano, Kazuaki; Watanabe, Masahito; Matsunari, Hitomi; Matsuda, Taisuke; Honda, Kasumi; Maehara, Miki; Kanai, Takahiro; Hayashida, Gota; Kobayashi, Mirina; Kuramoto, Momoko; Arai, Yoshikazu; Umeyama, Kazuhiro; Fujishiro, Shuh-hei; Mizukami, Yoshihisa; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi
2013-01-01
Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras. PMID:23626746
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Liu, Jiaen; Yang, Zhihong; Salem, Shala A; Rahil, Tayyab; Collins, Gary S; Liu, Xiaohong; Salem, Rifaat D
2012-01-01
Objective During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. Methods First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. Results Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). Conclusion While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation. PMID:22816070
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Progestin implants can rescue demi-embryo pregnancies in goats: a case study.
Beckett, D M; Oppenheim, S M; Moyer, A L; BonDurant, R H; Rowe, J D; Anderson, G B
1999-06-01
Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.
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Rotomskis, Ričardas; Jurgelėnė, Živilė; Stankevičius, Mantas; Stankevičiūtė, Milda; Kazlauskienė, Nijolė; Jokšas, Kęstutis; Montvydienė, Danguolė; Kulvietis, Vytautas; Karabanovas, Vitalijus
2018-09-01
Due to colloidal instability even with protective coatings, nanoparticles tend to aggregate in complex environments and possibly interact with biota. In this study, visualization of quantum dots (QDs) interaction with rainbow trout (Oncorhynchus mykiss) embryos was performed. Studies on zebrafish (Danio rerio) and pearl gourami (Trichogaster leerii) embryos have shown that QDs interact with embryos in a general manner and their affects are independent on the type of the embryo. It was demonstrated that carboxylated CdSe/ZnS QDs (4 nM) were aggregating in accumulation media and formed agglomerates on the surface of fish embryos under 1-12 days incubation in deep-well water. Detailed analysis of QDs distribution on fish embryos surface and investigation of the penetration of QDs through embryo's membrane showed that the chorion protects embryos from the penetration through the chorion and the accumulation of nanoparticles inside the embryos. Confocal microscopy and spectroscopy studies on rainbow trout embryos demonstrated that QDs cause chorion damage, due to QDs aggregation on the surface of chorion, even the formation of the agglomerates at the outer part of the embryos and/or with the mucus were detected. Aggregation of QDs and formation of agglomerates on the outer part of the embryo's membrane caused the intervention of the aggregates to the chorion and even partially destroyed the embryo's chorion. The incorporation of QDs in chorion was confirmed by two methods: in living embryos from a 3D reconstruction view, and in slices of embryos from a histology view. The damage of chorion integrity might have adverse effects on embryonic development. Moreover, for the first time the toxic effect of QDs was separated from the heavy metal toxicity, which is most commonly discussed in the literature to the toxicity of the QDs. Copyright © 2018 Elsevier B.V. All rights reserved.
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Code of Federal Regulations, 2013 CFR
2013-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...
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Code of Federal Regulations, 2013 CFR
2013-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...
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Code of Federal Regulations, 2012 CFR
2012-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...
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Code of Federal Regulations, 2011 CFR
2011-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...
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Code of Federal Regulations, 2012 CFR
2012-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...
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Code of Federal Regulations, 2010 CFR
2010-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...
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Code of Federal Regulations, 2014 CFR
2014-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...
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Code of Federal Regulations, 2014 CFR
2014-01-01
... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...
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2011-01-01
We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome. PMID:21527004
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Cryopreservation of embryos and oocytes in human assisted reproduction.
Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor
2014-01-01
Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.
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Dulay, Rich Milton R; Kalaw, Sofronio P; Reyes, Renato G; Alfonso, Noel F; Eguchi, Fumio
2012-01-01
This paper highlights the teratogenic and toxic effects of Ganoderma lucidum (Lingzhi or Reishi mushroom) extract on zebrafish embryos. Hatchability, malformations, and lethality rate of zebrafish embryos were assessed to provide valuable information regarding the potential teratogenic activity of G. lucidum. Hatching was completed 48 h post treatment application (hpta) at 1% or lower concentrations of extract and embryo water. The hatching rate of embryos treated with 5% or higher concentrations was significantly lower (p> 0.05) than the control. Tail malformation was the most marked morphological abnormality in embryos at 72 hpta, which was obviously caused by 1% extract (55.56% tail malformation) and was observed in all embryos exposed to 5% of extract. Growth retardation was evident in embryos exposed to 5%, 10%, and 20%. However, lethal effect of extract of G. lucidum was dependent on dose and time of exposure. Mortality rates of embryos treated with 5% (44.44%) or higher concentrations of the extract was significantly higher (p > 0.05) than that of the control embryos at 72 hpta. These results suggest that G. lucidum extract has lethal and sub-lethal effects on zebrafish embryos.
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Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.
Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred
2004-07-01
Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.
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Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.
Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D
Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.
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Is it time for a paradigm shift in understanding embryo selection?
Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H
2015-01-11
Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.
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A Technique for Facile and Precise Transfer of Mouse Embryos
Sarvari, Ali; Naderi, Mohammad Mehdi; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi
2013-01-01
Background Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. Methods In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). Conclusion In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium. PMID:23626878
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Laporta, J; Driver, A; Khatib, H
2011-08-01
Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Kelley, Rebecca L; Gardner, David K
2017-05-01
Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Dos Santos Neto, P C; Vilariño, M; Barrera, N; Cuadro, F; Crispo, M; Menchaca, A
2015-02-01
This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts. Copyright © 2014 Elsevier Inc. All rights reserved.
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Sartori, R; Sartor-Bergfelt, R; Mertens, S A; Guenther, J N; Parrish, J J; Wiltbank, M C
2002-11-01
Two experiments in two seasons evaluated fertilization rate and embryonic development in dairy cattle. Experiment 1 (summer) compared lactating Holstein cows (n = 27; 97.3 +/- 4.1 d postpartum [dppl; 40.0 +/- 1.5 kg milk/d) to nulliparous heifers (n = 28; 11 to 17 mo old). Experiment 2 (winter) compared lactating cows (n = 27; 46.4 +/- 1.6 dpp; 45.9 +/- 1.4 kg milk/d) to dry cows (n = 26). Inseminations based on estrus included combined semen from four high-fertility bulls. Embryos and oocytes recovered 5 d after ovulation were evaluated for fertilization, embryo quality (1 = excellent to 5 = degenerate), nuclei/embryo, and accessory sperm. In experiment 1, 21 embryos and 17 unfertilized oocytes (UFO) were recovered from lactating cows versus 32 embryos and no UFO from heifers (55% vs. 100% fertilization). Embryos from lactating cows had inferior quality scores (3.8 +/- 0.4 vs. 2.2 +/- 0.3), fewer nuclei/embryo (19.3 +/- 3.7 vs. 36.8 +/- 3.0) but more accessory sperm (37.3 +/- 5.8 vs. 22.4 +/- 5.5/embryo) than embryos from heifers. Sperm were attached to 80% of UFO (17.8 +/- 12.1 sperm/UFO). In experiment 2, lactating cows yielded 36 embryos and 5 UFO versus 34 embryos and 4 UFO from dry cows (87.8 vs. 89.5% fertilization). Embryo quality from lactating cows was inferior to dry cows (3.1 +/- 0.3 vs. 2.2 +/- 0.3), but embryos had similar numbers of nuclei (27.2 +/- 2.7 vs. 30.6 +/- 2.1) and accessory sperm (42.0 +/- 9.4 vs. 36.5 +/- 6.3). From 53% of the flushings from lactating cows and 28% from dry cows, only nonviable embryos were collected. Thus, embryos of lactating dairy cows were detectably inferior to embryos from nonlactating females as early as 5 d after ovulation, with a surprisingly high percentage of nonviable embryos. In addition, fertilization rate was reduced only in summer, apparently due to an effect of heat stress on the oocyte.
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Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming
2015-07-15
No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.
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PGS-FISH in reproductive medicine and perspective directions for improvement: a systematic review.
Zamora, Sandra; Clavero, Ana; Gonzalvo, M Carmen; de Dios Luna Del Castillo, Juan; Roldán-Nofuentes, Jose Antonio; Mozas, Juan; Castilla, Jose Antonio
2011-08-01
Embryo selection can be carried out via morphological criteria or by using genetic studies based on Preimplantation Genetic Screening. In the present study, we evaluate the clinical validity of Preimplantation Genetic Screening with fluorescence in situ hybridization (PGS-FISH) compared with morphological embryo criteria. A systematic review was made of the bibliography, with the following goals: firstly, to determine the prevalence of embryo chromosome alteration in clinical situations in which the PGS-FISH technique has been used; secondly, to calculate the statistics of diagnostic efficiency (negative Likelihood Ratio), using 2 × 2 tables, derived from PGS-FISH. The results obtained were compared with those obtained from embryo morphology. We calculated the probability of transferring at least one chromosome-normal embryo when it was selected using either morphological criteria or PGS-FISH, and considered what diagnostic performance should be expected of an embryo selection test with respect to achieving greater clinical validity than that obtained from embryo morphology. After an embryo morphology selection that produced a negative result (normal morphology), the likelihood of embryo aneuploidies was found to range from a pre-test value of 65% (prevalence of embryo chromosome alteration registered in all the study groups) to a post-test value of 55% (Confidence interval: 50-61), while after PGS-FISH with a negative result (euploid), the post-test probability was 42% (Confidence interval: 35-49) (p < 0.05). The probability of transferring at least one euploid embryo was the same whether 3 embryos were selected according to morphological criteria or whether 2, selected by PGS-FISH, were transferred. Any embryo selection test, if it is to provide greater clinical validity than embryo morphology, must present a LR-value of 0.40 (Confidence interval: 0.32-0.51) in single embryo transfer, and 0.06 (CI: 0.05-0.07) in double embryo transfer. With currently available technology, and taking into account the number of embryos to be transferred, the clinical validity of PGS-FISH, although superior to that of morphological criteria, does not appear to be clinically relevant.