A potential non-invasive approach to evaluating blastocyst quality using biodynamic imaging

NASA Astrophysics Data System (ADS)

Li, Zhe; Ehmke, Natalie; Machaty, Zoltan; Nolte, David

2018-02-01

Biodynamic imaging (BDI) is capable of capturing the intracellular dynamics of blastocysts within a relatively short time. Spectroscopic signatures of embryos in the 0.01 Hz - 1 Hz range display responses to external factors before morphology changes take place. Viability evaluation is consistent with results from other non-invasive methods. Biodynamic imaging is a potential tool for selecting high quality embryos in clinical IVF practices.

Reduced blastocyst formation in reduced culture volume.

PubMed

De Munck, N; Santos-Ribeiro, S; Mateizel, I; Verheyen, G

2015-09-01

The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation. Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7 μl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated. No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0 %). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25 μl droplets led to more embryos with a higher cell number when compared to 7 μl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25 μl group (75.0 versus 61.6 %; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5 %; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8 %; p = 0.043), per fertilized oocyte (50.7 versus 36.6 %; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7 %; p = 0.019) were all significantly higher in the 25 μl group. Reduced culture volume (7 μl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.

Antimüllerian hormone as a predictor of good-quality supernumerary blastocyst cryopreservation among women with levels <1 ng/mL versus 1-4 ng/mL.

PubMed

Kavoussi, Shahryar K; Odenwald, Kate C; Boehnlein, Lynn M; Summers-Colquitt, Roxanne B; Pool, Thomas B; Swain, Jason E; Jones, Jeffrey M; Lindstrom, Mary J; Lebovic, Dan I

2015-09-01

To determine whether antimüllerian hormone (AMH) levels predict the availability of good-quality supernumerary blastocysts for cryopreservation. Retrospective study. Two fertility centers. First fresh IVF cycles (n = 247) grouped as follows: 40 women <35 year old with AMH <1 ng/mL and 77 women with AMH 1-4 ng/mL; 62 women ≥35 year old with AMH <1 ng/mL, and 68 women with AMH 1-4 ng/mL. AMH level measured before IVF with ovarian stimulation protocols based on patient age and AMH level, including short gonadotropin-releasing hormone (GnRH) agonist, GnRH antagonist, or GnRH agonist microdose flare; supernumerary good-quality blastocysts cryopreserved on days 5 or 6 after retrieval. Supernumerary good-quality blastocysts for cryopreservation in relation to AMH levels. Among women <35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocysts for cryopreservation between the groups with AMH <1 ng/mL and AMH 1-4 ng/mL (30.0% vs. 58.4%) when adjusted for age. Among women ≥35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocyst cryopreservation between groups with AMH <1 ng/mL and AMH 1-4 ng/mL (16.1% vs. 42.6%), when adjusted for age. Low AMH levels are associated with a statistically significantly lower likelihood of blastocysts for cryopreservation as compared with higher AMH levels. This effect was seen among women both <35 and ≥35 years of age. Patient counseling should include realistic expectations for the probability of good-quality supernumerary blastocysts available for cryopreservation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Mitochondrial DNA variations in ova and blastocyst: implications in assisted reproduction.

PubMed

Shamsi, Monis Bilal; Govindaraj, Periyasamy; Chawla, Latika; Malhotra, Neena; Singh, Neeta; Mittal, Suneeta; Talwar, Pankaj; Thangaraj, Kumarasamy; Dada, Rima

2013-03-01

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission. Copyright © 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage

PubMed Central

Gupta, Alisha; Singh, Jaswant; Dufort, Isabelle; Robert, Claude; Dias, Fernanda Caminha Faustino

2017-01-01

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to

Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

PubMed

Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

2011-04-01

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts.

PubMed

Cervera, R P; Garcia-Ximénez, F

2003-10-01

The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

Physiology and culture of the human blastocyst.

PubMed

Gardner, David K; Lane, Michelle; Schoolcraft, William B

2002-01-01

The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

PubMed Central

Chan, Wen-Hsiung

2014-01-01

We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole. PMID:24933639

Statins inhibit blastocyst formation by preventing geranylgeranylation

PubMed Central

Alarcon, Vernadeth B.; Marikawa, Yusuke

2016-01-01

STUDY HYPOTHESIS Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway and prescription drugs that treat hypercholesterolemia, compromise preimplantation mouse development via modulation of HIPPO signaling. STUDY FINDING HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yes-associated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase. WHAT IS KNOWN ALREADY Previous studies have shown that treatment of mouse embryos with mevastatin prevents blastocyst formation, but how HMG-CoA reductase is involved in preimplantation development is unknown. HIPPO signaling regulates specification of the trophectoderm lineage of the mouse blastocyst by controlling the nuclear localization of YAP. In human cell lines, the mevalonate pathway regulates YAP to mediate self-renewal and survival through geranylgeranylation of RHO proteins. These studies suggest that in preimplantation development, statins may act through HIPPO pathway to interfere with trophectoderm specification and thereby inhibit blastocyst formation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Eight-cell stage (E2.5) mouse embryos were treated in hanging drop culture with chemical agents, namely statins (lovastatin, atorvastatin, cerivastatin and pravastatin), mevalonic acid (MVA), cholesterol, squalene, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranylgeranyltransferase inhibitor GGTI-298, RHO inhibitor I, and squalene synthase inhibitor YM-53601, up to the late blastocyst stage (E4.5). Efficiency of blastocyst formation was assessed based on gross morphology and the measurement of the cavity size using an image analysis software. Effects on cell lineages and HIPPO signaling were analyzed using immunohistochemistry with confocal microscopy based on the expression patterns of the

Research on Blastocyst Implantation Essential Factors (BIEFs).

PubMed

Yoshinaga, Koji

2010-06-01

Blastocyst implantation is a process of interaction between embryo and the uterus. To understand this process, this review tries to summarize what blastocyst implantation essential factors (BIEFs) play what roles, as well as where in the uterus and at what stage of implantation process. Addition of more new data to this kind of compilation of information will help the development of diagnosis and treatment of infertility caused by implantation failure. The major, important cells of the endometrial cells that interact with invading blastocyst (trophoblast) are luminal epithelial cells, stromal cells (decidual cells) and resident immune cells. BIEFs regulate these cells to successfully maintain pregnancy.

Obstetric and perinatal outcomes of singletons after single blastocyst transfer: is there any difference according to blastocyst morphology?

PubMed

Bouillon, Céline; Celton, Noémie; Kassem, Sandra; Frapsauce, Cynthia; Guérif, Fabrice

2017-08-01

A strong correlation between blastocyst morphology and implantation has been shown by many studies. The consequences and effects of assisted reproductive techniques on children's short and long-term health have always been a source of discussion. The obstetric and perinatal outcome of singletons according to blastocyst morphology has rarely been evaluated. The aim of this observational study is to determine whether a relationship exists between blastocyst morphology and obstetric and perinatal outcomes. A total of 799 singleton clinical pregnancies were analysed after transfer of a single fresh blastocyst on day 5 between 2006 and 2013. Blastocysts were divided into four groups based on their morphology on day 5: group 1 = good morphology blastocysts; group 2 = fair morphology blastocysts; group 3 = poor morphology blastocysts and group 4 = early (B1/B2) blastocysts. Obstetric and perinatal outcomes were compared between the four groups. After adjustment for some confounding variables, main obstetric and perinatal outcomes after transfer of blastocysts with poor morphological characteristics were not associated with increased adverse obstetric and perinatal events. Sex ratio was significantly higher in group 1 compared with groups 2, 3 and 4, and in Group 2 compared with Group 3 (P < 0.001) even after adjustment (P < 0.05). Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice.

PubMed

Kang, Hee Jung; Hwang, Soo Jin; Yoon, Jung Ah; Jun, Jin Hyun; Lim, Hyunjung Jade; Yoon, Tae Ki; Song, Haengseok

2011-10-01

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Comparing blastocyst quality and live birth rates of intravaginal culture using INVOcell™ to traditional in vitro incubation in a randomized open-label prospective controlled trial.

PubMed

Doody, Kevin J; Broome, E Jason; Doody, Kathleen M

2016-04-01

The purpose of this study is to to compare the efficacy of intravaginal culture (IVC) of embryos in INVOcell™ (INVO Bioscience, MA, USA) to traditional in vitro fertilization (IVF) incubators in a laboratory setting using a mild pre-determined stimulation regimen based solely on anti-mullerian hormone (AMH) and body weight with minimal ultrasound monitoring. The primary endpoint examined was total quality blastocysts expressed as a percentage of total oocytes placed in incubation. Secondary endpoints included percentage of quality blastocysts transferred, pregnancy, and live birth rates. In this prospective randomized open-label controlled single-center study, 40 women aged <38 years of age with a body mass index (BMI) of <36 and an AMH of 1-3 ng/mL were randomized prior to trigger to receive either IVC or IVF. Controlled ovarian stimulation was administered with human menopausal gonadotropin (hMG) in a fixed gonadotropin-releasing hormone (GnRH) agonist cycle based solely on AMH and body weight. A single ultrasound-monitoring visit was performed on the 10th day of stimulation. One or two embryos were transferred following 5 days of culture. IVF produced a greater percentage of total quality embryos as compared to IVC (50.6 vs. 30.7 %, p = 0.0007, respectively). There was no significant difference between in IVF and IVC in the percentage of quality blastocysts transferred (97.5 vs. 84.9 %, p = 0.09) or live birth rate (60 % IVF, 55 % IVC). IVF was shown to be superior to IVC in creating quality blastocysts. However, both IVF and IVC produced identical blastocysts for transfer resulting in similar live birth rates. IVC using INVOcell™ is effective and may broaden access to fertility care in selected patient populations by ameliorating the need for a traditional IVF laboratory setting. Further studies will help elucidate the potential physiological, psychological, geographic, and financial impact of IVC on the delivery of fertility care.

A comparison of the survival and implantation rates of blastocysts that were vitrified on post-fertilization day five, six and seven.

PubMed

Berrin, Avci; Isıl, Kasapoglu; Baris, Ata; Goktan, Kuspinar; Seda, Saribal; Gurkan, Uncu

2018-05-03

The goal of this retrospective cohort study was to compare survival, implantation, clinical and ongoing pregnancy rates between blastocysts that were vitrified on post-fertilization days 5, 6 and 7. Before vitrification, blastocysts were evaluated in terms of morphology and blastocyst expansion, inner cell mass and trophectoderm quality. They were thawed and transfered in a subsequent artificial cycle. Embryo implantation rates were 39%, 25% and 25% for blastocysts that were vitrified on days 5, 6, and 7, respectively (p = 0.006). Clinical and ongoing pregnancy rates were 19%, 12%, 13% (p = 0.100) and 9%, 7%, 12% (p = 0.99) for days 5, 6 and 7 blastocysts, respectively. Day 5 blastocysts had significantly higher full-collapsing score after assisted-hatching compared to days 6 and 7 blastocysts (p = 0.014). As blastocyst quality increased, implantation and clinical pregnancy rates increased in all groups and both parameters were statistically significantly higher on day 5 blastocysts than on days 6 or 7 (p = 0.001). It was clearly found that good quality blastocysts obtained on day 5 have higher implantation and clinical pregnancy rates than 6th and 7th day cryopreserved embryos. There were no statistically significant differences between the cryopreserved embryos on days 6 and 7 regarding the implantation, clinic and ongoing pregnancy rates.

Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion.

PubMed

Cuman, Carly; Van Sinderen, Michelle; Gantier, Michael P; Rainczuk, Kate; Sorby, Kelli; Rombauts, Luk; Osianlis, Tiki; Dimitriadis, Evdokia

2015-10-01

Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.

[The specificity of blastocysts (Rhizopoda: Lobosea)].

PubMed

Belova, L M; Krylov, M V

1994-01-01

The analysis of specificity of blastocysts was based on the data on host association and on experimental data. In experiments we failed to infect the geese (Anser anser) with Blastocystis galli taken from the fowl (Gallus gallus) and also failed to infect the fowl with B. suis taken from the pigs (Sus scrofa domestica). Experimental data and field observations of blastocysts distribution among different groups of hosts point out that the same species of blastocysts can not parasitize in hosts belonging to different classes and orders. The examination of 89 fish specimens belonging to 14 species of Osteichthyes taken from the Neman delta did not discover any blastocysts.

Blastocyst utilization rates after continuous culture in two commercial single-step media: a prospective randomized study with sibling oocytes.

PubMed

Sfontouris, Ioannis A; Kolibianakis, Efstratios M; Lainas, George T; Venetis, Christos A; Petsas, George K; Tarlatzis, Basil C; Lainas, Tryfon G

2017-10-01

The aim of this study is to determine whether blastocyst utilization rates are different after continuous culture in two different commercial single-step media. This is a paired randomized controlled trial with sibling oocytes conducted in infertility patients, aged ≤40 years with ≥10 oocytes retrieved assigned to blastocyst culture and transfer. Retrieved oocytes were randomly allocated to continuous culture in either Sage one-step medium (Origio) or Continuous Single Culture (CSC) medium (Irvine Scientific) without medium renewal up to day 5 post oocyte retrieval. Main outcome measure was the proportion of embryos suitable for clinical use (utilization rate). A total of 502 oocytes from 33 women were randomly allocated to continuous culture in either Sage one-step medium (n = 250) or CSC medium (n = 252). Fertilization was performed by either in vitro fertilization or intracytoplasmic sperm injection, and embryo transfers were performed on day 5. Two patients had all blastocysts frozen due to the occurrence of severe ovarian hyperstimulation syndrome. Fertilization and cleavage rates, as well as embryo quality on day 3, were similar in the two media. Blastocyst utilization rates (%, 95% CI) [55.4% (46.4-64.1) vs 54.7% (44.9-64.6), p = 0.717], blastocyst formation rates [53.6% (44.6-62.5) vs 51.9 (42.2-61.6), p = 0.755], and proportion of good quality blastocysts [36.8% (28.1-45.4) vs 36.1% (27.2-45.0), p = 0.850] were similar in Sage one-step and CSC media, respectively. Continuous culture of embryos in Sage one-step and CSC media is associated with similar blastocyst development and utilization rates. Both single-step media appear to provide adequate support during in vitro preimplantation embryo development. Whether these observations are also valid for other continuous single medium protocols remains to be determined. NCT02302638.

Blastocyst-like structures generated solely from stem cells.

PubMed

Rivron, Nicolas C; Frias-Aldeguer, Javier; Vrij, Erik J; Boisset, Jean-Charles; Korving, Jeroen; Vivié, Judith; Truckenmüller, Roman K; van Oudenaarden, Alexander; van Blitterswijk, Clemens A; Geijsen, Niels

2018-05-01

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

Expression of granulosa cell microRNAs, AVEN and ATRX are associated with human blastocyst development.

PubMed

O'Doherty, Alan M; O'Brien, Yvonne M; Browne, John A; Wingfield, Mary; O'Shea, Lynne C

2018-04-25

A greater understanding of the key molecules associated with embryo development during human-assisted reproduction is imperative for the development of advanced diagnostics. Previous studies have shown that follicular microRNAs (miRNAs) are reliable markers of the polycystic ovarian syndrome (PCOS). Leveraging the utility of miRNAs in PCOS, the aim of this study was to identify miRNAs in human granulosa cells that may be indicative of blastocyst development. Granulosa cells and oocytes were collected from the first follicle aspirated from patients undergoing oocyte retrieval for in vitro fertilization or intracytoplasmic sperm injection. The development of isolated oocytes was recorded, and granulosa cell samples in this study were separated as follows. Group 1-BLAST: granulosa cells from follicles containing an oocyte that fertilized and developed into a blastocyst, and Group 2-FERT: granulosa cells from oocytes that fertilized but failed to reach blastocyst. A panel of 84 miRNAs, related to development and cellular differentiation, was assessed between the two groups using a miScript PCR array. Fourteen miRNAs and one snoRNA were differentially expressed between the groups. In addition, two downstream candidate protein biomarkers, ATRX and AVEN, were also found to be differentially expressed between the groups. The findings of this pilot study reveal follicular abnormalities on a molecular level, which may affect oocyte competence and its potential to develop successfully as an embryo. We encourage additional studies to confirm and expand on our findings and to determine the usefulness of granulosa-borne miRNAs, ATRX, and AVEN as biomarkers. © 2018 Wiley Periodicals, Inc.

Comparison of the clinical outcomes between fresh blastocyst and vitrified-thawed blastocyst transfer.

PubMed

Ku, Pei-Yun; Lee, Robert Kuo-Kuang; Lin, Shyr-Yeu; Lin, Ming-Huei; Hwu, Yuh-Ming

2012-12-01

To compare the clinical outcomes between fresh and vitrified-thawed day 5 blastocyst transfers. Retrospective case control study. Tertiary referral center. Patients 38 years of age or less who underwent IVF/ICSI cycles with fresh or frozen-thawed blastocysts transferred from June 1, 2009 to November 30, 2011 Vitrification and thawing of day 5 blastocysts using the Cryotop method. (Kitazato BioPharma Co., Ltd., Fuji city, Shizuoka, Japan) Clinical pregnancy rate, implantation rate, ongoing pregnancy rate, and multiple pregnancy rates. Of the 118 cycles in the fresh transfer group, 234 blastocysts were transferred. The clinical pregnancy rate was 66.1 % and implantation rate was 50.9 %. The ongoing pregnancy rate was 56.8 % and the rates for singleton and twin pregnancies were 53.7 % and 44.8 %. Of the 59 cycles in the vitrified-thawed group, 111 blastocysts were transferred. The clinical pregnancy rate was 59.3 % and implantation rate was 43.2 %. The ongoing pregnancy rate was 47.5 % and the rates for singleton and twin pregnancies were 60.7 % and 39.3 %. The clinical pregnancy rate, implantation rate and ongoing pregnancy rate did not differ significantly between the two groups. The implantation rates were not significantly different between the fresh and the vitrified-thawed groups. Thus, single embryo transfer may be considered in fresh cycles to decrease multiple pregnancy rates. The surplus embryos should be vitrified for the frozen embryo transfer to improve the cumulative pregnancy rate.

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats.

PubMed

Romaguera, R; Moll, X; Morató, R; Roura, M; Palomo, M J; Catalá, M G; Jiménez-Macedo, A R; Hammami, S; Izquierdo, D; Mogas, T; Paramio, M T

2011-07-01

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

PubMed

Laughlin, Taylor D; Miles, Jeremy R; Wright-Johnson, Elane C; Rempel, Lea A; Lents, Clay A; Pannier, Angela K

2017-11-01

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17β-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

Rhein Induces Oxidative Stress and Apoptosis in Mouse Blastocysts and Has Immunotoxic Effects during Embryonic Development.

PubMed

Huang, Chien-Hsun; Chan, Wen-Hsiung

2017-09-20

Rhein, a glucoside chemical compound found in a traditional Chinese medicine derived from the roots of rhubarb, induces cell apoptosis and is considered to have high potential as an antitumor drug. Several previous studies showed that rhein can inhibit cell proliferation and trigger mitochondria-related or endoplasmic reticulum (ER) stress-dependent apoptotic processes. However, the side effects of rhein on pre- and post-implantation embryonic development remain unclear. Here, we show that rhein has cytotoxic effects on blastocyst-stage mouse embryos and induces oxidative stress and immunotoxicity in mouse fetuses. Blastocysts incubated with 5-20 μM rhein showed significant cell apoptosis, as well as decreases in their inner cell mass cell numbers and total cell numbers. An in vitro development assay showed that rhein affected the developmental potentials of both pre- and post-implantation embryos. Incubation of blastocysts with 5-20 μM rhein was associated with increased resorption of post-implantation embryos and decreased fetal weight in an embryo transfer assay. Importantly, in an in vivo model, intravenous injection of dams with rhein (1, 3, and 5 mg/kg body weight/day) for four days resulted in apoptosis of blastocyst-stage embryos, early embryonic developmental injury, and decreased fetal weight. Intravenous injection of dams with 5 mg/kg body weight/day rhein significantly increased the total reactive oxygen species (ROS) content of fetuses and the transcription levels of antioxidant proteins in fetal livers. Additional work showed that rhein induced apoptosis through ROS generation, and that prevention of apoptotic processes effectively rescued the rhein-induced injury effects on embryonic development. Finally, the transcription levels of the innate-immunity related genes, CXCL1 , IL-1 β and IL-8 , were down-regulated in the fetuses of dams that received intravenous injections of rhein. These results collectively show that rhein has the potential to

Obstetrical and perinatal outcomes following blastocyst transfer compared to cleavage transfer: a systematic review and meta-analysis.

PubMed

Martins, W P; Nastri, C O; Rienzi, L; van der Poel, S Z; Gracia, C R; Racowsky, C

2016-11-01

Is blastocyst transfer safe when compared to cleavage stage embryo transfer regarding obstetric and perinatal outcomes? The clinical equipoise between blastocyst and cleavage stage embryo transfer remains as the evidence associating blastocyst transfer with some adverse perinatal outcomes is of low/very low quality. Extended embryo culture to the blastocyst stage provides some theoretical advantages and disadvantages. While it permits embryo self-selection, it also exposes those embryos to possible harm due to the in vitro environment. Both effectiveness and safety should be weighed to permit evidence-based decisions in clinical practice. This is a systematic review and meta-analysis of randomized controlled trials (RCTs) and observational studies reporting perinatal outcomes for singletons comparing the deliveries resulting from blastocyst and cleavage stage embryo transfer. Observational studies were included because the primary outcomes, perinatal mortality and birth defects, are rare and require a large number of participants (>50 000) to be properly assessed. The last electronic searches were last run on 11 March 2016. There were 12 observational studies encompassing 195 325 singleton pregnancies included in the study. No RCT reported the studied outcomes. The quality of the included studies was evaluated according to the Newcastle-Ottawa Scale and the quality of the evidence was evaluated according to GRADE criteria. Blastocyst stage transfer was associated with increased risks of preterm birth (<37 weeks), very preterm birth (<32 weeks), large for gestational age and perinatal mortality, although the latter was only identified from one study. Conversely, blastocyst stage transfer was associated with a decrease in the risks of small for gestational age and vanishing twins, although the latter was reported by only one study. The observational nature of the included studies and some inconsistency and imprecision in the analysis contributed to decreasing our

Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles.

PubMed

Du, Qing-Yun; Wang, En-Yin; Huang, Yan; Guo, Xiao-Yi; Xiong, Yu-Jing; Yu, Yi-Ping; Yao, Gui-Dong; Shi, Sen-Lin; Sun, Ying-Pu

2016-04-01

To evaluate the independent effects of the degree of blastocoele expansion and re-expansion and the inner cell mass (ICM) and trophectoderm (TE) grades on predicting live birth after fresh and vitrified/warmed single blastocyst transfer. Retrospective study. Reproductive medical center. Women undergoing 844 fresh and 370 vitrified/warmed single blastocyst transfer cycles. None. Live-birth rate correlated with blastocyst morphology parameters by logistic regression analysis and Spearman correlations analysis. The degree of blastocoele expansion and re-expansion was the only blastocyst morphology parameter that exhibited a significant ability to predict live birth in both fresh and vitrified/warmed single blastocyst transfer cycles respectively by multivariate logistic regression and Spearman correlations analysis. Although the ICM grade was significantly related to live birth in fresh cycles according to the univariate model, its effect was not maintained in the multivariate logistic analysis. In vitrified/warmed cycles, neither ICM nor TE grade was correlated with live birth by logistic regression analysis. This study is the first to confirm that the degree of blastocoele expansion and re-expansion is a better predictor of live birth after both fresh and vitrified/warmed single blastocyst transfer cycles than ICM or TE grade. Copyright © 2016. Published by Elsevier Inc.

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts.

PubMed

Kubisch, H M; Larson, M A; Ealy, A D; Murphy, C N; Roberts, R M

2001-04-30

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

PubMed

Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

2018-04-19

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

PubMed

Wen, Duan-Cheng; Bi, Chun-Ming; Xu, Ying; Yang, Cai-Xia; Zhu, Zi-Yu; Sun, Qing-Yuan; Chen, Da-Yuan

2005-08-01

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids. Copyright (c) 2005 Wiley-Liss, Inc.

Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence.

PubMed

Choi, Y H; Love, L B; Varner, D D; Hinrichs, K

2007-11-01

The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells.

PubMed

Kumar, R; Ahlawat, S P S; Sharma, M; Verma, O P; Sai Kumar, G; Taru Sharma, G

2014-03-01

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations.

PubMed

Choi, Young-Ho; Ross, Pablo; Velez, Isabel C; Macías-García, B; Riera, Fernando L; Hinrichs, Katrin

2015-07-01

Equine embryos develop in vitro in the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31-46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively for POU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did. GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation in in vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse. © 2015 Society for Reproduction and Fertility.

Pluripotency and lineages in the mammalian blastocyst: an evolutionary view.

PubMed

Cañon, Susana; Fernandez-Tresguerres, Beatriz; Manzanares, Miguel

2011-06-01

Early mammalian development is characterized by a highly specific stage, the blastocyst, by which embryonic and extraembryonic lineages have been determined, but pattern formation has not yet begun. The blastocyst is also of interest because cell precursors of the embryo proper retain for a certain time the capability to generate all the cell types of the adult animal. This embryonic pluripotency is established and maintained by a regulatory network under the control of a small set of transcription factors, comprising Oct4, Sox2 and Nanog. This network is largely conserved in eutherian mammals, but there is scarce information about how it arose in vertebrates. We have analysed the conservation of gene regulatory networks controlling blastocyst lineages and pluripotency in the mouse by comparison with the chick. We found that few of elements of the network are novel to mammals; rather, most of them were present before the separation of the mammalian lineage from other amniotes, but acquired novel expression domains during early mammalian development. Our results strongly support the hypothesis that mammalian blastocyst regulatory networks evolved through rewiring of pre-existing components, involving the co-option and duplication of existing genes and the establishment of new regulatory interactions among them.

Development of Monozygotic Twin Mouse Embryos from the Time of Blastomere Separation at the Two-Cell Stage to Blastocyst1

PubMed Central

Katayama, Mika; Ellersieck, Mark R.; Roberts, R. Michael

2010-01-01

The development of blastomeres separated from two-cell stage murine embryos has been compared. Blastomeres were removed from the zona pellucida (ZP) and cultured individually; the twin embryos were compared during their progression to blastocyst in terms of development rate, cell number, morphology, conformation at the four-cell stage, and CDX2 and POU5F1 (also known as OCT4) expression. In general, twin embryos, whether obtained from superovulated or normally bred dams, displayed comparable cell numbers as they advanced. They formed morulae and blastocysts more or less synchronously with each other and with control embryos, although possessing about half of the latter's cell number. Despite this apparent synchrony, the majority of twin blastocysts differed in terms of their relative complements of POU5F1+/CDX2− cells, which represent inner cell mass (ICM), and POU5F1+/CDX2+ cells, which identify trophectoderm (TE). Many, but not all, exhibited a disproportionately small ICM. By contrast, demiembryos retained within their ZP and created by randomly damaging one of the two blastomeres in two-cell stage embryos exhibited a more normal ratio of ICM to TE cells at blastocyst and significantly less variance in ICM cell number. One possible explanation is that ZP-free demiembryos only infrequently adopt the same conformation as their partners, including the favorable tetrahedral form, at the four-cell stage, suggesting that such embryos exhibit a high degree of plasticity with regard to the orientation of their first two cleavage planes and that a significant number likely deviate from paths that provide an optimal geometric progression to blastocyst. These data could explain the difficulty of creating monozygotic twins from two-cell stage embryos. PMID:20181620

Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development.

PubMed

Ebner, T; Moser, M; Shebl, O; Sommergruber, M; Yaman, C; Tews, G

2008-06-01

Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus-oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo.

The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

PubMed Central

Takahashi, Kazumasa; Goto, Mayumi; Anzai, Mibuki; Ono, Natsuki; Shirasawa, Hiromitsu; Sato, Wataru; Miura, Hiroshi; Sato, Naoki; Sato, Akira; Kumazawa, Yukiyo; Terada, Yukihiro

2017-01-01

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality. PMID

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro.

PubMed

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.

Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

PubMed

Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

2014-08-01

Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, p<0.05), compact morulae formation (60.83 vs. 51.30%, p<0.05), and the blastomere apoptosis index (3.70 ± 1.41 vs. 4.43% ± 1.65, p<0.05) of bovine SCNT embryos. However, vitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, p<0.05) on day 7 and the hatching blastocysts formation rate on day 9 (26.51 vs. 50.65%, p<0.05) compared with that of the untreated group. Vitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids.

PubMed

Park, S P; Kim, E Y; Kim, D I; Park, N H; Won, Y S; Yoon, S H; Chung, K S; Lim, J H

1999-11-01

This study demonstrates that higher survival of vitrified-thawed bovine blastocysts can be obtained using electron microscope (EM) grids as embryo containers at freezing, rather than plastic straws. In-vitro produced day 7 bovine blastocysts after in-vitro fertilization (IVF) were vitrified on grids or in straws with EFS40 freezing solution and their survival after thawing was compared. Embryo survival was assessed as re-expanded and hatched rates at 24 and 48 h after thawing respectively. When the effects of exposure to vitrification solution and chilling injury from the freezing procedure were examined, embryo survival in the exposure group (24 h: 100, 48 h: 73.3%) was not different compared with that in the control group (100, 84.4%). After vitrification, the hatched rate of the EM grid group 48 h after thawing (67.8%) was significantly higher than that of the straw group (53.3%) (P < 0.05). Fast developing embryos (expanded blastocyst and early hatching blastocyst stage) showed better resistance to freezing than delayed ones (early blastocyst stage), irrespective of embryo containers (early: 24 h, 57.1 and 48 h, 24.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) (P < 0.001). When using expanded and early hatching blastocysts, embryo survival rates in the vitrification-EM grid group (67.8, 95.0% respectively) were significantly higher than that of the vitrification-straw group (53.0, 65.0%) at 48 h.

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

PubMed

Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

2014-06-20

Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles

PubMed Central

2014-01-01

Background Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Methods Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. Results A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

PubMed Central

McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

2010-01-01

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID

The negative influence of sperm cryopreservation on the quality and development of the embryo depends on the morphology of the oocyte.

PubMed

Braga, D P A F; Setti, A S; Figueira, R C S; Iaconelli, A; Borges, E

2015-07-01

The present case-control study aimed to identify the effect of sperm cryopreservation on the quality of the embryo and on the probability of blastocyst formation when oocytes free of dimorphisms are injected and when at least one dymorphism is present. The study included 22 186 zygotes, obtained from 2802 patients undergoing intracytoplasmic sperm injection cycles, in a private assisted reproduction center, using either fresh or cryopreserved sperm. The effect of sperm cryopreservation on the embryo quality on cleavage stage and blastocyst formation chance were evaluated when oocytes free of dimorphisms are injected and when at least one dymorphism is present. The quality of the embryo on cleavage stage as well as the chance for blastocyst formation was not influenced by the origin of the spermatozoa when the quality of the oocyte was not considered. When at least one oocyte defect was present, a negative influence of sperm cryopreservation on cleavage stage embryo quality and the chance for blastocyst formation was noted. In oocytes with extra-cytoplasmic dimorphisms, the injection of cryopreserved sperm did not affect the quality of the embryo during the cleavage stage, but did affect the chance for blastocyst formation. Conversely, in oocytes with intracytoplasmic defects, the quality of the embryos on cleavage stage and the chance of blastocyst formation were negatively influenced by the injection of cryopreserved sperm. The results suggest an oocyte quality-dependent negative effect of sperm cryopreservation on embryo quality and on the probability of blastocyst formation. © 2015 American Society of Andrology and European Academy of Andrology.

Decreased Blastocyst Production in Mice Exposed to Increased Rack Noise

PubMed Central

Zamora, Bernadette M; Jiang, Meisheng; Wang, Ying; Chai, Minghua; Lawson, P Timothy; Lawson, Gregory W

2009-01-01

This study was conducted to investigate the possible effect of rack type on the blastocyst yield of mouse embryo donors. The first phase of the study consisted of housing some mice (group A) in a ventilated rack and others (group B) in a static rack in the same room for 3 d, followed by euthanasia for blastocyst collection and corticosterone assay. Parametric tests were used to compare groups. The number of blastocysts per donor was lower in group A (5.0 ± 1.4 blastocysts) than group B (13.1 ± 3.7 blastocysts). Mean noise was higher in the ventilated rack (80.4 dBC) than in the static rack (69.2 dBC). Serum corticosterone concentrations did not differ between groups. For the second phase of the study, a third group of mice (group C) was housed in a static rack without a ventilated rack in the same room. The noise level for group C was even lower (45.18 ± 2.91 dBC), and the blastocyst count per donor (16.4 ± 2.4) was higher than that of group B. The mean noise levels of empty ventilated and static racks differed significantly between groups for 10 different sound frequencies. Plotting mean blastocyst production against mean rack noise revealed a negative linear relationship with good strength of correlation. These results support the earlier observation that decreased blastocyst count occurs following housing of bred C57BL/6 donor mice in ventilated cages. PMID:19807968

Molecular and cellular events during blastocyst implantation in the receptive uterus: clues from mouse models

PubMed Central

MATSUMOTO, Hiromichi

2017-01-01

The success of implantation is an interactive process between the blastocyst and the uterus. Synchronized development of embryos with uterine differentiation to a receptive state is necessary to complete pregnancy. The period of uterine receptivity for implantation is limited and referred to as the “implantation window”, which is regulated by ovarian steroid hormones. Implantation process is complicated due to the many signaling molecules in the hierarchical mechanisms with the embryo-uterine dialogue. The mouse is widely used in animal research, and is uniquely suited for reproductive studies, i.e., having a large litter size and brief estrous cycles. This review first describes why the mouse is the preferred model for implantation studies, focusing on uterine morphology and physiological traits, and then highlights the knowledge on uterine receptivity and the hormonal regulation of blastocyst implantation in mice. Our recent study revealed that selective proteolysis in the activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. Furthermore, in the context of blastocyst implantation in the mouse, this review discusses the window of uterine receptivity, hormonal regulation, uterine vascular permeability and angiogenesis, the delayed-implantation mouse model, morphogens, adhesion molecules, crosslinker proteins, extracellular matrix, and matricellular proteins. A better understanding of uterine and blastocyst biology during the peri-implantation period should facilitate further development of reproductive technology. PMID:28638003

Three-dimensional reconstruction of tetraploid↔diploid chimaeric mouse blastocysts

PubMed Central

EVERETT, CLARE A.; STARK, MARGARET H.; WEST, JOHN D.; DAVIDSON, DUNCAN; BALDOCK, RICHARD A.

2000-01-01

Studies of tetraploid↔diploid (4n↔2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett & West, 1996). In this study, sectioned 4n↔2n and 2n↔2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n↔2n and 2n↔2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n↔2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n↔2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts. PMID:10853956

Quantitative grading of a human blastocyst: optimal inner cell mass size and shape.

PubMed

Richter, K S; Harris, D C; Daneshmand, S T; Shapiro, B S

2001-12-01

To investigate the predictive value of quantitative measurements of blastocyst morphology on subsequent implantation rates after transfer. Prospective observational study. Private assisted reproductive technology center. One hundred seventy-four IVF patients receiving transfers of expanded blastocyst-stage embryos on day 5 (n = 112) or day 6 (n = 62) after oocyte retrieval. None. Blastocyst diameter, number of trophectoderm cells, inner cell mass (ICM) size, ICM shape, and implantation and pregnancy rates. Blastocyst diameter and trophectoderm cell numbers were unrelated to implantation rates. Day 5 expanded blastocysts with ICMs of >4,500 microm(2) implanted at a higher rate than did those with smaller ICMs (55% vs. 31%). Day 5 expanded blastocysts with slightly oval ICMs implanted at a higher rate (58%) compared with those with either rounder ICMs (7%) or more elongated ICMs (33%). Implantation rates were highest (71%) for embryos with both optimal ICM size and shape. Pregnancy rates were higher for day 5 transfers of optimally shaped ICMs compared with day 5 transfers of optimally sized ICMs. Quantitative measurements of the inner cell mass are highly indicative of blastocyst implantation potential. Blastocysts with relatively large and/or slightly oval ICMs are more likely to implant than other blastocysts.

Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse.

PubMed

Foss, R; Ortis, H; Hinrichs, K

2013-12-01

Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. In vitro experiment. Oocytes were recovered by transvaginal ultrasound-guided follicular aspiration either from dominant follicles 24 h after deslorelin administration (dominant stimulated follicle [DSF]), or from subordinate (immature) follicles at the same time. To mimic transport, DSF oocytes were incubated overnight under differing conditions before ICSI; immature oocytes were placed in varying conditions overnight before in vitro maturation, followed by ICSI. The rate of blastocyst production was compared among treatments. Blastocysts were produced in all groups. Dominant stimulated follicle oocytes held in sealed tubes in pre-equilibrated control maturation medium maintained at 37°C yielded blastocyst development equal to that obtained for control incubated oocytes (70%). Dominant stimulated follicle oocytes held similarly in a warm passive device yielded poor blastocyst development (10%). Immature oocytes held for one or 2 nights in modified M199 medium, or for one night in commercial embryo holding solution, in air at room temperature, yielded 35-37% blastocyst development per injected oocyte. A commercially available medium can be used for shipping immature oocytes at room temperature with good resulting blastocyst rates. Better blastocyst rates per oocyte are obtained from DSF oocytes; however, these require maintenance at 37°C and as they are already maturing at the time of collection, are more sensitive to delays. This new, practical information supporting transport of

Peroxisome proliferator-activated receptor δ improves porcine blastocyst hatching via the regulation of fatty acid oxidation.

PubMed

Guo, Jing; Lu, Wen-Fa; Liang, Shuang; Choi, Jeong-Woo; Kim, Nam-Hyung; Cui, Xiang-Shun

2017-03-01

Peroxisome proliferator-activated receptor δ (Pparδ) is a nuclear receptor that plays critical roles in lipid metabolism, glucose metabolism, and cell growth and differentiation. Several recent studies have shown that Pparδ promotes blastocyst hatching in vitro. However, the mechanism by which it promotes preimplantation embryonic development in vitro remains unclear. In this study, oocytes and parthenotes were treated with a specific agonist of PPARδ, GW501516. The activation of PPARδ had no effect on oocyte maturation for 1 μM and 10 μM GW501516 compared with the control group. Additionally, the PPARδ agonist did not affect blastocyst formation (77.79 ± 3.59% [10 μM], 79.00 ± 5.53% [50 μM], and 79.64 ± 6.00% [100 μM] vs. 81.69 ± 2.61% [control]). However, the blastocyst hatching rate was significantly greater for parthenotes treated with 10 and 50 μM agonist, and did not differ between those treated with 100 μM agonist and the control group (61.80 ± 3.03% [10 μM], 65.10 ± 5.25% [50 μM], and 38.85 ± 7.45% [100 μM] vs. 41.77 ± 10.88% [0 μM]). Activation of PPARδ also increased blastocyst quality and cell number, as well as ATP production. There were no clear differences in mitochondrial membrane potential, mitochondrion copy number, or glucose consumption between the treatment and control groups. However, PPARδ activation enhanced lipid accumulation via Fabp3 and Fabp5. Fatty acid oxidation also increased in response to treatment with the agonist via the rate-limiting gene Cpt2. Reactive oxygen species were modified and REDOX maintenance-related gene expression increased significantly in GW501516-exposed blastocysts. In addition, the activation of PPARδ resulted in changes in miRNA content. After treatment with the PPARδ agonist, miR-99 increased and miR-32 decreased. These data showed that PPARδ has a positive impact on blastocyst hatching via the regulation of lipid metabolism. Copyright © 2016 Elsevier

Risk of ectopic pregnancy lowest with transfer of single frozen blastocyst.

PubMed

Li, Z; Sullivan, E A; Chapman, M; Farquhar, C; Wang, Y A

2015-09-01

with fresh blastocyst transfer, the likelihood of ectopic pregnancy was 30% higher for fresh cleavage stage embryo transfers (AOR 1.30, 95% CI 1.07-1.59) and was consistent across subfertility groups. Transfer of frozen blastocyst was associated with a significantly decreased risk of ectopic pregnancy (AOR 0.70, 95% CI 0.54-0.91) compared with transfer of fresh blastocyst. A limitation of this population-based study is the lack of information available on clinical- specific protocols and processes for embryo transfer (i.e. embryo quality, cryopreservation protocol, transfer techniques, etc.) and the potential impact on outcomes. The lowest risk of ectopic pregnancy was associated with the transfer of a single frozen blastocyst. This finding adds to the increasing evidence of better perinatal outcomes following frozen embryo transfers. The approach of freezing all embryos in the initiated fresh cycle and transfer of a single frozen blastocyst in the subsequent thaw cycle may improve the overall pregnancy and birth outcomes following ART treatment, in part by reducing the ectopic pregnancy rate. There is no funding for this study. Authors declared no competing interest related to this study. © 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.

The low fertility of repeat-breeder cows during summer heat stress is related to a low oocyte competence to develop into blastocysts.

PubMed

Ferreira, R M; Ayres, H; Chiaratti, M R; Ferraz, M L; Araújo, A B; Rodrigues, C A; Watanabe, Y F; Vireque, A A; Joaquim, D C; Smith, L C; Meirelles, F V; Baruselli, P S

2011-05-01

It was hypothesized the lower fertility of repeat-breeder (RB) Holstein cows is associated with oocyte quality and this negative effect is enhanced during summer heat stress (HS). During the summer and the winter, heifers (H; n=36 and 34, respectively), peak-lactation (PL; n=37 and 32, respectively), and RB (n=36 and 31, respectively) Holstein cows were subjected to ovum retrieval to assess oocyte recovery, in vitro embryonic developmental rates, and blastocyst quality [terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and total cell number]. The environmental temperature and humidity, respiration rate, and cutaneous and rectal temperatures were recorded in both seasons. The summer HS increased the respiration rate and the rectal temperature of PL and RB cows, and increased the cutaneous temperature and lowered the in vitro embryo production of Holstein cows and heifers. Although cleavage rate was similar among groups [H=51.7% ± 4.5 (n=375), PL=37.9% ± 5.1 (n=390), RB=41.9% ± 4.5 (n=666)], blastocyst rate was compromised by HS, especially in RB cows [H=30.3% ± 4.8 (n=244) vs. 23.3% ± 6.4 (n=150), PL=22.0% ± 4.7 (n=191) vs. 14.6% ± 7.6 (n=103), RB=22.5% ± 5.4 (n=413) vs. 7.9% ± 4.3 (n=177)]. Moreover, the fragmentation rate of RB blastocysts was enhanced during the summer, compared with winter [4.9% ± 0.7 (n=14) vs. 2.2% ± 0.2 (n=78)] and other groups [H=2.5% ± 0.7 (n=13), and PL=2.7% ± 0.6 (n=14)] suggesting that the association of RB fertility problems and summer HS may potentially impair oocyte quality. Our findings provide evidence of a greater sensitivity of RB oocytes to summer HS. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (blastocyst through the perforation and formation of an incomplete demi-embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

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.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator.

PubMed

Costa-Borges, Nuno; Bellés, Marta; Meseguer, Marcos; Galliano, Daniela; Ballesteros, Agustin; Calderón, Gloria

2016-03-01

To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). Prospective cohort study on sibling donor oocytes. University-affiliated in vitro fertilization (IVF) center. Embryos from 59 patients. Culture in a TLI in a single medium with or without renewal of the medium on day-3. Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study.

PubMed

Li, Z; Wang, Y A; Ledger, W; Edgar, D H; Sullivan, E A

2014-12-01

What are the clinical efficacy and perinatal outcomes following transfer of vitrified blastocysts compared with transfer of fresh or of slow frozen blastocysts? Compared with slow frozen blastocysts, vitrified blastocysts resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes at population level. Although vitrification has been reported to be associated with significantly increased post-thaw survival rates compared with slow freezing, there has been a lack of general consensus over which method of cryopreservation (vitrification versus slow freezing) is most appropriate for blastocysts. A population-based cohort of autologous fresh and initiated thaw cycles (a cycle where embryos were thawed with intention to transfer) performed between January 2009 and December 2011 in Australia and New Zealand was evaluated retrospectively. A total of 46 890 fresh blastocyst transfer cycles, 12 852 initiated slow frozen blastocyst thaw cycles and 20 887 initiated vitrified blastocyst warming cycles were included in the data analysis. Pairwise comparisons were made between the vitrified blastocyst group and slow frozen or fresh blastocyst group. A Chi-square test was used for categorical variables and t-test was used for continuous variables. Cox regression was used to examine the pregnancy outcomes (clinical pregnancy rate, miscarriage rate and live delivery rate) and perinatal outcomes (preterm delivery, low birthweight births, small for gestational age (SGA) births, large for gestational age (LGA) births and perinatal mortality) following transfer of fresh, slow frozen and vitrified blastocysts. The 46 890 fresh blastocyst transfers, 11 644 slow frozen blastocyst transfers and 19 978 vitrified blastocyst transfers resulted in 16 845, 2766 and 6537 clinical pregnancies, which led to 13 049, 2065 and 4955 live deliveries, respectively. Compared with slow frozen blastocyst transfer cycles, vitrified blastocyst transfer cycles

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods.

PubMed

Ha, A-Na; Lee, Sang-Ryeul; Jeon, Jeong-Seon; Park, Han-Seul; Lee, Sang-Ho; Jin, Jong-In; Sessions, Benjamin R; Wang, Zhongde; White, Kenneth L; Kong, Il-Keun

2014-02-01

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading. Copyright © 2013 Elsevier Inc. All rights reserved.

Planar embryos have poor prognosis in terms of blastocyst formation and implantation.

PubMed

Ebner, T; Maurer, M; Shebl, O; Moser, M; Mayer, R B; Duba, H C; Tews, G

2012-09-01

compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular biopsy (5.4%; P<0.01). Embryos from the study group showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (survival to day 5 of preimplantation development) was higher in the normally cleaved control group (P<0.001) and so was blastocyst quality; however, the latter parameter did not reach level of significance. This was also reflected in a significantly higher implantation rate in the control group (P<0.01). Based on present data, it may be postulated that, in planar embryos, the mitotic spindle (which involves the sperm centrosome) might have been affected, which in turn might have led to an incomplete cleavage. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Detection and quantitation of chromosomal mosaicism in human blastocysts using copy number variation sequencing.

PubMed

Ruttanajit, Tida; Chanchamroen, Sujin; Cram, David S; Sawakwongpra, Kritchakorn; Suksalak, Wanwisa; Leng, Xue; Fan, Junmei; Wang, Li; Yao, Yuanqing; Quangkananurug, Wiwat

2016-02-01

Currently, our understanding of the nature and reproductive potential of blastocysts associated with trophectoderm (TE) lineage chromosomal mosaicism is limited. The objective of this study was to first validate copy number variation sequencing (CNV-Seq) for measuring the level of mosaicism and second, examine the nature and level of mosaicism in TE biopsies of patient's blastocysts. TE biopy samples were analysed by array comparative genomic hybridization (CGH) and CNV-Seq to discriminate between euploid, aneuploid and mosaic blastocysts. Using artificial models of TE mosaicism for five different chromosomes, CNV-Seq accurately and reproducibly quantitated mosaicism at levels of 50% and 20%. In a comparative 24-chromosome study of 49 blastocysts by array CGH and CNV-Seq, 43 blastocysts (87.8%) had a concordant diagnosis and 6 blastocysts (12.2%) were discordant. The discordance was attributed to low to medium levels of chromosomal mosaicism (30-70%) not detected by array CGH. In an expanded study of 399 blastocysts using CNV-Seq as the sole diagnostic method, the proportion of diploid-aneuploid mosaics (34, 8.5%) was significantly higher than aneuploid mosaics (18, 4.5%) (p < 0.02). Mosaicism is a significant chromosomal abnormality associated with the TE lineage of human blastocysts that can be reliably and accurately detected by CNV-Seq. © 2015 John Wiley & Sons, Ltd.

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

PubMed Central

Wang, Hehai; Ding, Tianbing; Brown, Naoko; Yamamoto, Yasutoshi; Prince, Lawrence S.; Reese, Jeff; Paria, B. C.

2008-01-01

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells. PMID:18423437

Penetration of chemicals into the oocyte, uterine fluid, and preimplantation blastocyst

PubMed Central

Fabro, Sergio

1978-01-01

Chemicals, including commonly used drugs (e.g., penicillin, meprobamate, pyridium, and mercaptomerin) penetrate and persist for some time in the ovarian follicular fluid at concentrations approximately similar to that of the serum. Information as to the penetration of chemicals into the granulosa cells and into the oocyte is scanty, although there are some indications that these structures are also permeable to foreign chemicals. Similarly, caffeine, nicotine, thiopental, salicylic acid, antipyrine, barbital, and isoniazid enter the uterine secretion and penetrate the preimplantation blastocyst of mice, rats and rabbits. The pattern of distribution of compounds among ovarian follicular fluid, uterine luminal fluid, blastocyst and plasma varies from compound to compound and appears to be related to the molecular weight and degree of ionization of the compound and differs in pregnant and nonpregnant animals. Thus, nicotine and DDT accumulate in the uterine luminal fluid of pregnant but not in that of nonpregnant rabbits. The penetration of foreign chemicals into the oocyte, uterine luminal fluid, and preimplantation blastocyst may exert adverse effects on fertilization, implantation, and/or further development of the conceptus. The possible toxicological importance of this process to eutherian reproduction is discussed. PMID:17539150

Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

PubMed

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

2015-01-15

Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

[Investigation of neural stem cell-derived donor contribution in the inner ear following blastocyst injection].

PubMed

Volkenstein, S; Brors, D; Hansen, S; Mlynski, R; Dinger, T C; Müller, A M; Dazert, S

2008-03-01

Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5-15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months POST PARTUM for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.

Predictive value of serum HCG concentrations in pregnancies achieved after single fresh or vitrified-warmed blastocyst transfer.

PubMed

Oron, Galia; Shavit, Tal; Esh-Broder, Efrat; Weon-Young, Son; Tulandi, Togas; Holzer, Hananel

2017-09-01

Possible differences between serum HCG levels in pregnancies achieved after transfer of a single fresh or a vitrified-warmed blastocyst were evaluated. Out of 1130 single blastocyst transfers resulting in positive HCG results, 789 were single fresh blastocyst transfers and 341 single vitrified-warmed blastocyst transfers. The initial serum HCG levels of 869 clinical intrauterine pregnancies were evaluated, 638 after the transfer of a single fresh blastocysts and 231 after the transfer of a single vitrified-warmed blastocysts. The HCG levels from cycles resulting in a clinical intrauterine pregnancy were significantly higher after the transfer of a single vitrified-warmed blastocyst (383 ± 230 IU/l) versus a fresh transfer (334 ± 192 IU/l; P = 0.01). Threshold values for predicting a clinical pregnancy for a fresh blastocyst were 111 IU/l and for a vitrified-warmed blastocyst 137 IU/l. Our study shows that the overall beta-HCG levels are comparable after the transfer of a fresh or vitrified-warmed blastocyst, suggesting that vitrification most probably does not affect the ability of the embryos to produce beta-HCG. This study further shows that when clinicians counsel patients, they should take into account that higher HCG levels are needed after a vitrified-warmed blastocyst transfer to predict a clinical intrauterine pregnancy. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas.

PubMed

Powell, Susan A; Smith, Bradford B; Timm, Karen I; Menino, Alfred R

2007-11-01

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts.

PubMed

Hwang, Jae Yeon; Choi, Kwang-Hwan; Lee, Dong-Kyung; Kim, Seung-Hun; Kim, Eun Bae; Hyun, Sang-Hwan; Lee, Chang-Kyu

2015-01-01

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.

[Pregnancy and delivery with transfer of vitrified blastocysts following trophectoderm biopsy].

PubMed

Mátyás, Szabolcs; Varga, Tünde; Kovács, Péter; Kónya, Márton; Rajczy, Klára; Babenko, Éva; Szabó, Barbara; Kaali, G Steven; Szentirmay, Zoltán

2015-11-01

Application of preimplantation genetic diagnosis makes it possible to transfer only embryos unaffected by a certain genetic disorder. The authors have applied the combination of trophectoderm biopsy and vitrification in order to detect a monogenic disorder. Previously diagnosed type 1 neurofibromatosis of the woman was the indication for genetic examination. In vitro fertilisation and embryo culture was performed using sequential culture mediums. Seven blastocysts could be sampled on the fifth day and were vitrified subsequently. Two blastocysts turned out to be genetically normal based on the result of genetic examination using polimerase chain reaction. A healthy boy was delivered following the transfer of warmed blastocysts and an uneventful singleton pregnancy.

Glucose and pyruvate metabolism in preimplantation blastocysts from normal and diabetic rats.

PubMed

Dufrasnes, E; Vanderheyden, I; Robin, D; Delcourt, J; Pampfer, S; De Hertogh, R

1993-05-01

Glucose metabolism was analysed in day-5 rat blastocysts incubated in the presence of [5-3H]-, [6-14C]- or [U-14C]glucose. Glycolysis, quantified by 3H2O recovery rate, was the main pathway of glucose utilization by fresh (11.5 +/- 0.36 pmol per embryo h-1) or cultured (24 h) blastocysts (20.4 +/- 0.6 pmol per embryo h-1). Glucose consumption rate was almost saturated at a medium glucose concentration of 0.28 mmol l-1 (Km: 0.17 mmol l-1; Vmax: 23 pmol per embryo h-1). A further 10% increase in glucose utilization was obtained with a tenfold higher glucose concentration (3 mmol l-1). Phloretin completely abolished the rapid component of glucose utilization kinetics, suggesting the existence of a Na(+)-independent glucose transport system. Less than 1% of [6-14C]glucose consumed by cultured blastocysts was oxidized through the Krebs cycle. [1-14C]pyruvate, however, was oxidized at a rate of 2 pmol per embryo h-1 by fresh blastocysts. The pentose-phosphate pathway accounted for about 2% of glucose utilization. One to two per cent of the total glucose metabolized in 24 h was retained in macromolecules. Insulin had no effect on glucose uptake, utilization, incorporation and turnover, or on pyruvate oxidation. Blastocysts from diabetic mothers utilized glucose at a rate similar to that of normal blastocysts. These results show that glucose is actively taken up by rat blastocysts and utilized mainly through the Embden-Meyerhof pathway, which is rapidly saturated at low glucose concentrations. Retention of glucose-derived products in macromolecules, although relatively small, may modulate the effect of high glucose concentrations on embryo growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

PubMed

Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina

2015-04-01

Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture. Copyright © 2015 Elsevier Inc. All rights reserved.

Rho-associated kinase activity is required for proper morphogenesis of the inner cell mass in the mouse blastocyst.

PubMed

Laeno, Arlene May A; Tamashiro, Dana Ann A; Alarcon, Vernadeth B

2013-11-01

The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

Blastocyst classification systems used in Latin America: is a consensus possible?

PubMed Central

Puga-Torres, Tatiana; Blum-Rojas, Xavier; Blum-Narváez, Medardo

2017-01-01

Objective To identify different blastocyst classification systems used by embryologists in Latin American countries and evaluate the possibility of establishing a consensus among these countries. Methods An E-mail survey was carried out through the Latin American Network of Assisted Reproduction (REDLARA) aimed at embryologists from assisted reproduction centers in Latin countries. Results Sixty surveys were collected from 12 Latin American countries, of which 66.7% had >10years of professional practice as embryologists. Seven different blastocyst classification systems were reported, of which 5 have previously been described in the literature. Conclusion Although the group of embryologists surveyed use different blastocyst classification systems, most in this group consider that the embryo score system should be unified in their countries as well as in the region. PMID:28837032

FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.

PubMed

Capalbo, Antonio; Wright, Graham; Elliott, Thomas; Ubaldi, Filippo Maria; Rienzi, Laura; Nagy, Zsolt Peter

2013-08-01

Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)? Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy. The effectiveness of chromosome aneuploidy screening is limited by the technologies available and chromosome mosaicism in the embryo. Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select diploid embryos for transfer. The study was performed between January 2011 and August 2011. In the first part, a new ICM isolation method was developed and tested on 20 good morphology blastocysts. In the main phase of the study, fluorescence in situ hybridization (FISH) was used to reanalyse the ICMs and TEs separated from 70 embryos obtained from 26 patients undergoing blastocyst stage array comparative genome hybridization (aCGH) PGS cycles. The isolated ICM and TE fractions were characterized by immunostaining for KRT18. Then, non-transferrable cryopreserved embryos were selected for the FISH reanalysis based on previous genetic diagnosis obtained by TE aCGH analysis. Blastocysts either diploid for chromosome copy number (20) or diagnosed as single- (40) or double aneuploid (10) were included after preparing the embryo into one ICM and three equal-sized TE sections. Accuracy of the aCGH was measured based on FISH reanalysis. Chromosomal segregations resulting in diploid/aneuploid mosaicism were classified as 'low-', 'medium-' and 'high-' grade and categorized with respect to their distribution (1TE, 2TE, 3TE, ICM or ALL embryo). Linear regression model was used to test the relationship between the distributions and the proportion of aneuploid cells across the four embryo sections. Fisher's exact test was used to test for random allocation of aneuploid cells between TE and

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection.

PubMed

Choi, Y H; Love, L B; Varner, D D; Hinrichs, K

2006-09-01

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.

Near-infrared laser irradiation improves the development of mouse pre-implantation embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoo, Masaki; Mori, Miho

The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing ofmore » blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. - Highlights: • Irradiation of

Delayed blastulation, multinucleation, and expansion grade are independently associated with live-birth rates in frozen blastocyst transfer cycles.

PubMed

Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea; Attaran, Marjan; Goldberg, Jeffrey M; Austin, Cynthia; Falcone, Tommaso

2016-11-01

To identify blastocyst features independently predictive of successful pregnancy and live births with vitrified-warmed blastocysts. Retrospective study. Academic hospital. Women undergoing a cycle with transfer of blastocysts vitrified using the Rapid-i closed carrier (n = 358). None. Clinical pregnancy and live-birth rates analyzed using logistic regression analysis. A total of 669 vitrified-warmed blastocysts were assessed. The survival rate was 95%. A mean of 1.7 ± 0.5 embryos were transferred. The clinical pregnancy, live-birth, and implantation rates were 55%, 46%, and 43%, respectively. The odds of clinical pregnancy (odds ratio [OR] 3.08; 95% confidence interval [CI], 1.88-5.12) and live birth (OR 2.93; 95% CI, 1.79-4.85) were three times higher with day-5 blastocysts versus slower-growing day-6 vitrified blastocysts, irrespective of patient age at cryopreservation. Blastocysts from multinucleated embryos were half as likely to result in a live birth (OR 0.46; 95% CI, 0.22-0.91). A four -fold increase in live birth was observed if an expanded blastocyst was available for transfer. The inner cell mass-trophectoderm score correlated to positive outcomes in the univariate analysis. The implantation rate was statistically significantly higher for day-5 versus day-6 vitrified blastocysts (50% vs. 29%, respectively). The blastocyst expansion grade after warming was predictive of successful outcomes independent of the inner cell mass or trophectoderm score. Delayed blastulation and multinucleation were independently associated with lower live-birth rates in frozen cycles. Implantation potential of the frozen blastocysts available should be included in the decision-making process regarding embryo number for transfer. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Vitamin K2 improves developmental competency and cryo-tolerance of in vitro derived ovine blastocyst.

PubMed

Sefid, Fatemeh; Ostadhosseini, S; Hosseini, S M; Ghazvini Zadegan, F; Pezhman, M; Nasr Esfahani, Mohammad Hossein

2017-08-01

Vitamin K2 (VK2), acts as an electron carrier in mitochondria and thereby effects reactive oxygen species (ROS) and ATP production. This study evaluates role of VK2 on in vitro developmental competency and cryo-survival of pre-implantation ovine embryos. Initially the optimal and beneficial concentration of VK2 on compaction and blastocyst formation rates was defined (0.1 μM). Subsequently, it was shown that 0.1 μM VK2, at blastocyst stage, reduces H2O2 production, increase the expression of mitochondrial related gene and improved embryos quality. We further assessed presence VK2 supplementation before and/or after vitrification of in vitro derived blastocysts. Our results reveal that presence of VK2 before and after vitrification improves rates of blastocysts re-expansion (88.19± 3.37% vs 73.68± 1.86%, P < 0.05) and hatching (49.55± 4.37% vs 32.7± 3.32%) compared to control group. These observation were consistent with reduction in H2O2 production and improved in expression of mitochondrial related genes. However, VK2 before or after vitrification, not only had no positive effect on these two parameters, but also significantly reduced these parameters. Therefore, in concordance with pervious report in bovine, we show that VK2 supplementation post genomic activation (Day 3-7) improved developmental competency of ovine in vitro derived embryos. We also showed that presence of VK2 after vitrification improves the cryo-survival of ovine embryos. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

PubMed

Salilew-Wondim, Dessie; Saeed-Zidane, Mohammed; Hoelker, Michael; Gebremedhn, Samuel; Poirier, Mikhaël; Pandey, Hari Om; Tholen, Ernst; Neuhoff, Christiane; Held, Eva; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Fournier, Eric; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

2018-06-01

Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels

Female bovine blastocysts are more prone to apoptosis than male ones.

PubMed

Ghys, Emmanuelle; Dallemagne, Matthew; De Troy, Delphine; Sauvegarde, Caroline; Errachid, Abdelmounaim; Donnay, Isabelle

2016-03-01

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro-produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid-based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts. Copyright © 2016 Elsevier Inc. All rights reserved.

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

PubMed Central

2012-01-01

Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

PubMed

Tong, Guo Qing; Cao, Shan Ren; Wu, Xun; Zhang, Jun Qiang; Cui, Ji; Heng, Boon Chin; Ling, Xiu Feng

2012-10-05

This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

Increased lectin binding capacity of trophoblastic cells of late day 5 rat blastocysts.

PubMed Central

Stein, B A; Shaw, T J; Turner, V F; Murphy, C R

1994-01-01

The binding of lectins to the trophoblast of rat blastocysts has been studied using quantitative ultrastructural cytochemistry. Rat blastocysts from early, mid and late d 5 of gestation were stained using biotinylated lectins (Phytolacca americana [Phy am], fucose binding protein [FBP] and soybean agglutinin [SBA]) and a sensitive avidin-ferritin cytochemical method. Electron micrographs of ferritin particles along the membrane were processed to produce images for which grey scale levels could be established and the ferritin particles automatically counted. The ferritin:membrane ratio was then calculated. Increased binding with Phy am (which detects short chain oligosaccharides) was found after midday of d 5, i.e. after hatching. Binding of FBP and SBA did not alter during the period studied. The increased concentration of oligosaccharides on the blastocyst surface membrane after hatching may have important implications for blastocyst attachment to the endometrium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7649802

Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity.

PubMed

Daikoku, Takiko; Cha, Jeeyeon; Sun, Xiaofei; Tranguch, Susanne; Xie, Huirong; Fujita, Tomoko; Hirota, Yasushi; Lydon, John; DeMayo, Francesco; Maxson, Robert; Dey, Sudhansu K

2011-12-13

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs. Copyright © 2011 Elsevier Inc. All rights reserved.

Conditional deletion of MSX homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity

PubMed Central

Daikoku, Takiko; Cha, Jeeyeon; Sun, Xiaofei; Tranguch, Susanne; Xie, Huirong; Fujita, Tomoko; Hirota, Yasushi; Lydon, John; DeMayo, Francesco; Maxson, Robert; Dey, Sudhansu K.

2011-01-01

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs. PMID:22100262

Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

PubMed

Itoi, Fumiaki; Asano, Yukiko; Shimizu, Masashi; Nagai, Rika; Saitou, Kanako; Honnma, Hiroyuki; Murata, Yasutaka

2017-04-01

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Control of interferon-tau secretion by in vitro-derived bovine blastocysts during extended culture and outgrowth formation.

PubMed

Kubisch, H M; Larson, M A; Kiesling, D O

2001-04-01

A series of experiments was conducted to examine the pattern of interferon-tau (IFN-tau) secretion by bovine blastocysts during extended culture in vitro. In the first experiment, blastocysts were cultured individually for three 48-hour periods. The day of blastocyst formation affected how much IFN-tau was produced during the first two culture periods, but not during the third period. The overall secretion of IFN-tau during the 6-day period increased significantly and well beyond what could be accounted for by the concomitant increase in cell numbers. In the second experiment, blastocysts were initially cultured in individual droplets for 48 hr, then plated into 48-well plates. Medium concentrations of IFN-tau were determined after 48 hr and again after 6 and 12 days of culture. Initial IFN-tau secretion did not affect the ability to form outgrowths or their final size, and initial differences in secretion between groups of blastocysts had disappeared by the second and third analyses. In the third experiment, blastocysts were cultured individually for 48 hr in droplets containing the medium that had been flushed through the uteri of non-pregnant sheep on days 10, 12, and 15 of the estrous cycle. Culture in the medium obtained from the Day 15 flush significantly increased the number of cells that blastocysts contained, as well as IFN-tau secretion.

Improved quality of porcine embryos cultured with hyaluronan due to the modification of the mitochondrial membrane potential and reactive oxygen species level.

PubMed

Romek, Marek; Gajda, Barbara; Krzysztofowicz, Ewa; Kucia, Marcin; Uzarowska, Agnieszka; Smorag, Zdzislaw

2017-10-15

Although considerable progress has been made in pig embryo culture systems, the developmental competence and quality of the produced embryos are still lower than their in vivo-derived counterparts. Because hyaluronan (HA) regulates various cellular processes and possesses antioxidant properties, this glycosaminoglycan seems to be a promising supplement in culture media. However, until now, its beneficial influence on in vitro pig embryo development has been debatable. Hence, we aimed to investigate the effect of 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL concentrations of HA on the developmental potential and quality of cultured porcine embryos. We found that 1 mg/mL HA supplementation significantly increased the obtained percentages of cleaved embryos to ∼95%, morulae to ∼87% and blastocysts to ∼77%. At 0.5 mg/mL and 1 mg/mL HA concentrations, we observed a significantly improved blastocyst quality, expressed as the total number of cells per blastocyst, number of cells in the inner cell mass, number of TUNEL-positive nuclei per blastocyst, the TUNEL index and the blastocyst diameter. Because the inner mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level are important for proper embryo development, for the first time, we measured these two parameters in cultured embryos at various HA concentrations and during their development up to the expanded blastocyst stage. For blastocysts cultured with 1 mg/mL HA, the ΔΨm and ROS level were ∼1.6 and 2.7 times lower, respectively, than those of the control blastocysts. Both ΔΨm and the ROS level were increased in parallel during in vitro embryo development with and without HA, but this increase was less pronounced in the presence of HA. Hence, our quantitative data unequivocally show that supplementation of NCSU-23 culture medium with 1 mg/mL HA improves the developmental potential and quality of pig embryos. This effect results from a significant decrease in the ROS level induced by

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals

PubMed Central

Hosseini, Sayyed-Morteza; Moulavi, Fariba; Tanhaie-Vash, Nima; Asgari, Vajihe; Ghanaei, Hamid-Reza; Abedi-Dorche, Maryam; Jafarzadeh, Naser; Gourabi, Hossein; Shahverdi, Abdol-Hossein; Dizaj, Ahmad Vosough; Shirazi, Abolfazl; Nasr-Esfahani, Mohammad-Hossein

2016-01-01

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with

Selenium and vitamin E improve the in vitro maturation, fertilization and culture to blastocyst of porcine oocytes.

PubMed

Tareq, K M A; Akter, Quzi Sharmin; Khandoker, M A M Yahia; Tsujii, Hirotada

2012-01-01

Selenium (Se) and vitamin E (Vit-E), as integral parts of antioxidant systems, play important roles for sperm and embryos in vitro. In this study, the effects of Se and Vit-E on the maturation, in vitro fertilization and culture to blastocysts of porcine oocytes and accumulation of ammonia in the culture medium during different development stages were investigated. The maturation was performed in modified tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, the fertilization medium was modified Tyrode's albumin lactate pyruvate (mTALP), and the embryo culture medium was modified North Carolina State University (mNCSU)-23. Se in the form of sodium selenite (SS) and seleon-L-methionine (SeMet) and Vit-E at different concentrations were also used. The incorporation and oxidation of (14)C(U)-glucose were assessed with a liquid scintillation counter. In this study, SeMet and SeMet+Vit-E increased oocyte maturation, fertilization and incorporation and oxidation of (14)C(U)-glucose significantly (P<0.05) compared with the control and other treatments. In addition, embryo development, specifically in terms of the numbers of morulae and blastocysts, significantly increased (P<0.05) with SeMet and SeMet+Vit-E. In contrast, the accumulation of ammonia was reduced with SeMet and SeMet+Vit-E compared with other treatments. These findings indicate that SeMet and SeMet+Vit-E may play important roles in reducing the accumulation of ammonia and subsequently in increasing the rate of maturation of porcine oocytes and fertilization, as well as development of the blastocyst and utilization of glucose in in vitro maturation, fertilization and culture to blastocysts of porcine oocytes.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

PubMed

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.

Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

PubMed

Yokoo, Masaki; Mori, Miho

2017-05-27

The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights

H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression.

PubMed

Nikdel, K; Aminafshar, M; Mohammadi-Sangcheshmeh, A; EmamJomeh-Kashan, N; Seyedjafari, E

2017-05-20

In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 μM H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 μM H2O2 had the lowest GSH level. Treatment of oocytes with 1000 μM H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 μM H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.

Effect of body condition and season on yield and quality of in vitro produced bovine embryos.

PubMed

Chrenek, Peter; Kubovičová, Elena; Olexíková, Lucia; Makarevich, Alexander V; Toporcerová, Silvia; Ostró, Alexander

2015-12-01

The aim of our study was to examine the effects of cow's body condition score (BCS; scale 1-5) and season on the quality of bovine in vitro produced embryos. The proportion of good quality oocytes (Q1 and Q2) was higher (P < 0.05) in the BCS 2 (57.60%) and BCS 3 (60.90%) groups compared with the BCS 1 (43.60%) group. There were no statistical differences in embryo cleavage and blastocyst rate among the BCS groups. The highest total cell number (TCN, DAPI stain) of blastocysts (P < 0.05), recorded in BCS 1 (122.27 ± 6.90) in comparison with BCS 2 (101.8 ± 3.60) or BCS 3 (105.44 ± 3.70) groups, was related to higher dead cell (DCI, TUNEL) index in this group (7.07%) when compared with BCS 2 (6.54%) or BCS 3 (6.06%), respectively. The yield of good quality oocytes during spring was lower (P < 0.05) compared with the summer season. There were significant differences (P < 0.05) in maturation and cleavage rates between autumn and summer (73.42%, 76.2% vs. 85.0%, 41.8%, respectively). The highest (P < 0.01) blastocyst rate was noted during spring and summer months. Significant difference (P < 0.05) in the TCN among spring (99.38 ± 3.90), autumn (110.1 ± 4.58) or summer (108.96 ± 3.52) was observed. The highest proportion of embryos with the best (grade I) actin cytoskeleton (phalloidin-TRITC) quality was noted during the summer months. Our results indicate that body condition affects the initial quality of oocytes, but does not affect embryo cleavage, blastocyst rate and actin quality. This finding may suggest that development in vitro can mask the influence of BCS. The season affects yield and quality of blastocysts in the way that the autumn period is more favorable for embryo development.

Equine cloning: in vitro and in vivo development of aggregated embryos.

PubMed

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.

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

PubMed

Rule, Kiersten; Chosed, Renee J; Arthur Chang, T; David Wininger, J; Roudebush, William E

2018-06-04

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25 μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R 2  = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R 2 = 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Xenogeneic chimera-Generated by blastocyst complementation-As a potential unlimited source of recipient-tailored organs.

PubMed

Oldani, Graziano; Peloso, Andrea; Lacotte, Stéphanie; Meier, Raphael; Toso, Christian

2017-07-01

Blastocyst complementation refers to the injection of cells into a blastocyst. The technology allows for the creation of chimeric animals, which have the potential to be used as an unlimited source of organ donors. Pluripotent stem cells could be generated from a patient in need of a transplantation and injected into a large animal blastocyst (potentially of a pig), leading to the creation of organ(s) allowing immunosuppression-free transplantation. Various chimera combinations have already been generated, but one of the most recent steps leads to the creation of human-pig chimeras, which could be studied at an embryo stage. Although still far from clinical reality, the potential application is almost unlimited. The present review illustrates the historical steps of intra- and interspecific blastocyst complementation in rodents and large animals, specifically looking at its potential for generation of organ grafts. We also speculate on how it could change transplant indications, on its economic impact, and on the linked ethical concerns. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program.

PubMed

Scott, L F; Sundaram, S G; Smith, S

1993-09-01

To define both the limits of a mouse embryo bioassay for quality control in an assisted reproductive technology (ART) program and the areas where it can be effectively used. Embryos at the pronuclear and two-cell stage from three different strains of mice were used to assess the effectiveness of this assay for media quality control using five different media routinely used in ART. Pronuclear and two-cell embryos from CD-1 mice were used to test the ability of a mouse embryo bioassay to control for water quality, contaminants in the culture system, and fluctuations in the environmental conditions using a medium, culture system, and scoring technique that were optimized for this strain. The mouse embryo bioassay is not effective in differentiating media appropriate for supporting human embryo development since the development of mouse embryos in vitro is strain, stage, and media related. However, CD-1 embryos were shown to be sensitive to variations in water quality, pH, temperature, incubator conditions, and contaminants in the system when grown in a protein-free medium optimized for their development. Both total blastocyst number and the cell count in the blastocysts were affected. Pronuclear embryos were more sensitive to perturbations in the culture system than two-cell embryos. A mouse embryo bioassay can be effectively used as a means of quality control of water, chemicals, and contact materials and for technique standardization and training in an assisted reproduction program. All the conditions of the test should be defined, pronuclear embryos should be used, and the end point should be fully expanded blastocysts and/or cell numbers in these blastocysts where appropriate.

Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

PubMed

Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

2015-06-01

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes.

PubMed

Cremades, N; Sousa, M; Silva, J; Viana, P; Sousa, S; Oliveira, C; Teixeira da Silva, J; Barros, A

2004-02-01

Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.

Chromosomal analysis of blastocyst derived from monopronucleated ICSI zygotes: approach by double trophectoderm biopsy.

PubMed

Mateo, Silvia; Vidal, Francesca; Coll, Lluc; Veiga, Anna; Boada, Montserrat

2017-09-01

This study aims to increase the knowledge about monopronucleated ICSI-derived blastocysts, analyzing trophectoderm biopsies by aCGH and FISH to evaluate their chromosome constitution. Fifteen monopronucleated ICSI-derived blastocysts were studied. Double trophectoderm biopsy was performed and analyzed by FISH and aCGH. The blastocysts were classified according to chromosome constitution. Disagreements between the two techniques were assessed. Results obtained after FISH and aCGH analyses showed the following: 20% (3/15) and 60% (9/15) diploid females, respectively; 26.7% (4/15) and 26.7% (4/15) diploid males, respectively; and 53.3% (8/15) and 13.3% (2/15) mosaics, respectively. No mosaic male embryos were found using FISH or aCGH. There were disagreements in 40% (6/15) of the cases due to the higher detection of mosaicism by FISH compared to aCGH. The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.

Comparison of two closed carriers for vitrification of human blastocysts in a donor program.

PubMed

Guerrero, Jaime; Gallardo, Miguel; Rodríguez-Arnedo, Adoración; Ten, Jorgen; Bernabeu, Rafael

2018-04-01

The survival of human blastocysts to vitrification with two different carriers is compared. Both vitrification carriers used in this study are in the category of closed carriers, as they completely isolate the samples from direct contact with liquid nitrogen or its vapours during cooling and storage, until warming. This characteristic is appealing because it reduces or eliminates the theoretical risk of cross-contamination during that period of time. The two closed vitrification systems used present very different design and features: in the High Security Vitrification device, the carrier straw containing the embryos is encapsulated inside an external straw before plunging in liquid nitrogen, resulting in thermal insulation during cooling. On the other hand, in the SafeSpeed carrier embryos are loaded in a thin-walled, narrow capillary designed to maximize the thermal transference. Both closed carriers achieved comparable outcomes in terms of survival of blastocysts to the vitrification process, with 97.5% vs. 96.1% survival with HSV and SafeSpeed, respectively. In conclusion, the cooling and warming rates at which these carriers operate, in combination with the cytosolic solute concentration in the cells of the cryopreserved blastocysts attained after a cryoprotectant-loading protocol, result in successful vitrification of human blastocysts for human assisted reproduction. Copyright © 2018. Published by Elsevier Inc.

Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

PubMed

Bolnick, Alan; Abdulhasan, Mohammed; Kilburn, Brian; Xie, Yufen; Howard, Mindie; Andresen, Paul; Shamir, Alexandra M; Dai, Jing; Puscheck, Elizabeth E; Secor, Eric; Rappolee, Daniel A

2017-12-01

This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Effect of sericin on preimplantation development of bovine embryos cultured individually.

PubMed

Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

2012-09-01

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress. Copyright © 2012 Elsevier Inc. All rights reserved.

Four simple rules that are sufficient to generate the mammalian blastocyst

PubMed Central

Nissen, Silas Boye; Perera, Marta; Gonzalez, Javier Martin; Morgani, Sophie M.; Jensen, Mogens H.; Sneppen, Kim; Brickman, Joshua M.

2017-01-01

Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs in the absence of maternal information and in the presence of transcriptional stochasticity. How does the preimplantation embryo ensure robust, reproducible development in this context? It utilizes a versatile toolbox that includes complex intracellular networks coupled to cell—cell communication, segregation by differential adhesion, and apoptosis. Here, we ask whether a minimal set of developmental rules based on this toolbox is sufficient for successful blastocyst development, and to what extent these rules can explain mutant and experimental phenotypes. We implemented experimentally reported mechanisms for polarity, cell—cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells’ competence of fibroblast growth factor (FGF)/extracellular signal—regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network. PMID:28700688

Blastocyst culture using single versus sequential media in clinical IVF: a systematic review and meta-analysis of randomized controlled trials.

PubMed

Sfontouris, Ioannis A; Martins, Wellington P; Nastri, Carolina O; Viana, Iara G R; Navarro, Paula A; Raine-Fenning, Nick; van der Poel, Sheryl; Rienzi, Laura; Racowsky, Catherine

2016-10-01

The purpose of this study was to undertake a review of the available evidence comparing the use of a single medium versus sequential media for embryo culture to the blastocyst stage in clinical IVF. We searched the Cochrane Central, PubMed, Scopus, ClinicalTrials.gov, Current Controlled Trials and WHO International Clinical Trials Registry Platform to identify randomized controlled trials comparing single versus sequential media for blastocyst culture and ongoing pregnancy rate. Included studies randomized either oocytes/zygotes or women. Eligible oocyte/zygote studies were analyzed to assess the risk difference (RD) and 95 % confidence intervals (CI) between the two media systems; eligible woman-based studies were analyzed to assess the risk ratio (RR) and 95 % CI for clinical pregnancy rate. No differences were observed between single and sequential media for either ongoing pregnancy per randomized woman (relative risk (RR) = 0.9, 95 % CI = 0.7 to 1.3, two studies including 246 women, I 2  = 0 %) or clinical pregnancy per randomized woman (RR = 1.0, 95 % CI = 0.7 to 1.4, one study including 100 women); or miscarriage per clinical pregnancy: RR = 1.3, 95 % CI = 0.4 to 4.3, two studies including 246 participants, I 2  = 0 %). Single media use was associated with an increase blastocyst formation per randomized oocyte/zygote (relative distribution (RD) = +0.06, 95 % CI = +0.01 to +0.12, ten studies including 7455 oocytes/zygotes, I 2  = 83 %) but not top/high blastocyst formation (RD = +0.05, 95 % CI = -0.01 to +0.11, five studies including 3879 oocytes/zygotes, I 2  = 93 %). The overall quality of the evidence was very low for all these four outcomes. Although using a single medium for extended culture has some practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the culture of

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PubMed

Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

2016-10-01

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10 3 and 10 4 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10 4 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10 4 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10 3 b-ATMSCs/mL (p = 0.051), group 10 4 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10 4 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

PubMed

Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2017-10-01

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst.

PubMed

Lin, Lin; Yu, Yutao; Zhang, Xiuqing; Yang, Huanming; Bolund, Lars; Callesen, Henrik; Vajta, Gábor

2011-01-01

Minimum volume vitrification may provide extremely high cooling and warming rates if the sample and the surrounding medium contacts directly with the respective liquid nitrogen and warming medium. However, this direct contact may result in microbial contamination. In this work, an earlier aseptic technique was applied for minimum volume vitrification. After equilibration, samples were loaded on a plastic film, immersed rapidly into factory derived, filter-sterilized liquid nitrogen, and sealed into sterile, pre-cooled straws. At warming, the straw was cut, the filmstrip was immersed into a 39 degree C warming medium, and the sample was stepwise rehydrated. Cryosurvival rates of porcine blastocysts produced by parthenogenetical activation did not differ from control, vitrified blastocysts with Cryotop. This approach can be used for minimum volume vitrification methods and may be suitable to overcome the biological dangers and legal restrictions that hamper the application of open vitrification techniques.

Trophinin expression in the mouse uterus coincides with implantation and is hormonally regulated but not induced by implanting blastocysts.

PubMed

Suzuki, N; Nadano, D; Paria, B C; Kupriyanov, S; Sugihara, K; Fukuda, M N

2000-11-01

Trophinin mediates apical cell adhesion between two human cell lines, trophoblastic teratocarcinoma and endometrial adenocarcinoma. In humans, trophinin is specifically expressed in cells involved in implantation and early placentation. The present study was undertaken to establish trophinin expression by the mouse uterus. In the pregnant mouse uterus, trophinin transcripts are expressed during the time which coincides with the timing of blastocyst implantation. Trophinin is also expressed in the nonpregnant mouse uterus at estrus stage. Uteri from ovariectomized mice did not express trophinin, whereas strong expression was induced by estrogen but not by progesterone. Trophinin transcripts and protein were found in the pseudopregnant mouse uterus. No differences were detected in trophinin expression by the uteri in the pregnant, pseudopregnant, and pseudopregnant received blastocysts. In delayed implantation model, trophinin proteins were found in both luminal and glandular epithelium, whereas dormant blastocysts were negative for trophinin. Upon activation with estrogen, however, no significant changes were detected either in the blastocyst or in the uterus. These results indicate that ovarian hormones regulate trophinin expression by the mouse uterus, and that an implanting blastocyst has no effect on trophinin expression in the surrounding endometrial luminal epithelial cells.

Blastocyst transfer in human in vitro fertilization. A solution to the multiple pregnancy epidemic.

PubMed

Vidaeff, A C; Racowsky, C; Rayburn, W F

2000-07-01

Since the 1950s, the incidence of twin gestation has doubled and the incidence of triplets has increased approximately sevenfold in the United States. Of extreme concern is the fact that many of these multiple pregnancies are iatrogenic: 35% of twin gestations and 77% of higher-order pregnancies are the result of some form of infertility therapy. Anything that can be done to reduce the number of these multiple pregnancies would benefit our patients and society. Great hope is placed on emerging blastocyst technology, which has the potential of achieving higher pregnancy rates per embryo transfer while reducing the risk of multiple pregnancy. We present the evolution of the blastocyst transfer concept and the technical aspects involved. The article also outlines the experience with blastocyst culture and transfer at Brigham and Women's Hospital, Boston, and describes identifiers for application of blastocyst transfer. The number of eight-cell embryos on day 3 is an independent marker for the selection of patients who would benefit from transfer on day 5. With no eight-cell embryos on day 3, 0% and 33% pregnancies resulted from day 5 vs. day 3 transfers, suggesting that these cases would not benefit from day 5 transfer. When at least one eight-cell embryo is available, there is no difference in ongoing pregnancy rates between day 5 and day 3 transfers, but there is a significant decrease in multiple gestations with day 5 transfers.

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway.

PubMed

Yang, Jing; Zhang, Dan; Yu, Ying; Zhang, Run-Ju; Hu, Xiao-Ling; Huang, He-Feng; Lu, Yong-Chao

2015-01-01

Fibroblast growth factors (FGF1, FGF2 and FGF4) and fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3 and FGFR4) have been reported to be expressed in preimplantation embryos and be required for their development. However, the functions of these molecules in trophectoderm cells (TEs) that lead to the formation of the blastocyst as well as the underlying mechanism have not been elucidated. The present study has demonstrated for the first time that endogenous FGF2 secreted by TEs can regulate protein expression and distribution in TEs via the FGFR2-mediated activation of PKC and p38, which are important for the development of expanded blastocysts. This finding provides the first explanation for the long-observed phenomenon that only high concentrations of exogenous FGFs have effects on embryonic development, but in vivo the amount of endogenous FGFs are trace. Besides, the present results suggest that FGF2/FGFR2 may act in an autocrine fashion and activate the downstream PKC/p38 pathway in TEs during expanded blastocyst formation.

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes.

PubMed

Zhang, Zhiguo; Wang, Tianjuan; Hao, Yan; Panhwar, Fazil; Chen, Zhongrong; Zou, Weiwei; Ji, Dongmei; Chen, Beili; Zhou, Ping; Zhao, Gang; Cao, Yunxia

2017-02-01

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8.

PubMed

Vandaele, Leen; Wesselingh, Wendy; De Clercq, Kris; De Leeuw, Ilse; Favoreel, Herman; Van Soom, Ann; Nauwynck, Hans

2011-01-24

Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 10(3.8) or 10(4.9) TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

Early in vitro fertilization improves development of bovine ova heat stressed during in vitro maturation.

PubMed

Schrock, G E; Saxton, A M; Schrick, F N; Edwards, J L

2007-09-01

The objectives were to examine the development of embryos derived from control (38.5 degrees C) or heat-stressed ova [41.0 degrees C during the first 12 h of in vitro maturation (hIVM)] when in vitro fertilization (IVF) was performed at 16, 18, 20, 24, or 30 hIVM. Effects of heat stress in compromising ovum development depended on when IVF was performed (in vitro maturation temperature x IVF time interaction). When IVF was performed at 24 or 30 hIVM, fewer heat-stressed ova developed to the blastocyst stage compared with the respective controls. In contrast, when IVF was performed at 16, 18, or 20 hIVM, more heat-stressed ova developed to the blastocyst stage compared with the respective controls. Performing IVF earlier than usual was beneficial, because the ability of heat-stressed ova to develop to the blastocyst stage was improved when IVF was performed at 18 or 20 vs. 24 hIVM. Blastocyst stage and quality were equivalent to non-heat-stressed controls regardless of IVF time. Control ova undergoing IVF at 20, 24, 30, or 32 hIVM and heat-stressed ova undergoing IVF at 16, 18, 20, or 24 hIVM were compared for blastocyst development by multisource regression. Although linear and quadratic slopes were similar, heat stress reduced the peak and shifted the developmental response of ova by 7.3 h. In other words, obtaining optimal blastocyst development from heat-stressed ova would depend on performing IVF at 19.5 hIVM compared with 26.7 hIVM for non-heat-stressed controls. Heat-induced reductions in peak blastocyst development significantly reduced the window of time available to perform IVF and obtain > or = 20% blastocyst development. In summary, results support an effect of heat stress to hasten developmentally important events during oocyte maturation. The inability of earlier IVF to fully restore the development of heat-stressed ova to that of non-heat-stressed controls highlights the importance of further study.

Noninferiority, randomized, controlled trial comparing embryo development using media developed for sequential or undisturbed culture in a time-lapse setup.

PubMed

Hardarson, Thorir; Bungum, Mona; Conaghan, Joe; Meintjes, Marius; Chantilis, Samuel J; Molnar, Laszlo; Gunnarsson, Kristina; Wikland, Matts

2015-12-01

To study whether a culture medium that allows undisturbed culture supports human embryo development to the blastocyst stage equivalently to a well-established sequential media. Randomized, double-blinded sibling trial. Independent in vitro fertilization (IVF) clinics. One hundred twenty-eight patients, with 1,356 zygotes randomized into two study arms. Embryos randomly allocated into two study arms to compare embryo development on a time-lapse system using a single-step medium or sequential media. Percentage of good-quality blastocysts on day 5. Percentage of day 5 good-quality blastocysts was 21.1% (standard deviation [SD] ± 21.6%) and 22.2% (SD ± 22.1%) in the single-step time-lapse medium (G-TL) and the sequential media (G-1/G-2) groups, respectively. The mean difference (-1.2; 95% CI, -6.0; 3.6) between the two media systems for the primary end point was less than the noninferiority margin of -8%. There was a statistically significantly lower number of good-quality embryos on day 3 in the G-TL group [50.7% (SD ± 30.6%) vs. 60.8% (SD ± 30.7%)]. Four out of the 11 measured morphokinetic parameters were statistically significantly different for the two media used. The mean levels of ammonium concentration in the media at the end of the culture period was statistically significantly lower in the G-TL group as compared with the G-2 group. We have shown that a single-step culture medium supports blastocyst development equivalently to established sequential media. The ammonium concentrations were lower in the single-step media, and the measured morphokinetic parameters were modified somewhat. NCT01939626. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

PubMed

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard; Callesen, Henrik

2015-06-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

Parental contributions to early embryo development: influences of urinary phthalate and phthalate alternatives among couples undergoing IVF treatment.

PubMed

Wu, Haotian; Ashcraft, Lisa; Whitcomb, Brian W; Rahil, Tayyab; Tougias, Ellen; Sites, Cynthia K; Pilsner, J Richard

2017-01-01

Are preconception urinary concentrations of phthalates and phthalate alternatives associated with diminished early stage embryo quality in couples undergoing IVF? Male, but not female, urinary concentrations of select metabolites of phthalates and phthalate alternatives are associated with diminished blastocyst quality. Although phthalates are endocrine disrupting compounds associated with adverse reproductive health, they are in widespread use across the world. Male and female preconception exposures to select phthalates have been previously associated with adverse reproductive outcomes in both the general population and in those undergoing IVF. This prospective cohort included 50 subfertile couples undergoing IVF in western Massachusetts. This study includes the first 50 couples recruited from the Baystate Medical Center's Fertility Center in Springfield, MA, as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Relevant data from both partners, including embryo quality at the cleavage (Day 3) and blastocyst (Day 5) stages, were collected by clinic personnel during the normal course of an IVF cycle. A spot urine sample was collected from both male and female partners on the same day as semen sample procurement and oocyte retrieval. Concentrations of 17 urinary metabolite were quantified by liquid chromatography mass spectrometry and normalized via specific gravity. Generalized estimating equations were used to estimate odds ratios (OR) and 95% CI, with urinary phthalates and phthalate alternatives fitted as continuous variables and embryo quality as a binary variable. The 50 couples contributed 761 oocytes, of which 423 progressed to the cleavage stage, 261 were high-quality cleavage stage embryos, 137 were transferrable quality blastocysts and 47 were high-quality blastocysts. At the cleavage stage, male urinary monoethyl phthalate concentrations were positively associated with high-quality cleavage stage embryos (OR = 1.20, 95% CI 1

Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization.

PubMed

Montero-Pardo, A; Hernández-Cerón, J; Rojas-Maya, S; Valencia, J; Rodríguez-Cortez, A; Gutiérrez, C G

2011-05-01

Treatment with bovine somatotropin (bST) during estrous synchronization increased fertility and prolificacy in sheep. In the present study, a single dose of bST 5 days before the end of progestin treatment improved cleavage and embryo development. Stage of estrous cycle was synchronized in ewes (n=32) with progestin and superovulation was induced by use of FSH. Five days before the end of progestin treatment, ewes were randomly assigned to two groups: bST group (n=16) received a depot injection of 125 mg of bST sc (Lactotropina, Elanco, México) and the control group (n=16) received saline solution. Estrous was detected with rams fitted with an apron every 2 h and estrous sheep were mated every 8 h whilst in estrous. Embryos were recovered on Day 7 post mating, assessed microscopically and fixed in 4% paraformaldehyde. Cell number in blastocysts was counted after Hoechst 33342 staining. Plasma concentrations of IGF-I, insulin and progesterone were determined in eight sheep per group from the day of bST treatment to the day of embryo recovery. Cleavage rate, percentage of transferable embryos (transferable embryos/cleaved) and percentage of embryos reaching the blastocyst stage (blastocyst/cleaved) were compared between groups by logistic regression. IGF-I, insulin and progesterone plasma concentrations were analyzed by ANOVA for repeated measurements and cell number by ANOVA. Cleavage rate was greater (P<0.01) in bST treatment group (86%) than in the control group (62%). Similarly, the proportion of embryos reaching the blastocyst stage (bST=68.7 vs control=42.5) and the number of cells per blastocyst (bST group 91.8±5.5 compared to control group 75±6) were greater (P<0.01) in the bST-treated sheep. Plasma concentrations of IGF-I and insulin were greater (P<0.01) in the bST-treated group. No changes were observed in progesterone concentrations (P=0.5). It is concluded that bST injection 5 days before progestin removal increases cleavage rate and the proportion of

Effects of lead on the male mouse as investigated by in vitro fertilization and blastocyst culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johansson, L.; Sjoeblom, P.; Wide, M.

1987-02-01

Long-term exposure of male mice to inorganic lead (lead chloride, 1 g/liter) in the drinking water reduces their fertility. The cause of this reduction, expressed as a decrease in the number of mated females showing inplantations, was investigated, using an in vivo fertilization method. It was found that spermatozoa from lead-exposed males had a significantly lower ability to fertilize mouse eggs than those from unexposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males, were examined. No morphologically abnormal embryos were found. However, whenmore » cultured in vitro over the implantation period, blastocysts of the group mated with lead-exposed males showed an increased frequency of delayed hatching from the zona pellucida or an inability to hatch. Among blastocysts from this group a decreased frequency of inner cell mass development was also found.« less

The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis.

PubMed Central

Massip, A; Mulnard, J; Vanderzwalmen, P; Hanzen, C; Ectors, F

1982-01-01

The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching; discontinuous expansion interrupted by few contractions and followed by hatching; discontinuous expansion interrupted by several rapid contractions without hatching. This demonstrated that the pulsatile activity of the blastocyst is not a necessary condition of hatching but also that only a moderate pulsatile activity is compatible with normal hatching. The time of hatching in vitro corresponded approximately with the time of zona loss in vivo (9-10 days). Rupture of the zona occurred at any point of the trophoblast layer. Hatching by herniation through a reduced opening of the zona was occasionally observed. The behavior of the embryos from a particular animal was very similar but differences were noted between embryos from different animals. Images Fig. 3 PMID:7076563

A caprine chimera produced by injection of embryonic germ cells into a blastocyst.

PubMed

Jia, W; Yang, W; Lei, A; Gao, Z; Yang, C; Hua, J; Huang, W; Ma, X; Wang, H; Dou, Z

2008-02-01

This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo.

Influence of cryopreservation on perinatal outcome after blastocyst- vs cleavage-stage embryo transfer: systematic review and meta-analysis.

PubMed

Alviggi, C; Conforti, A; Carbone, I F; Borrelli, R; de Placido, G; Guerriero, S

2018-01-01

To compare the perinatal outcomes of singleton pregnancies resulting from blastocyst- vs cleavage-stage embryo transfer and to assess whether they differ between fresh and frozen embryo transfer cycles. A systematic review of the literature was carried out using the Scopus, MEDLINE and ISI Web of Science databases with no time restriction. We included only peer-reviewed articles involving humans, in which perinatal outcomes of singleton pregnancies after blastocyst-stage embryo transfer were compared with those after cleavage-stage embryo transfer. Primary outcomes were preterm birth before 37 weeks and low birth weight (< 2500 g). Secondary outcomes were very preterm birth before 32 weeks, very low birth weight (< 1500 g), small-for-gestational-age (SGA), large-for-gestational-age (LGA), perinatal mortality and congenital anomaly. A meta-analysis was performed using a random-effects model. Three subgroups were evaluated: fresh only, frozen only and fresh plus frozen embryo transfer cycles. From a total of 3928 articles identified, 14 were selected for qualitative/quantitative analysis. Significantly higher incidences of preterm birth < 37 weeks (11 studies, n = 106 629 participants; risk ratio (RR), 1.15 (95% CI, 1.05 - 1.25); P = 0.002) and very preterm birth < 32 weeks (seven studies, n = 103 742; RR, 1.16 (95% CI, 1.02-1.31); P = 0.03) were observed after blastocyst- than after cleavage-stage embryo transfer in fresh cycles. However, the risk of preterm and very preterm birth was similar after blastocyst- and cleavage-stage transfers in frozen and fresh plus frozen cycles. Overall effect size analysis revealed fewer SGA deliveries after blastocyst- compared with cleavage-stage transfer in fresh cycles but a similar number in frozen cycles. Conversely, more LGA deliveries were observed after blastocyst- compared with cleavage-stage transfer in frozen cycles (two studies, n = 39 044; RR, 1.18 (95% CI, 1

Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.

PubMed

Hara, Hiromasa; Goto, Teppei; Takizawa, Akiko; Sanbo, Makoto; Jacob, Howard J; Kobayashi, Toshihiro; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi

2016-04-01

Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.

The prevalence of chromosomal deletions relating to developmental delay and/or intellectual disability in human euploid blastocysts.

PubMed

He, Wenyin; Sun, Xiaofang; Liu, Lian; Li, Man; Jin, Hua; Wang, Wei-Hua

2014-01-01

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34-149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

PubMed

Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

2015-07-01

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.

PubMed

Roels, K; Smits, K; Ververs, C; Govaere, J; D'Herde, K; Van Soom, A

2018-06-01

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected. © 2018 Blackwell Verlag GmbH.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

PubMed

Liu, Weiqiang; Yin, Yifei; Long, Xiaolin; Luo, Yumei; Jiang, Yonghua; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Li, Qing; Chen, Xinjie; Liao, Baoping; Xiao, Guohong; Wang, Weihua; Sun, Xiaofang

2009-04-01

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

PubMed Central

Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

2010-01-01

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.

PubMed Central

Brûlet, P; Babinet, C; Kemler, R; Jacob, F

1980-01-01

Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells. Images PMID:6933460

Modified natural cycle for embryo transfer using frozen-thawed blastocysts: A satisfactory option.

PubMed

Le, Quoc V; Abhari, Sina; Abuzeid, Omar M; DeAnna, Jennifer; Satti, Mohamed A; Abozaid, Tarek; Khan, Iqbal; Abuzeid, Mostafa I

2017-06-01

To describe pregnancy outcomes of frozen-thawed blastocysts cycles using modified natural cycle frozen embryo transfers (NC-FET) and down-regulated hormonally controlled frozen embryo transfers (HC-FET) protocols. This retrospective cohort study included all patients undergoing either modified NC-FET or down-regulated HC-FET using frozen-thawed day 5 embryos. Cycles with donor blastocysts were excluded. Four hundred twenty eight patients underwent a total of 493 FET cycles. Patients with regular menses and evidence of ovulation underwent modified NC-FET. These patients were given hCG 10,000 IU IM on the day of LH-surge. Vaginal progesterone (P4) was started two days later and blastocyst transfer was planned seven days after detecting the LH surge. Anovulatory patients and some ovulatory patients underwent down-regulated HC-FET. These patients were placed on medroxy-progesterone acetate (10mg) for 10days to bring on menses and were also given a half-dose of GnRH-agonist (GnRH-a) on the third day of medroxy-progesterone acetate. Exogenous estradiol was initiated on the third day of menses. Once serum E2 levels reached >500pg/mL and endometrial lining reached >8mm, intramuscular (IM) P4 in oil was administered. Blastocyst FET was planned 6days after initiating P4. The primary outcomes included clinical pregnancy and delivery rates. There were 197 patients in the modified NC-FET protocol and 181 in the down-regulated HC-FET protocol. Mean age (years), day-3 FSH levels (mIU/mL) and percentage of patients with male factor infertility were significantly higher and mean BMI (kg/m 2 ) was significantly lower in modified NC-FET compared to HC-FET, respectively. Analysis of the first cycle pregnancy outcomes revealed no significant differences in clinical pregnancy rate (54.3% vs. 52.5%) and delivery rate (47.2% vs. 43.6%) between modified NC-FET and HC-FET. Logistic regression analysis showed age (OR=0.939, 95% CI 0.894-0.989, p=0.011), number of blastocysts transferred (OR

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide

USDA-ARS?s Scientific Manuscript database

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu du...

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.

PubMed

Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A; Nakauchi, Hiromitsu

2013-03-19

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield.

PubMed

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-03-01

To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 invitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored.

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield

PubMed Central

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-01-01

Objective To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. Methods This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 in-vitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Results Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Conclusion Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored. PMID:29338139

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

PubMed

Lee, Y L; Lee, K F; Xu, J S; Kwok, K L; Luk, J M; Lee, W M; Yeung, W S B

2003-02-01

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PubMed

Gómez, M C; Serrano, M A; Pope, C Earle; Jenkins, J A; Biancardi, M N; López, M; Dumas, C; Galiguis, J; Dresser, B L

2010-09-01

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4

Double-quality control reveals high-level toxicity in gloves used for operator protection in assisted reproductive technology.

PubMed

Lierman, Sylvie; De Sutter, Petra; Dhont, Marc; Van der Elst, Josiane

2007-10-01

To submit different glove brands to double-quality control tests using mouse embryo assay (MEA) and the human sperm motility assay (HuSMA). Operator protection against infectious body fluid contamination is a safety issue in assisted reproductive technology (ART). When using gloves in the ART laboratory, toxic substances can be transmitted to culture media, even during brief contact. Quality control study of gloves in ART. University hospital-based infertility center. Seven- to 8-week-old female B6D2F1 hybrid mice. We tested two surgical, two cleanroom, and six examination glove brands. Only gloves brands that passed both HuSMA and MEA were submitted to further QC using zona-free and/or cryopreserved MEA. Sperm motility index, two-cell and blastocyst development, blastocyst total cell number. Quality control by MEA and HuSMA identified two glove brands to be nontoxic. Our study shows that gloves used in ART can be toxic and should be tested as part of an ongoing quality control program.

Day 6 blastocyst is associated with increased birth weight in full-term singleton newborns after frozen-thawed transfer.

PubMed

Cai, Jiali; Liu, Lanlan; Xu, Yingpei; Liu, Zhenfang; Jiang, Xiaoming; Li, Ping; Sha, Aiguo; Ren, Jianzhi

2018-06-13

The purpose of the study is to compare the newborns weight in singleton term birth following transfer of thawed blastocysts-frozen on either day 5 or day 6 after in vitro fertilization. The retrospective study included 1444 frozen-thawed blastocyst transfer (FBT) cycles resulting in live singleton births between Jan 2013 and Dec 2016. The main outcomes measured were absolute birth weight, z-score adjusted for gestational age and gender, and incidence of large-for-gestational-age (LGA) newborns. Generalized linear model (GLM) and logistic regression were used in multivariate analyses. Both the absolute birth weight (3416.49 ± 404.74 vs 3349.22 ± 416.17) and the z-score (0.6 ± 0.93 vs 0.41 ± 0.93) were significantly higher on day 6 FBT in comparison with day 5 FBT. The incidence of LGA newborns was also increased on day 6 FBT (22.8 vs 14.7%, P = 0.006). Adjusted for maternal age, BMI, PCOS diagnosis, present of vanishing twin, and embryo quality, the odds ratio (95% confidence interval) for LGA on day 6 FBT comparing with day 5 FBT was 1.76 (1.18-2.64). Day 6 FBT is associated with increased birth weight and contributes to the incidence of LGA newborns in FBT.

Neutral effect of body mass index on implantation rate after frozen-thawed blastocyst transfer.

PubMed

Insogna, Iris G; Lee, Malinda S; Reimers, Rebecca M; Toth, Thomas L

2017-11-01

To examine the effects of body mass index (BMI) on implantation rate after uniform protocol frozen-thawed blastocyst transfer in women with a homogenous uterine environment. Retrospective cohort study. Single IVF clinic at a large academic institution. Four hundred sixty-one infertile women treated at a large academic institution from January 2007 to January 2014. All women underwent standardized slow frozen-thawed blastocyst transfers with good-quality day 5-6 embryos, following an identical hormonal uterine preparation, with comparison groups divided according to BMI category: underweight (<18.5 kg/m 2 ), normal weight (18.5-24.9 kg/m 2 ), overweight (25.0-29.9 kg/m 2 ), and obese (≥30.0 kg/m 2 ). Implantation rate. There were no statistically significant differences identified when comparing implantation rates among the four BMI cohorts. The implantation rate was 38.2% in normal weight patients, 41.7% in underweight patients, 45.1% in overweight patients, and 34.7% in obese patients. Adjusted odds ratios (OR) demonstrated no association between the main outcome, implantation rate, and BMI. Compared with the normal weight patients, the adjusted OR of implantation was 1.70 (95% confidence interval [CI], 0.40-7.72) for underweight patients, 1.61 (95% CI, 0.97-2.68) for overweight patients, and 0.92 (95% CI, 0.49-1.72) for obese patients. Secondary outcomes, including rates of miscarriage, clinical pregnancy, ongoing pregnancy, and live birth, were not significantly different between cohorts. While powered to detect a 16% difference between overweight and normal weight women, the study was underpowered to detect differences in the underweight and obese women, and no definitive conclusions can be drawn for these small cohorts. Patients with transfers that required the longest amount of time, greater than 200 seconds, had the highest average BMI of 27.5 kg/m 2 . Under highly controlled circumstances across 7 years of data from a single institution, using a

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs

PubMed Central

Matsunari, Hitomi; Nagashima, Hiroshi; Watanabe, Masahito; Umeyama, Kazuhiro; Nakano, Kazuaki; Nagaya, Masaki; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Sumazaki, Ryo; Herzenberg, Leonard A.; Nakauchi, Hiromitsu

2013-01-01

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals. PMID:23431169

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

PubMed

Xu, Hui-Yan; Yang, Xiao-Gan; Lu, Sheng-Sheng; Liang, Xing-Wei; Lu, Yang-Qing; Zhang, Ming; Lu, Ke-Huan

2018-06-01

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation

Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified-warmed blastocysts.

PubMed

Ueno, Satoshi; Uchiyama, Kazuo; Kuroda, Tomoko; Yabuuchi, Akiko; Ezoe, Kenji; Okimura, Tadashi; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

2018-06-01

A retrospective cohort study of 8736 autologous single vitrified-warmed blastocyst transfer cycles was conducted in a single centre to investigate the effect of cryostorage on clinical and neonatal outcomes. Cryostorage duration was classified into three groups: (A) 0-2 months (n = 4702); (B) 2-13 months (n = 2853) and (C) 13-97 months (n = 1181). Blastocysts were vitrified using the Cryotop method. No significant differences were observed in live birth rates: (A) 37.3%; (B) 34.9%; (C) (35.2%). Gestational period was significantly shorter in group C: (A) 38.7 ± 1.8; (B) 38.6 ± 1.6; (C) 38.1 ± 1.7; P < 0.05. This was clinically unimportant as the average gestational age was more than 38 weeks. No significant differences between groups were observed in birth weight: (A) 3060 ± 455 g; (B) 3052 ± 449 g; (C) 2992 ± 445 g, or congenital malformation rates: (A) 2.2%; (B) 1.9%; (C) 1.8%. The limitation of this study was that maximum storage duration was 8 years; most blastocysts were in cryostorage for much shorter periods. Long-term storage of blastocysts that are vitrified using an open device vitrification system has no negative effect on pregnancy and neonatal outcomes. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

[Effect of spermatozoa from different sources on normal fertilization of oocytes and embryo quality and development in intracytoplasmic sperm injection cycles].

PubMed

Xie, Duo; Qiu, Zhuolin; Luo, Chen; Chu, Qingjun; Quan, Song

2014-06-01

To evaluate the impact of spermatozoa from different sources on normal fertilization of oocytes, embryo quality and embryo developmental potential in intracytoplasmic sperm injection (ICSI) cycles. A retrospective analysis was conducted among 197 patients undergoing ICSI cycles in our center. The patients were classified into 3 groups according to the sources of semen, namely ejaculated spermatozoa group (n=102), percutaneous epididymal sperm aspiration (PESA) group (n=68), and testicular sperm aspiration (TESA) group (n=27). The ejaculated spermatozoa group was further classified into oligoasthenoteratozoospermia (n=67) and cryptozoospermia (n=35) subgroups. The normal fertilization, high-quality embryo, implantation and clinical pregnancy rates were compared among the groups; the rate of high-quality blastocyst formation in in-vitro culture of non-top quality embryos was also observed. The patients with PESA showed significantly higher normal fertilization rate (75.6%) than those in oligoasthenoteratozoospermia (64.8%), cryptozoospermia (62.1%), and TESA (61.6%) groups (P<0.05). No significant differences were found in the high-quality embryo, implantation, and clinical pregnancy rates among the groups (P>0.05). The rate of high-quality blastocyst formation in the in-vitro culture of non-top quality embryos was also comparable among the groups (P>0.05). Although spermatozoa obtained with by PESA is associated with a higher normal fertilization rate, the sources of spermatozoa do not significantly affect the embryonic quality and developmental potential in ICSI cycles.

Female age, serum antimüllerian hormone level, and number of oocytes affect the rate and number of euploid blastocysts in in vitro fertilization/intracytoplasmic sperm injection cycles.

PubMed

La Marca, Antonio; Minasi, Maria Giulia; Sighinolfi, Giovanna; Greco, Pierfrancesco; Argento, Cindy; Grisendi, Valentina; Fiorentino, Francesco; Greco, Ermanno

2017-11-01

To study the relative role of female age and ovarian reserve, measured through serum antimüllerian hormone (AMH) in determining the rate and number of euploid blastocysts in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Retrospective analysis of cycles performed in 2014-2015. Tertiary referral IVF center. A total of 578 infertile couples undergoing IVF/ICSI and preimplantation genetic screening (PGS) analysis. All embryos were cultured and biopsied at the blastocyst stage. The method involved whole-genome amplification followed by array comparative genome hybridization. Serum AMH was measured by means of the modified Beckman Coulter AMH Gen II assay. The rate and number of euploid blastocysts and their correlation with ovarian reserve and response to stimulation. The mean (±SD) age of patients was 37.6 ± 4.1 years, and the mean number of blastocysts per patient was 3.1 ± 2. The total number of blastocysts available to the analysis was 1,814, and 36% of them were euploid after PGS. Age and serum AMH were significantly and independently related to the rate of euploid blastocysts available for patients. As an effect of the cohort size, the number of mature oocytes positively affected the total number of euploid blastocysts per patient. A strong positive age-independent relationship between AMH level and the rate of euploid blastocysts was found. This confirms that the measurement of ovarian reserve by means of AMH has high relevance when counseling infertile patients. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial.

PubMed

Kieslinger, Dorit C; Hao, Zhenxia; Vergouw, Carlijn G; Kostelijk, Elisabeth H; Lambalk, Cornelis B; Le Gac, Séverine

2015-03-01

To compare the development of human embryos in microfluidic devices with culture in standard microdrop dishes, both under static conditions. Prospective randomized controlled trial. In vitro fertilization laboratory. One hundred eighteen donated frozen-thawed human day-4 embryos. Random allocation of embryos that fulfilled the inclusion criteria to single-embryo culture in a microfluidics device (n = 58) or standard microdrop dish (n = 60). Blastocyst formation rate and quality after 24, 28, 48, and 72 hours of culture. The percentage of frozen-thawed day-4 embryos that developed to the blastocyst stage did not differ significantly in the standard microdrop dishes and microfluidic devices after 28 hours of culture (53.3% vs. 58.6%) or at any of the other time points. The proportion of embryos that would have been suitable for embryo transfer was comparable after 28 hours of culture in the control dishes and microfluidic devices (90.0% vs. 93.1%). Furthermore, blastocyst quality was similar in the two study groups. This study shows that a microfluidic device can successfully support human blastocyst development in vitro under static culture conditions. Future studies need to clarify whether earlier stage embryos will benefit from the culture in microfluidic devices more than the tested day-4 embryos because many important steps in the development of human embryos already take place before day 4. Further improvements of the microfluidic device will include parallel culture of single embryos, application of medium refreshment, and built-in sensors. NTR3867. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Complete method to obtain, culture, and transfer mouse blastocysts nonsurgically to study implantation and development.

PubMed

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

Duration of blastulation may be associated with ongoing pregnancy rate in single euploid blastocyst transfer cycles.

PubMed

Mumusoglu, Sezcan; Ozbek, Irem Y; Sokmensuer, Lale K; Polat, Mehtap; Bozdag, Gurkan; Papanikolaou, Evangelos; Yarali, Hakan

2017-12-01

Not all euploid embryos implant, necessitating additional tools to select viable blastocysts in preimplantation genetic screening cycles. In this retrospective cohort study, 129 consecutive patients who underwent 129 single euploid blastocyst transfers in cryopreserved embryo transfer cycles were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophoectoderm biopsy. Twenty-three time-lapse morphokinetic variables were tested among patients with (n = 68) or without (n = 61) ongoing pregnancy. All 23 time-lapse morphokinetic variables, apart from duration of blastulation (tB-tSB), were comparable between patients with or without ongoing pregnancy. Duration of blastulation was significantly shorter in patients with ongoing pregnancy (8.1 ± 3.2 versus 9.5 ± 3.4 h; P = 0.014); shorter duration of blastulation remained an independent predictor for ongoing pregnancy, when tested by logistic regression analysis (OR 0.81; 95% CI 0.70 to 0.93). One important limitation of this study, and a reason for caution, is the use of multiple comparisons, which can lead to differences at the 0.05 level simply by chance or random variation. Nonetheless, the study suggests that when more than one euploid blastocyst is available, priority might be given to those with a shorter duration of blastulation. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Comparison of the interferon-tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts.

PubMed

Talbot, Neil C; Powell, Anne M; Ocón, Olga M; Caperna, Thomas J; Camp, Mary; Garrett, Wesley M; Ealy, Alan D

2008-02-01

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 70/5 (93%); NT 67/1 (99%)] and less (P < .05) for P blastocysts [65/27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day. 2007 Wiley-Liss, Inc

Effects of sorbitol on porcine oocyte maturation and embryo development in vitro.

PubMed

Lin, Tao; Zhang, Jin Yu; Diao, Yun Fei; Kang, Jung Won; Jin, Dong-Il

2015-04-01

In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.

In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment.

PubMed

Leoni, Giovanni Giuseppe; Succu, Sara; Satta, Valentina; Paolo, Mereu; Bogliolo, Luisa; Bebbere, Daniela; Spezzigu, Antonio; Madeddu, Manuela; Berlinguer, Fiammetta; Ledda, Sergio; Naitana, Salvatore

2009-01-01

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus-oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean +/- s.e.m., 89.67 +/- 5.74 and 26.69 +/- 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90beta and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

A Method Based on Artificial Intelligence To Fully Automatize The Evaluation of Bovine Blastocyst Images.

PubMed

Rocha, José Celso; Passalia, Felipe José; Matos, Felipe Delestro; Takahashi, Maria Beatriz; Ciniciato, Diego de Souza; Maserati, Marc Peter; Alves, Mayra Fernanda; de Almeida, Tamie Guibu; Cardoso, Bruna Lopes; Basso, Andrea Cristina; Nogueira, Marcelo Fábio Gouveia

2017-08-09

Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.

Evaluation of foal production following intracytoplasmic sperm injection and blastocyst culture of oocytes from ovaries collected immediately before euthanasia or after death of mares under field conditions.

PubMed

Hinrichs, Katrin; Choi, Young-Ho; Norris, Jody D; Love, Linda B; Bedford-Guaus, Sylvia J; Hartman, David L; Velez, Isabel C

2012-10-15

To evaluate the efficiency of foal production following intracytoplasmic sperm injection (ICSI) and blastocyst culture of oocytes from mares that died or were euthanized under field conditions. Prospective case series. 16 mares (age, 3 to 19 years) that died or were euthanized for various causes. Ovaries were collected immediately before euthanasia (n = 10) or after death (6). Ovaries were transported to the laboratory for oocyte recovery (15 mares), or oocytes were recovered at a remote location and shipped to the laboratory (1). Oocytes underwent ICSI, and presumptive zygotes were cultured for 7 to 10 days. Blastocysts were shipped to embryo transfer facilities for transcervical transfer to recipient mares. Ovaries were processed 30 minutes to 12 hours (mean ± SD, 4.6 ± 3.3 hours) after mares' deaths. A mean of 14.1 ± 8.6 oocytes/mare were cultured, and 110 of 225 (49%) matured. Twenty-one blastocysts developed after ICSI and were transferred to recipient mares. Thirteen pregnancies were established; 10 healthy foals were produced from 6 donor mares. The number of blastocysts produced per mare and number of live foals produced per mare were significantly correlated with the number of oocytes recovered. Foals were produced from mares after death or euthanasia under field conditions. Proportions of foals born overall (10 foals/16 mares) and mares from which ≥ 1 foal was produced (6/16) were greater than those reported following recovery and oviductal transfer of oocytes to inseminated recipients after death of donor mares under field conditions.

Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts.

PubMed

Fielder, Thomas J; Yi, Charles S; Masumi, Juliet; Waymire, Katrina G; Chen, Hsiao-Wen; Wang, Shuling; Shi, Kai-Xuan; Wallace, Douglas C; MacGregor, Grant R

2012-12-01

To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8%) compared with B6NTac chimeric males (7/9, 78%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86%) B6J male chimeras were fertile compared with 6 of 11 (55%) B6NTac male chimeras. Ten of 12 (83%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42 ± 1.73, n = 12) compared to B6NTac chimeras (2.17 ± 1.33, n = 6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64%) compared with chimeras produced using B6NTac blastocysts (4/11; 36%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.

Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone reverses the adverse effects of diet-induced obesity on oocyte quality.

PubMed

Minge, Cadence E; Bennett, Brenton D; Norman, Robert J; Robker, Rebecca L

2008-05-01

Obesity and its physiological consequences are increasingly prevalent among women of reproductive age and are associated with infertility. To investigate, female mice were fed a high-fat diet until the onset of insulin resistance, followed by assessments of ovarian gene expression, ovulation, fertilization, and oocyte developmental competence. We report defects to ovarian function associated with diet-induced obesity (DIO) that result in poor oocyte quality, subsequently reduced blastocyst survival rates, and abnormal embryonic cellular differentiation. To identify critical cellular mediators of ovarian responses to obesity induced insulin resistance, DIO females were treated for 4 d before mating with an insulin-sensitizing pharmaceutical: glucose and lipid-lowering AMP kinase activator, 5-aminoimidazole 4-carboxamide-riboside, 30 mg/kg.d; sodium salicylate, IkappaK inhibitor that reverses insulin resistance, 50 mg/kg.d; or peroxisome proliferator activated receptor-gamma agonist rosiglitazone, 10 mg/kg.d. 5-aminoimidazole 4-carboxamide-riboside or sodium salicylate treatment did not have significant effects on the reproductive parameters examined. However, embryonic development to the blastocyst stage was significantly improved when DIO mice were treated with rosiglitazone, effectively repairing development rates. Rosiglitazone also normalized DIO-associated abnormal blastomere allocation to the inner cell mass. Such improvements to oocyte quality were coupled with weight loss, improved glucose metabolism, and changes in ovarian mRNA expression of peroxisome proliferator activated receptor-regulated genes, Cd36, Scarb1, and Fabp4 cholesterol transporters. These studies demonstrate that peri-conception treatment with select insulin-sensitizing pharmaceuticals can directly influence ovarian functions and ultimately exert positive effects on oocyte developmental competence. Improved blastocyst quality in obese females treated with rosiglitazone before mating

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

PubMed

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

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

PubMed

Moro, L N; Hiriart, M I; Buemo, C; Jarazo, J; Sestelo, A; Veraguas, D; Rodriguez-Alvarez, L; Salamone, D F

2015-07-01

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos. © 2015 Society for Reproduction and Fertility.

Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study

PubMed Central

2012-01-01

Background Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET. Methods First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups. Results For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies. Conclusion Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used

Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development.

PubMed

Jee, Byung Chul; Youm, Hye Won; Lee, Jae Ho; Kim, Jee Hyun; Suh, Chang Suk; Kim, Seok Hyun

2013-05-01

We performed this study to investigate the effect of ketorolac (a non-steroidal anti-inflammatory drug) administration around ovarian stimulation on in vivo and in vitro fertilization process. Sixty-four female mice (ICR) were injected with ketorolac (0, 7.5, 15 and 30 µg/d) for 3 d starting from the day of eCG treatment. In experiment 1, 41 mice were triggered by hCG and then mated; two-cell embryos were obtained and in vitro development up to blastocyst was observed. In experiment 2, 23 mice were triggered by hCG and mature oocytes were collected; in vitro fertilization rate and subsequent embryo development up to blastocyst was recorded. In experiment 1, the blastocyst-forming rates per in vivo fertilized two-cell embryo showed an inverse relationship with a dosage of ketorolac (97.6%, 64.2%, 35.4% and 25.9%). In experiment 2, degenerated oocytes were frequently observed in a dose-dependent manner (4.3%, 22.9%, 22.4% and 75.0%). Lower fertilization rates were noted in all the three ketorolac-treating groups; blastocyst-forming rate was significantly lower in 30-µg-treating group when compared with the control group. Administration of ketorolac around ovarian stimulation significantly affects the development of in vivo fertilized embryo in a dose-dependent manner. High-dose ketorolac could result in a poor oocyte quality and decreased embryo developmental competence.

Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo

PubMed Central

Schulz, Laura C.; Roberts, R. Michael

2011-01-01

The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally-derived leptin from the oocyte becomes differentially distributed among blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased towards a particular fate as early as the 2-cell stage. Here, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 % and 76.9 %, respectively) collected following superovulation, but was reduced, particularly in CF1 embryos (29.8 %; p < 0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division, and was not affected by removal of the zona pellucida. Presence or absence of leptin polarization was not linked to differences in ability of embryos to develop normally to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm but upon blastocoel expansion it was lost from cells. Throughout development leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development. PMID:21444625

Better Quality and More Usable Embryos Obtained on Day 3 Cultured in 5% Than 20% Oxygen: A Controlled and Randomized Study Using the Sibling Oocytes.

PubMed

Peng, Huixia; Shi, Wenhao; Zhang, Wei; Xue, Xia; Li, Na; Li, Wei; Shi, Juanzi

2016-03-01

To evaluate the effect of atmospheric oxygen (O2) concentration on embryonic development, a controlled and randomized study using the sibling oocytes was carried out. A total of 147 patients were studied. Embryos were cultured in O2 concentration 20% versus 5% during the gamete, zygote, and first 3 days. The mean cell numbers of embryo (7.69 ± 1.91 vs 7.20 ± 1.82, P = .011) and rate of clinically useable embryos (81.62% vs 77.22%, P = .017) were significantly higher in 5% O2 than in 20% O2. There was no difference in the zygote developmental stage, day 2, day 4, and blastocyst stage. The quality of blastocyst (both inner cell mass and trophectoderm) showed no difference. Also, there was no increase in embryos fragmentation and uneven cells in 20% O2 culture condition. In conclusion, 20% O2 reduced the mean cell numbers of embryo and the number of clinically useable embryos on day 3. However, there was no subsequent negative impact on development of day 4 and blastocyst stage. © The Author(s) 2015.

In vitro fertilization outcomes after fresh and frozen blastocyst transfer in South Asian compared with Caucasian women.

PubMed

Shah, Meera Sridhar; Caballes, Marissa; Lathi, Ruth Bunker; Baker, Valerie Lynn; Westphal, Lynn Marie; Milki, Amin A

2016-06-01

To study pregnancy outcomes between South Asian and Caucasian women undergoing frozen blastocyst transfer cycles. Retrospective cohort study. Not applicable. Caucasian and South Asian patients undergoing frozen blastocyst transfer between January 2011 and December 2014. Not applicable. Live birth rate. A total of 196 Caucasian and 117 South Asian women were included in our study. Indians were on average 2.2 years younger than Caucasian women (34.9 vs. 37.1 years), and were more likely to be nulliparous (59% vs. 43%). All other baseline characteristics were similar. In women undergoing their first frozen ET cycle, implantation rate (49% vs. 47%), clinical pregnancy rate (PR; 54% vs. 49%), and live birth rate (43% vs. 43%) were similar between South Asians and Caucasians, respectively. In patients who underwent a prior fresh blastocyst transfer, the live birth rate was significantly lower in South Asian versus Caucasian women (21% vs. 37%). Our data demonstrate that IVF outcomes are better in frozen versus fresh cycles among South Asian women. The IVF clinics may wish to consider these findings when counseling South Asian patients about the timing of ET. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Morbid obesity and pregnancy outcomes after single blastocyst transfer: a retrospective, North American study.

PubMed

Russo, Miguel; Ates, Senem; Shaulov, Talya; Dahan, Michael H

2017-04-01

Maternal obesity has been shown to affect reproductive function and pregnancy outcomes following in vitro fertilization. More recently, studies have demonstrated lower live birth rates after single blastocyst transfer (SBT) in patients who are overweight or obese. However, the impact of morbid obesity on pregnancy outcomes after SBT has not been well elucidated. The present study aimed to determine whether morbid obesity has a detrimental impact on pregnancy outcomes after SBT in a North American population. A retrospective, cohort study including 520 nulliparous and multiparous women undergoing top-quality SBT between August 2010 and March 2014 at a University Health Centre in North America was conducted. Primary outcomes included: miscarriage rate, clinical pregnancy rate, and live birth rate. Subjects were divided into different BMI categories (kg/m 2 ), including <20, 20-24.9, 25.0-29.9, 30-40, and 40 or more. The miscarriage rate per pregnancy for each group, respectively, was 36, 64, 59, 61, and 50% (p = 0.16); the clinical pregnancy (per patient) rate per group was 36, 52, 38, 26, and 10% (p = 0.009); and the live birth rate (per patient) per group was 35, 50, 38, 26 and 10% (p = 0.03). Morbid obesity is a strong and independent predictor of poor pregnancy outcomes in patients undergoing top-quality SBT.

Massage therapy improves in vitro fertilization outcome in patients undergoing blastocyst transfer in a cryo-cycle.

PubMed

Okhowat, Jasmin; Murtinger, Maximilian; Schuff, Maximilian; Wogatzky, Johannes; Spitzer, Dietmar; Vanderzwalmen, Pierre; Wirleitner, Barbara; Zech, Nicolas Herbert

2015-01-01

Massage therapy is increasingly used to relieve physical and mental discomfort and is suggested as a safe therapeutic modality, without any significant risks or any known side effects. Although a multitude of complementary therapies, such as acupuncture, are applied in reproductive medicine, no information is available with regard to the application of massage as an adjuvant therapy in assisted-reproduction techniques (ARTs). This study was intended to assess the effectiveness of a deep relaxation (andullation) therapy based on oscillating vibrations when used prior to embryo transfer (ET) in in vitro fertilization (IVF) cryo-cycles. The research team designed a retrospective, observational study. Participants willing to undergo the massage treatment were allocated to the intervention (andullation) group. The study was performed at the IVF Centers Prof. Zech-Bregenz in Bregenz, Austria. A total of 267 IVF patients, with a mean age of 36.3 y, participated in this single-center study. All patients receiving a transfer of vitrified and warmed blastocysts between January and December 2012 were included in the evaluation. Prior to ET, the andullation group received a standardized program of therapy-a 30-min, deep relaxation massage on an oscillating (vibrating) device, whereas the control group did not. To determine efficacy, the primary outcomes that the study measured were (1) pregnancy rates (PRs), by testing urine and obtaining a positive β-human chorionic gonadotropin (β-hCG); and (2) ongoing, pregnancies (oPR), by observation of fetal heartbeat and birth rates (BR) as well as miscarriage rates. The patients' medical histories and types of infertility as well as the quality of the embryo transfers (ETs) were evaluated. In patients using the massage therapy prior to ET, significantly higher PRs, oPRs, and BRs were observed compared with the control group-PR: 58.9% vs 41.7%, P<.05; oPR: 53.6% vs 33.2%, P<.01; and BR: 32.0% vs 20.3%, P<.05. No differences were

Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

PubMed Central

Chan, Wen-Hsiung

2007-01-01

The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen.

PubMed

Lee, Hae-Lee; Kim, Sue-Hee; Ji, Dong-Beom; Kim, Yong-Jun

2009-09-01

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/ propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

PubMed

Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

2017-10-01

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation

Facilitated glucose transporters play a crucial role throughout mouse preimplantation embryo development.

PubMed

Leppens-Luisier, G; Urner, F; Sakkas, D

2001-06-01

The role of glucose fluctuates during preimplantation mouse embryo development, indicating that a specific interplay exists between glucose metabolism and uptake. In this study, attempts were made to characterize the role of the Na(+)-coupled active and the facilitated glucose transporters (GLUT) during preimplantation development by using specific glucose analogues and transport inhibitors and by examining the expression of GLUT1. One-cell outbred mouse embryos were cultured in medium M16 (5.5 mmol/l glucose), M16 without glucose (M16-G), M16-G + 2-deoxyglucose, M16-G + 3-O-methylglucose, M16 + phlorizin and M16 + phloretin and development to the blastocyst stage assessed. The absence of glucose, or the presence of 3-O-methylglucose, which is taken up but not metabolized, did not inhibit blastocyst development. 2-Deoxyglucose, which is phosphorylated but not metabolized, inhibited blastocyst development. Culture in M16 supplemented with phlorizin, an inhibitor of Na(+)-coupled active glucose transport did not inhibit blastocyst formation. Phloretin had no effect on the cleavage of two-cell embryos to the four-cell stage, but inhibited the morula/blastocyst transition. Both phloretin and phlorizin inhibited glucose uptake in two-cell embryos. Finally, GLUT1 expression was 10-fold less in blastocysts cultured in M16 compared to in-vivo blastocysts and those cultured in M16-G. The results show that both types of glucose transporters influence preimplantation embryo development and that the embryo has an innate ability to control the uptake of glucose by regulating the expression of GLUT1.

Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles

PubMed Central

2013-01-01

Background In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles. Methods First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups. Results There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05). Conclusions While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the

Progesterone is critical for the development of mouse embryos.

PubMed

Zhang, Cong; Murphy, Bruce D

2014-08-01

Infertility affects approximately 10-15 % of reproductive-aged couples, and embryo loss due to preimplantation death is common to many mammals. Previous studies showed that a complex series of interactive molecular events are associated with this process, especially hormones (progesterone and estrogens) and growth factors, and are important for the cleavage and differentiation of the blastocysts. Yet, the mechanism of preimplantation embryo development is unclear. Using conditional knockout mice (CKO), we showed the development of blastocyst is tightly controlled by the level of progesterone (P4); furthermore, we found that the time when P4 should increase is also crucial for the formation of blastocysts. In CKO mice whose Lrh1 (liver receptor homolog 1) is deleted under the expression of Cre recombinase driven by progesterone receptor promoter, which reduced P4 synthesis, few of their embryos can reach blastocyst stage. When these CKO mice were supplied with P4 in the afternoon of dpc 1 (day post copulation), most of the embryos can form blastocysts; when CKO mice were supplied with P4 from the morning of dpc1, one-third of the embryos can reach blastocyst stage; however, the supplement of P4 in the morning of dpc 2 made very few of the embryos become blastocysts. We conclude that early exposure to P4 is essential for timely progression of early embryogenesis in the mouse.

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance.

PubMed

Rienzi, Laura; Gracia, Clarisa; Maggiulli, Roberta; LaBarbera, Andrew R; Kaser, Daniel J; Ubaldi, Filippo M; Vanderpoel, Sheryl; Racowsky, Catherine

2017-03-01

Successful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice. The objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method. A Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI. One RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05-7.51; P = 0.039; 48 and 30 cycles, respectively, per transfer (RR = 1

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance

PubMed Central

Rienzi, Laura; Gracia, Clarisa; Maggiulli, Roberta; LaBarbera, Andrew R.; Kaser, Daniel J.; Ubaldi, Filippo M.; Vanderpoel, Sheryl; Racowsky, Catherine

2017-01-01

Abstract BACKGROUND Successful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice. OBJECTIVE AND RATIONALE The objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method. SEARCH METHODS A Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI. OUTCOMES One RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05–7

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

PubMed Central

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

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

PubMed

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Dual effects of fluoxetine on mouse early embryonic development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Chang-Woon; Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723; Choe, Changyong

2012-11-15

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetinemore » (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from Ca

A mathematical model of in vivo bovine blastocyst developmental to gestational Day 15.

PubMed

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

Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos.

PubMed

Hosoe, Misa; Inaba, Yasushi; Hashiyada, Yutaka; Imai, Kei; Kajitani, Kenji; Hasegawa, Yuichi; Irie, Mamoru; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

2017-02-01

Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible. © 2016 Japanese Society of Animal Science.

Aflatoxin B1 impairs sperm quality and fertilization competence.

PubMed

Komsky-Elbaz, A; Saktsier, M; Roth, Z

2018-01-15

, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted. Copyright © 2017 Elsevier B.V. All rights reserved.

Frozen-thawed blastocyst transfer in natural cycle: feasibility in everyday clinical practice.

PubMed

Cardellicchio, Lucia; Reschini, Marco; Paffoni, Alessio; Guarneri, Cristina; Restelli, Liliana; Somigliana, Edgardo; Vegetti, Walter

2017-06-01

Transfer of frozen-thawed embryos in natural cycle is gaining consensus but evidence on this approach is scanty. The aim of this study is reporting on the feasibility of this type of policy in everyday clinical practice. We retrospectively selected all women undergoing the procedure between July 2013 and December 2014. During the study period, women were systematically scheduled for natural cycle if they referred regular menstrual cycles. Hormone replacement therapy (HRT) was conversely prescribed if the woman had irregular menstrual cycles or if the monitoring of the natural cycle failed. The analysis exclusively focussed on the first cycle per woman. Overall, 251 women were selected. HRT was initially chosen in 52 women, leaving 199 women suitable for the natural cycle. This procedure could be performed in 194 of these women (97%, 95% CI 95-99%). Two additional women initially allocated to HRT ultimately performed the blastocyst transfer with natural cycle. Overall, 196 were thus treated with natural cycle (78%, 95% CI 73-83%). The basal characteristics of the women who did and did not undergo natural cycles were similar with the exceptions of serum FSH (p < 0.001) and AMH (p = 0.03). The live birth rate did not also differ (34% versus 31%, p = 0.63). Characteristics of women treated with the natural cycle who did (n = 67) and did not (n = 129) achieve a live birth did not differ. Frozen-thawed blastocyst transfer in natural cycle can be successfully performed in the vast majority of women.

Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern.

PubMed

Mohapatra, Sushil K; Sandhu, Anjit; Neerukattu, Venkata S; Singh, Karn P; Selokar, Naresh L; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

2015-04-01

We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB-). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB- oocytes (43.41 ± 2.54 vs. 22.74 ± 1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB- blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB- blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB- blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB- blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

PubMed

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).

Derivation, characterization and differentiation of a new human embryonic stem cell line from a Chinese hatched blastocyst assisted by a non-contact laser system.

PubMed

Wu, Rongrong; Xu, Chenming; Jin, Fan; Tan, Zhou; Gu, Bin; Chen, Liangbiao; Yao, Xing; Zhang, Ming

2010-08-01

Currently worldwide attention has focused on the derivation of human embryonic stem cells (hESCs) for future therapeutic medicine. However, the majority of existing hESCs are directly or indirectly exposed to non-human materials during their derivation and/or propagation, which greatly restrict their therapeutic potential. Besides the efforts to improve culture systems, the derivation procedure, especially blastocyst manipulation, needs to be optimized. We adopted a non-contact laser-assisted hatching system in combination with sequential culture process to obtain hatched blastocysts as materials for hESC derivation, and derived a hESC line ZJUhES-1 of a Chinese population without exposure to any non-human materials during blastocyst manipulation. ZJUhES-1 satisfies the criteria of pluripotent hESCs: typically morphological characteristics; the expression of alkaline phosphatase, human telomerase reverse transcriptase and multiple hESC-specific markers including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, Nanog, Rex-1, Sox-2, UTF-1, Connexins 43 and 45, TERF-1 and TERF-2, Glut-1, BCRP-1/ABCG-2, GDF3, LIN28, FGF4, Thy-1, Cripto1/TDGF1, AC133 as well as SMAD1/2/3/5; extended proliferative capacity; maintenance of a stable male karyotype after long-term cultivation; and robust multiple-lineage developmental potentials both in vivo and in vitro. Moreover, ZJUhES-1 has distinct identity revealed from DNA fingerprinting. Our xeno-free blastocyst manipulation procedure may promote the progression toward clinical-grade hESC derivation. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle.

PubMed

Yamanaka, Ken-Ichi; Kaneda, Masahiro; Inaba, Yasushi; Saito, Koji; Kubota, Kaiyu; Sakatani, Miki; Sugimura, Satoshi; Imai, Kei; Watanabe, Shinya; Takahashi, Masashi

2011-08-01

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors.

PubMed Central

Paria, B C; Dey, S K

1990-01-01

We have established a model that shows cooperative interaction among preimplantation embryos and the role of growth factors on their development and growth. Two-cell mouse embryos cultured singly in 25-microliters microdrops had inferior development to blastocysts and lower cell numbers per blastocyst compared with those cultured in groups of 5 or 10. The inferior development of singly cultured embryos was markedly improved by addition of epidermal growth factor (EGF) or transforming growth factor alpha or beta 1 (TGF-alpha or TGF-beta 1) to the culture medium. The stage of embryonic development, primarily affected by these treatments, was between eight-cell/morula and blastocyst. Furthermore, blastocysts developed from eight-cell embryos cultured in groups or singly in the presence of EGF showed a higher incidence of zona hatching compared with those cultured singly in the absence of EGF. Detection of EGF receptors on the embryonic cell surface at eight-cell/morula and blastocyst stages suggests beneficial effects of EGF or TGF-alpha on preimplantation embryo development and blastocyst functions. Insulin-like growth factor I (IGF-I) had no influence on embryo development. To further document the cooperative interactions among embryos, the volume of the culture medium was doubled to 50 microliters. This increase in culture volume was even more detrimental to the development of singly cultured embryos. However, this detrimental effect was significantly reversed by EGF and reversed even more markedly by a combination of EGF and TGF-beta 1 but not by TGF-beta 1 alone. Although TGF-beta 1 plus IGF-I caused a modest improvement of embryo development, the response was not as great as shown by EGF alone. Furthermore, IGF-I had no additive effect on EGF-induced embryonic development. The study presents clear evidence that specific growth factors of embryonic and/or reproductive tract origin participate in preimplantation embryo development and blastocyst functions in an

Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced by In Vitro Fertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

PubMed Central

Mohapatra, Sushil K.; Sandhu, Anjit; Neerukattu, Venkata S.; Singh, Karn P.; Selokar, Naresh L.; Singla, Suresh K.; Chauhan, Manmohan S.; Manik, Radhey S.

2015-01-01

Abstract We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB−). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB− oocytes (43.41±2.54 vs. 22.74±1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB− blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB− blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB− blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB− blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC. PMID:25826727

Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

PubMed Central

Srirattana, Kanokwan; St. John, Justin C.

2017-01-01

The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA. PMID:28500053

Dual effects of fluoxetine on mouse early embryonic development.

PubMed

Kim, Chang-Woon; Choe, Changyong; Kim, Eun-Jin; Lee, Jae-Ik; Yoon, Sook-Young; Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee; Kang, Dawon

2012-11-15

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50μM) for different durations. When late 2-cells were incubated with 5μM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5μM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Copyright © 2012 Elsevier Inc. All rights reserved.

Acute dietary zinc deficiency before conception compromises oocyte epigenetic programming and disrupts embryonic development.

PubMed

Tian, X; Diaz, F J

2013-04-01

Recent findings show that zinc is an important factor necessary for regulating the meiotic cell cycle and ovulation. However, the role of zinc in promoting oocyte quality and developmental potential is not known. Using an in vivo model of acute dietary zinc deficiency, we show that feeding a zinc deficient diet (ZDD) for 3-5 days before ovulation (preconception) dramatically disrupts oocyte chromatin methylation and preimplantation development. There was a dramatic decrease in histone H3K4 trimethylation and global DNA methylation in zinc deficient oocytes. Moreover, there was a 3-20 fold increase in transcript abundance of repetitive elements (Iap, Line1, Sineb1, Sineb2), but a decrease in Gdf9, Zp3 and Figla mRNA. Only 53% and 8% of mature eggs reached the 2-cell stage after IVF in animals receiving a 3 and 5 days ZDD, respectively, while a 5 day ZDD in vivo reduced the proportion of 2-cells to 49%. In vivo fertilized 2-cell embryos cultured in vitro formed fewer (38%) blastocysts compared to control embryos (74%). Likewise, fewer blastocyst and expanded blastocyst were collected from the reproductive tract of zinc deficient animals on day 3.5 of pregnancy. This could be due to a decrease in Igf2 and H19 mRNA in ZDD blastocyst. Supplementation with a methyl donor (SAM) during IVM restored histone H3K4me3 and doubled the IVF success rate from 17% to 43% in oocytes from zinc deficient animals. Thus, the terminal period of oocyte development is extremely sensitive to perturbation in dietary zinc availability. Copyright © 2013 Elsevier Inc. All rights reserved.

[Electroacupuncture Combined with Clomiphene Promotes Pregnancy and Blastocyst Implantation Possibly by Up-regulating Expression of Insulin Receptor and Insulin Receptor Substrate 1 Proteins in Endometrium in Rats with PCOS].

PubMed

Lai, Mao-Hua; Ma, Hong-Xia; Song, Xing-Hua

2016-10-25

To observe the effect of electroacupuncture (EA) intervention combined with clomiphene critate (CC) on the blastocyst implantation and pregnancy rate and expression of insulin receptor (INSR) and insulin receptor substrate 1 (IRS 1) proteins in the endometrium in rats with polycystic ovary syndrome (PCOS), so as to reveal its mechanisms underlying improvement of PCOS. One hundred and twenty-five female SD rats were randomly divided into normal control, PCOS model, medication (CC), EA and EA+CC groups ( n =25 in each group, 15 for checking blastocyst implantation, and 10 for Western blot). The PCOS model was established by subcutaneous injection of Dehydroepiandrosterone (DHEA) and fed with high-fat diet. Rats of the normal control group were treated by subcutaneous injection of sesame oil and fed with the normal forage. EA stimulation was applied to "Zhongwan" (CV 12), "Guanyuan" (CV 4) and bilateral "Tianshu" (ST 25) for 30 min, 3 times a week, 5 weeks altogether. Rats of the CC and EA+CC groups were fed with CC (100 mg·kg -1 ·d -1 ) for 2 days after regular restriction (30 min, 3 times a week, 5 weeks altogether). The pregnancy was determined by vaginal smear tests and the number of blastocyst implantation determined by examination of the uterus after execution. The expression of INSR and IRS 1 proteins in the endometrium was detected by Western blot. The pregnancy rate and the number of blastocyst implantation were significantly lower in the model group than in the normal control group ( P <0.05), and remarkably increased after EA and EA+CC interventions ( P <0.05). The effects of EA+CC were obviously superior to those of simple EA and simple medication in raising the pregnancy rate and the number of blastocyst implantation ( P <0.05). No significant differences were found between the EA and CC groups in the pregnancy rate and the number of blastocyst implantation ( P >0.05). The relative expression levels of both INSR and IRS 1 proteins were markedly lower in

Morphology vs morphokinetics: a retrospective comparison of inter-observer and intra-observer agreement between embryologists on blastocysts with known implantation outcome.

PubMed

Adolfsson, Emma; Andershed, Anna Nowosad

2018-06-18

Our primary aim was to compare the morphology and morphokinetics on inter- and intra-observer agreement for blastocyst with known implantation outcome. Our secondary aim was to validate the morphokinetic parameters' ability to predict pregnancy using a previous published selection algorithm, and to compare this to standard morphology assessments. Two embryologists made independent blinded annotations on two occasions using time-lapse images and morphology evaluations using the Gardner Schoolcraft criteria of 99 blastocysts with known implantation outcome. Inter- and intra-observer agreement was calculated and compared using the two methods. The embryos were grouped based on their morphological score, and on their morphokinetic class using a previous published selection algorithm. The implantation rates for each group was calculated and compared. There was moderate agreement for morphology, with agreement on the same embryo score in 55 of 99 cases. The highest agreement rate was found for expansion grade, followed by trophectoderm and inner cell mass. Correlation with pregnancy was inconclusive. For morphokinetics, almost perfect agreement was found for early and late embryo development events, and strong agreement for day-2 and day-3 events. When applying the selection algorithm, the embryo distributions were uneven, and correlation to pregnancy was inconclusive. Time-lapse annotation is consistent and accurate, but our external validation of a previously published selection algorithm was unsuccessful.

The effect of air bubble position after blastocyst transfer on pregnancy rates in IVF cycles.

PubMed

Friedman, Brooke E; Lathi, Ruth B; Henne, Melinda B; Fisher, Stephanie L; Milki, Amin A

2011-03-01

To investigate the relationship between air bubble position after blastocyst transfer (BT) and pregnancy rates (PRs). Retrospective cohort study. University-based infertility center. Three hundred fifteen consecutive nondonor BTs by a single provider. Catheters were loaded with 25 μL of culture media, 20 μL of air, 25 μL of media containing the blastocysts, 20 μL of air, and a small amount of additional media. The distance from the air bubble to the fundus, as seen on abdominal ultrasound examination, was measured at the time of transfer. Air bubble location was categorized as <10 mm, 10-20 mm, and >20 mm from the fundus. Clinical pregnancy rate. After controlling for age, parity, FSH and frozen transfers, and accounting for repeated cycles per patient, the PRs for both the >20-mm (38.3%) and the 10-20-mm (42.0%) from the fundus group were significantly reduced compared with the group in which the bubble was <10 mm from the fundus (62.5%). This study is the first to suggest that BT closer to the fundus is associated with higher PR. Although no ectopic pregnancies occurred in the <10-mm group, this outcome should be monitored closely in larger studies. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts

PubMed Central

Jiang, Zongliang; Harrington, Patrick; Zhang, Ming; Marjani, Sadie L.; Park, Joonghoon; Kuo, Lynn; Pribenszky, Csaba; Tian, Xiuchun (Cindy)

2016-01-01

High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. PMID:26883277

Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations.

PubMed

Walker, S K; Hill, J L; Kleemann, D O; Nancarrow, C D

1996-09-01

The effects of supplementing synthetic oviductal fluid (SOF) with amino acids, at oviductal fluid concentrations, on the development of ovine in vitro-matured/in vitro-fertilized embryos was examined in three experiments. In the first, embryo development in SOF, SOF + 2% human serum (HS), SOF + 20% HS, and SOF + BSA, with and without amino acid supplementation, was examined. Development of zygotes to the blastocyst and hatching blastocyst stages was highest in medium containing 20% HS (64.8% and 54.4%, respectively) irrespective of amino acid supplementation. However, supplementation was significantly beneficial in all other media, with up to 42.1% of zygotes developing into hatching blastocysts. In these media, supplementation also significantly increased the mean number of nuclei per newly formed blastocyst (up to a mean of 70.8) and reduced the time during which blastocysts formed. Experiment 2 was an examination of the effect on embryo development of three amino acid preparations (oviduct amino acid concentrations vs. Eagle's Basal Medium (BME) essential + Minimum Essential Medium (MEM) nonessential vs. MEM essential + MEM nonessential concentrations) and the presence or absence of BSA. Both the amino acid and BSA treatments significantly influenced the percentage of zygotes that developed to the hatching blastocyst stage but not to the blastocyst stage. The preferred medium contained amino acids at oviductal fluid concentrations and BSA (54.5% hatching rate). The amino acid treatments did not significantly influence the mean number of nuclei per newly formed blastocyst, but the addition of BSA had a significant effect (70.7 +/- 1.14 vs. 75.7 +/- 1.13). In experiment 3, embryo development to Day 13 was examined after culture in SOF containing amino acids at oviductal fluid concentrations. Embryos were cultured in the presence of either BSA, polyvinyl alcohol (PVA), or no additional supplement and were transferred to recipient ewes on either Day 0 (after in

Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

PubMed

Mio, Yasuyuki; Maeda, Kazuo

2008-12-01

The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.

The influence of female age on the cumulative live-birth rate of fresh cycles and subsequent frozen cycles using vitrified blastocysts in hyper-responders.

PubMed

Wu, Cheng-Hsuan; Lee, Tsung-Hsien; Chen, Hsiu-Hui; Chen, Chung-I; Huang, Chun-Chia; Lee, Maw-Sheng

2015-10-01

The aim of this research was to study the influence of female age on the cumulative live-birth rate of fresh and subsequent frozen cycles using vitrified blastocysts of the same cohort in hyper-responders. This was a retrospective study of 1137 infertile women undergoing their first in vitro fertilization treatment between 2006 and 2013. The main outcome measure was cumulative live births among the fresh and all vitrified blastocyst transfers combined after the same stimulation cycle. The results were also analyzed according to age (i.e., <35 years, 35-39 years, and ≥ 40 years). The mean number of retrieved oocytes was 19.9 ± 8.5 oocytes. The cumulative pregnancy rate was 89.2% and the cumulative live-birth rate was 73.3%. The cumulative live-birth rate declined from 73.9% for women younger than 35 years old to 67.3% for women 35-39 years old to 57.9% for women 40 years or older. Combined fresh and vitrified blastocyst transfer cycles can result in a high cumulative live-birth rate. The cumulative live-birth rates among older women are lower than the rates among younger women when autologous oocytes are used. Copyright © 2015. Published by Elsevier B.V.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

PubMed Central

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

PubMed

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Pretreatment of bovine sperm with dithiobutylamine (DTBA) significantly improves embryo development after ICSI

PubMed Central

SUTTIROJPATTANA, Tayita; SOMFAI, Tamas; MATOBA, Satoko; NAGAI, Takashi; PARNPAI, Rangsun; GESHI, Masaya

2016-01-01

We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work. PMID:27523189

Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

PubMed

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.

Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres.

PubMed

Saito, S; Niemann, H

1991-05-01

The effects of extracellular matrices and growth factors on the development of isolated blastomeres derived from intact 4-, 8-, and 16-cell porcine embryos (termed, respectively, 1/4, 1/8, and 1/16 blastomeres) were investigated in vitro and in vivo. Blastomeres were incubated in extracellular matrix components fibronectin (FIN) or swine skin gelatin (SSG)-precoated culture dishes containing either modified Krebs' Ringer Bicarbonate solution (mKRB) supplemented with 10% heat-inactivated lamb serum, or Hanks' solution supplemented with 10% heat-inactivated newborn calf serum (NBCS) or Waymouth medium supplemented with 10% NBCS or in noncoated dishes in mKRB supplemented with either insulin (10, 100, or 1,000 micrograms/ml), transferrin (10, 100, or 1,000 micrograms/ml), or cAMP (0.2 or 2.0 micrograms/ml). Cultures observed at 24-h intervals and morphological development was recorded. Blastomeres were classified into three categories according to their morphology: (1) regular blastocysts, (2) trophectodermal vesicles, or (3) no development. After 96 h, culture was determined; the overall diameter of the blastocysts was determined and the nuclei were counted. Blastomeres/blastocysts did not adhere to the bottom of the culture dishes coated with extracellular matrices. Blastocyst formation rate was highest when FIN/mKRB was used and reached 44.3%, 41.8%, and 36.5% for 1/4, 1/8, and 1/16 blastomeres, respectively. The respective blastocysts contained an average of 31.2 +/- 5.8, 58.2 +/- 8.4, and 18.5 +/- 3.5 nuclei and had an overall diameter of 250.0 +/- 10.1, 235.0 +/- 12.8, and 172.5 +/- 13.7 microns, 1/8 blastomeres displayed a better (p less than 0.05) growth rate than 1/4 and 1/16 blastomeres, and 1/8 blastomeres in FIN/mKRB grew better (p less than 0.01) when cultured in an open system than in a microdrop under oil (35.5% vs. 5.0% blastocysts). Neither cAMP nor transferrin had a significant stimulating effect on blastocyst development of 1/8 blastomeres when m

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

PubMed Central

Rodríguez, Lleretny; Navarrete, Felipe I.; Tovar, Heribelt; Cox, José F.

2008-01-01

Purpose In order to simplify cloning, a new method that does not require micromanipulators was used. We aimed to evaluate the developmental potential of two bovine cell lines upon cloning. Materials and methods In vitro matured bovine oocytes, were released from zona pellucida, enucleated, fused to foetal or adult somatic donor cells. The reconstructed embryos were reprogrammed, activated and cultured until blastocyst stage. No micromanipulators were used. Blastocyst rate and quality was scored. Some expanded (d7) blastocysts were transferred to recipient cattle and collected back at d17 to assess elongation. Results High developmental potential in vitro of cloned embryos to expanded (d7) blastocysts was achieved (52.6%). In one cell line, 65.7% of blastocysts was scored. Most blastocysts (87.4%) were graded as excellent. In vivo development to elongation (day-17) in temporary recipient cows also showed a high developmental potential (11/18 transferred blastocysts elongated). Conclusions Hand-made cloning is an efficient alternative for cloning in cattle. PMID:18205035

Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender.

PubMed

Ebner, Thomas; Tritscher, Katja; Mayer, Richard B; Oppelt, Peter; Duba, Hans-Christoph; Maurer, Maria; Schappacher-Tilp, Gudrun; Petek, Erwin; Shebl, Omar

2016-01-01

Prolonged in vitro culture is thought to affect pre- and postnatal development of the embryo. This prospective study was set up to determine whether quality/size of inner cell mass (ICM) (from which the fetus ultimately develops) and trophectoderm (TE) (from which the placenta ultimately develops) is reflected in birth and placental weight, healthy live-birth rate, and gender after fresh and frozen single blastocyst transfer. In 225 patients, qualitative scoring of blastocysts was done according to the criteria expansion, ICM, and TE appearance. In parallel, all three parameters were quantified semi-automatically. TE quality and cell number were the only parameters that predicted treatment outcome. In detail, pregnancies that continued on to a live birth could be distinguished from those pregnancies that aborted on the basis of TE grade and cell number. Male blastocysts had a 2.53 higher chance of showing TE of quality A compared to female ones. There was no correlation between the appearance of both cell lineages and birth or placental weight, respectively. The presented correlation of TE with outcome indicates that TE scoring could replace ICM scoring in terms of priority. This would automatically require a rethinking process in terms of blastocyst selection and cryopreservation strategy.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

PubMed

Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

2008-03-01

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

PubMed

Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

The expression of fibroblast growth factor receptors during early bovine conceptus development and pharmacological analysis of their actions on trophoblast growth in vitro.

PubMed

Ozawa, Manabu; Yang, Qi-En; Ealy, Alan D

2013-02-01

The overall aim of this work was to examine the expression profiles for fibroblast growth factor receptors (FGFRs) and describe their biological importance during bovine pre- and peri-implantation conceptus development. FGFR1 and FGFR2 mRNAs were detected at 1-, 2-, 8-cell, morula and blastocyst stages whereas FGFR3 and FGFR4 mRNAs were detected after the 8-cell stage but not earlier. The abundance of FGFR1, FGFR3, and FGFR4 mRNAs increased at the morula and blastocyst stages. Immunofluorescence microscopy detected FGFR2 and FGFR4 exclusively in trophoblast cells whereas FGFR1 and FGFR3 were detected in both trophoblast cells and inner cell mass in blastocysts. Neither transcripts for FGF10 nor its receptor (FGFR2b) were temporally related to interferon τ (IFNT) transcript profile during peri- and postimplantation bovine conceptus development. A series of studies used a chemical inhibitor of FGFR kinase function (PD173074) to examine FGFR activation requirements during bovine embryo development. Exposing embryos to the inhibitor (1 μM) beginning on day 5 post-fertilization did not alter the percentage of embryos that developed into blastocysts or blastocyst cell numbers. The inhibitor did not alter the abundance of CDX2 mRNA but decreased (P<0.05) the relative abundance of IFNT mRNA in blastocysts. Exposing blastocysts to the inhibitor from days 8 to 11 post-fertilization reduced (P<0.05) the percentage of blastocysts that formed outgrowths after transfer to Matrigel-coated plates. In conclusion, each FGFR was detected in bovine embryos, and FGFR activation is needed to maximize IFNT expression and permit outgrowth formation.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

PubMed

Loi, Pasqualino; Galli, Cesare; Lazzari, Giovanna; Matsukawa, Kazutsugu; Fulka, Josef; Goeritz, Frank; Hildebrandt, Thomas B

2018-04-13

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

[Effect of phytohemagglutinin (PHA) from Yunnan white kidney bean on development of mouse embryos].

PubMed

Zhang, Lifen; Wang, Changmei; Yang, Mingjie; Zhang, Tian; Wang, Minkang

2011-06-01

To study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development. In experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage. Low concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death. Depending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes.

PubMed

Do, L T K; Namula, Z; Luu, V V; Sato, Y; Taniguchi, M; Isobe, T; Kikuchi, K; Otoi, T

2014-04-01

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation. © 2014 Blackwell Verlag GmbH.

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium.

PubMed

Grisart, B; Massip, A; Dessy, F

1994-07-01

Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three-four-cell, five-eight-cell, 9-16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11-62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula-blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three-four-cell and five-eight-cell), the higher the probability that they developed into morula-blastocyst: 70% of the embryos reaching the two-cell stage before 30-31 h after insemination developed into morula-blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel aspects of endometrial function: a biological sensor of embryo quality and driver of pregnancy success.

PubMed

Sandra, Olivier; Mansouri-Attia, Nadéra; Lea, Richard G

2011-01-01

Successful pregnancy depends on complex biological processes that are regulated temporally and spatially throughout gestation. The molecular basis of these processes have been examined in relation to gamete quality, early blastocyst development and placental function, and data have been generated showing perturbations of these developmental stages by environmental insults or embryo biotechnologies. The developmental period falling between the entry of the blastocyst into the uterine cavity to implantation has also been examined in terms of the biological function of the endometrium. Indeed several mechanisms underlying uterine receptivity, controlled by maternal factors, and the maternal recognition of pregnancy, requiring conceptus-produced signals, have been clarified. Nevertheless, recent data based on experimental perturbations have unveiled unexpected biological properties of the endometrium (sensor/driver) that make this tissue a dynamic and reactive entity. Persistent or transient modifications in organisation and functionality of the endometrium can dramatically affect pre-implantation embryo trajectory through epigenetic alterations with lasting consequences on later stages of pregnancy, including placentation, fetal development, pregnancy outcome and post-natal health. Developing diagnostic and prognostic tools based on endometrial factors may enable the assessment of maternal reproductive capacity and/or the developmental potential of the embryo, particularly when assisted reproductive technologies are applied.

Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos.

PubMed

Yao, Tatsuma; Suzuki, Rie; Furuta, Natsuki; Suzuki, Yuka; Kabe, Kyoko; Tokoro, Mikiko; Sugawara, Atsushi; Yajima, Akira; Nagasawa, Tomohiro; Matoba, Satoko; Yamagata, Kazuo; Sugimura, Satoshi

2018-05-10

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

The loss of imprinted DNA methylation in mouse blastocysts is inflicted to a similar extent by in vitro follicle culture and ovulation induction.

PubMed

Saenz-de-Juano, M D; Billooye, K; Smitz, J; Anckaert, E

2016-06-01

Does in vitro follicle culture (IFC) have an effect on maintenance of imprinted DNA methylation in preimplantation mouse embryos? We report similar alterations in the methylation pattern of H19 imprinted maternally expressed transcript (H19), small nuclear ribonucleoprotein polypeptide N (Snrpn) and mesoderm specific transcript (Mest) imprinted genes in mouse blastocysts obtained after ovulation induction and IFC. Furthermore, we observed no differences in the gene expression of maternal effect proteins related with imprinting maintenance between superovulated in vivo grown or IFC oocytes. Assisted reproductive technology is associated with adverse post-natal outcomes such as increased risk of premature birth, altered birthweight, congenital anomalies and genomic imprinting syndromes in human and in animal models. Previous studies have shown that ovulation induction allowed normal imprinting establishment in mouse oocytes, but interfered with imprinting maintenance during preimplantation . Normal imprinting establishment was also observed in mouse oocytes derived from a standardized IFC from the early pre-antral follicle stage. The methylation profiles of differentially methylated regions (DMRs) of three key imprinted genes (H19, Snrpn and Mest) were compared at hatched blastocyst stage between embryos obtained from IFC or superovulated oocytes, each subjected to IVF and preimplantation in vitro culture (IVC); in non-manipulated in vivo produced late blastocyst (control) and in in vivo produced 2-cell embryos that were in vitro cultured until the hatched blastocyst stage (to assess the effect of IVC). Two different mice strains (Mus musculus C57BL/6J X CBA/Ca and Mus musculus B6 (CAST7)) were used to discriminate between maternal and paternal alleles of imprinted genes. Additionally, a limiting-dilution bisulfite-sequencing technique was carried out on individual embryos in order to avoid amplification bias. To assess whether IFC and ovulation induction

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage.

PubMed

Yang, Yu; Bolnick, Alan; Shamir, Alexandra; Abdulhasan, Mohammed; Li, Quanwen; Parker, G C; Puscheck, Elizabeth E; Rappolee, D A

2017-08-01

Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.

Poor Centrosomal Function of Cat Testicular Spermatozoa Impairs Embryo Development In Vitro after Intracytoplasmic Sperm Injection1

PubMed Central

Comizzoli, Pierre; Wildt, David E.; Pukazhenthi, Budhan S.

2007-01-01

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h post-activation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages. PMID:16687647

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation.

PubMed

Eswari, S; Sai Kumar, G; Sharma, G Taru

2013-05-01

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

PubMed

Sakagami, N; Nishida, K; Akiyama, K; Abe, H; Hoshi, H; Suzuki, C; Yoshioka, K

2015-01-01

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥ 0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (< 0.59) were transferred, became pregnant. These results suggest that the viability of in vivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos.

PubMed

Cuello, C; Gomis, J; Almiñana, C; Maside, C; Sanchez-Osorio, J; Gil, M A; Sánchez, A; Parrilla, I; Vazquez, J M; Roca, J; Martinez, E A

2013-01-30

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus-oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10μM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification. Copyright © 2012 Elsevier B.V. All rights reserved.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange

PubMed Central

LOI, Pasqualino; GALLI, Cesare; LAZZARI, Giovanna; MATSUKAWA, Kazutsugu; FULKA, Josef; GOERITZ, Frank; HILDEBRANDT, Thomas B.

2018-01-01

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation. PMID:29445070

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

PubMed

George, Aman; Sharma, Ruchi; Singh, Karn P; Panda, Sudeepta K; Singla, Suresh K; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S

2011-06-01

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes.

PubMed

Diaw, Mouhamadou; Salgado, Renato M; Canesin, Heloísa S; Gridley, Nell; Hinrichs, Katrin

2018-04-15

Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at cold (∼4 °C) temperatures. If so, this might allow longer holding periods that would ease shipping requirements. In this study, we compared oocyte maturation rates, as well as cleavage and blastocyst rates after ICSI, for COCs held at either room or cold temperatures overnight before the onset of in vitro maturation. In Exp. 1, COCs were shipped overnight in a commercial embryo holding medium, ViGRO (Vg), in insulated containers designed to hold at either room temperature (RT, ∼22 °C) or cold temperatures (Cold, ∼7 °C). Subsequent rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (39%, 90% and 41%, respectively) than in the Cold treatment (23%, 60% and 17%, respectively, P < .05). In Exp. 2, we compared Vg medium with a second commercial embryo holding medium, SYNGRO (Sy). There was no significant difference between Vg and Sy groups in any evaluated parameter within either RT or Cold treatments. Within each medium group and for both media combined, the rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (42%, 81% and 42%, respectively for the combined media) than in the Cold treatment (29%, 54% and 10%, respectively for the combined media, P < .05). We conclude that shipment of immature equine COCs at cold temperatures (∼7 °C) is

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

PubMed

Ottolini, Christian S; Kitchen, John; Xanthopoulou, Leoni; Gordon, Tony; Summers, Michael C; Handyside, Alan H

2017-08-29

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

PubMed

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.

In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

PubMed

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.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

PubMed

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

. 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

Endometrial thickness as a predictor of the reproductive outcomes in fresh and frozen embryo transfer cycles: A retrospective cohort study of 1512 IVF cycles with morphologically good-quality blastocyst.

PubMed

Zhang, Tao; Li, Zhou; Ren, Xinling; Huang, Bo; Zhu, Guijin; Yang, Wei; Jin, Lei

2018-01-01

To evaluate the relationship between endometrial thickness during fresh in vitro fertilization (IVF) cycles and the clinical outcomes of subsequent frozen embryo transfer (FET) cycles.FET cycles using at least one morphological good-quality blastocyst conducted between 2012 and 2013 at a university-based reproductive center were reviewed retrospectively. Endometrial ultrasonographic characteristics were recorded both on the oocyte retrieval day and on the day of progesterone supplementation in FET cycles. Clinical pregnancy rate, spontaneous abortion rate, and live birth rate were analyzed.One thousand five hundred twelve FET cycles was included. The results showed that significant difference in endometrial thickness on day of oocyte retrieval (P = .03) was observed between the live birth group (n = 844) and no live birth group (n = 668), while no significant difference in FET endometrial thickness was found (P = .261) between the live birth group and no live birth group. For endometrial thickness on oocyte retrieval day, clinical pregnancy rate ranged from 50.0% among patients with an endometrial thickness of ≤6 mm to 84.2% among patients with an endometrial thickness of >16 mm, with live birth rate from 33.3% to 63.2%. Multiple logistic regression analysis of factors related to live birth indicated endometrial thickness on oocyte retrieval day was associated with improved live birth rate (OR was 1.069, 95% CI: 1.011-1.130, P = .019), while FET endometrial thickness did not contribute significantly to pregnancy outcomes following FET cycles. The ROC curves revealed the cut-off points of endometrial thickness on oocyte retrieval day was 8.75 mm for live birth.Endometrial thickness during fresh IVF cycles was a better predictor of endometrial receptivity in subsequent FET cycles than FET cycle endometrial thickness. For those females with thin endometrium in fresh cycles, additional estradiol stimulation might be helpful for adequate

Antiestrogenic and Anti-Inflammatory Potential of n-Hexane Fraction of Vitex negundo Linn Leaf Extract: A Probable Mechanism for Blastocyst Implantation Failure in Mus musculus.

PubMed

Jivrajani, Mehul; Ravat, Nirav; Anandjiwala, Sheetal; Nivsarkar, Manish

2014-01-01

The anti-implantation potential of different fractions of Vitex negundo Linn leaf extract was evaluated in female Swiss Albino mice. Animals from different groups were dosed orally either with 0.2% agar (vehicle) or with fractions of V. negundo leaf extract (n-hexane, chloroform, n-butanol, and remnant fractions) at 10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant females from each group were sacrificed on different days of pregnancy (n = 6), and uterus was excised and used for estimation of lipid peroxidation and assay of superoxide dismutase activity as a marker for blastocyst implantation. Animals treated with n-hexane fraction showed altered level of superoxide anion radical and superoxide dismutase activity as compared to control animals. The probable mechanism by which this extract exhibits inhibition of blastocyst implantation is through the anti-inflammatory and antiestrogenic potential.

Berberine impairs embryonic development in vitro and in vivo through oxidative stress-mediated apoptotic processes.

PubMed

Huang, Chien-Hsun; Huang, Zi-Wei; Ho, Feng-Ming; Chan, Wen-Hsiung

2018-03-01

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H 2 O 2 -induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine-triggering or preventing apoptosis-has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5-10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine-induced injury of mouse blastocysts appeared to be attributable to oxidative stress-triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo. © 2017 Wiley Periodicals, Inc.

HCG administration after endogenous LH rise negatively influences pregnancy rate in modified natural cycle for frozen-thawed euploid blastocyst transfer: a pilot study.

PubMed

Litwicka, Katarzyna; Mencacci, Cecilia; Arrivi, Cristiana; Varricchio, Maria Teresa; Caragia, Alina; Minasi, Maria Giulia; Greco, Ermanno

2018-03-01

The aim of the present study was to evaluate whether in a modified natural cycle (modified-NC) for a frozen-thawed single euploid blastocyst transfer, a critical LH value, above which human chorionic gonadotropin (hCG) administration should be avoided, may be defined. One hundred and sixty-seven patients underwent modified natural cycle in order to transfer a single frozen-thawed euploid blastocyst. All embryos were obtained by intracytoplasmic sperm injection and were biopsied at the blastocyst stage and analyzed by means of array comparative genomic hybridization (aCGH). Ovulation was induced using 10.000 IU hCG when the mean follicle diameter was at least of 17 mm, independently from LH values. The primary end points were the hCG-positive test and clinical pregnancy. The interim analysis showed that LH value ≥ 13 mIU/ml on the day of hCG injection may negatively influence the clinical results, suggesting that in this condition, it should be advisable waiting for spontaneous ovulation. Among patients who received hCG for ovulation induction, the hCG-positive test and clinical pregnancy rates in modified-NC were significantly lower in cycles with LH ≥ 13 mIU/ml in respect to those with LH < 13 mIU/ml (45.4 vs 73.3 and 36.4 vs 65.9%, in LH ≥ 13 and LH < 13 groups, respectively). In patients with LH value ≥ 13 mIU/ml, hCG administration led to significantly lower rates of hCG-positive test (45.4 vs 74.5% in hCG administration and spontaneous ovulation groups, respectively) and clinical pregnancy (36.4 vs 64.7% in hCG administration and spontaneous ovulation groups, respectively). The baseline patient characteristics were comparable in all groups. The findings of this study highlight that LH elevation ≥ 13 mIU/ml prior to hCG administration may negatively affect clinical pregnancy rates in modified-NC for single euploid blastocyst transfer. The LH determination should be routinely performed during follicular monitoring. In the

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization.

PubMed

Osychenko, Alina A; Zalessky, Alexandr D; Kostrov, Andrey N; Ryabova, Anastasia V; Krivokharchenko, Alexander S; Nadtochenko, Viktor A

2017-12-01

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization

NASA Astrophysics Data System (ADS)

Osychenko, Alina A.; Zalessky, Alexandr D.; Kostrov, Andrey N.; Ryabova, Anastasia V.; Krivokharchenko, Alexander S.; Nadtochenko, Viktor A.

2017-12-01

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion.

Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj

Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20 mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophinmore » (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5 mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20 mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome. - Highlights: • Methyl parathion induces severe cytoplasmic abnormalities in oocytes. • Inhibits nuclear maturation and spindle damage • Poor blastocyst quality and

Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage.

PubMed

Fathi, Mohamed; Moawad, Adel R; Badr, Magdy R

2018-01-01

Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop. Viable oocytes were cultured in vitro for 30 h. Matured oocytes were fertilized with epididymal spermatozoa and then cultured in vitro in modified KSOMaa medium for 7 days. Survival and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%, respectively) than those exposed to VS1 (68.2% and 48.0%, respectively) and VS2 (79.3% and 56.9%, respectively). Although recovery rates were significantly lower (P ≤ 0.05) in SSV and Cryotop vitrified oocytes (66.9% to 71.1%) than those vitrified by straws with VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in the SSV and Cryotop groups (90.7% to 94.8%) than in the straw and OPS groups (68.2% to 86.5%). Among vitrified groups, maturation and fertilization rates were the highest in the Cryotop-VS2 group (51.8% and 39.2%, respectively). These values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and Cryotop groups than in straws. No significant differences were observed in these parameters between the Cryotop and control groups. We report for the first time that dromedary oocytes vitrified at the GV-stage have the ability to be matured, fertilized and subsequently develop in vitro to produce blastocysts at frequencies comparable to those obtained using fresh oocytes.

Pregnancy and delivery after in vitro maturation of naked ICSI-GV oocytes with GH and transfer of a frozen thawed blastocyst: case report.

PubMed

Menezo, Yves J R; Nicollet, Bernard; Rollet, Jacques; Hazout, André

2006-01-01

To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37 degrees C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

PubMed Central

Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

2016-01-01

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture.

PubMed

Keskintepe, L; Morton, P C; Smith, S E; Tucker, M J; Simplicio, A A; Brackett, B G

1997-08-01

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection.

PubMed

Choi, Young-Ho; Gibbons, John R; Canesin, Heloísa S; Hinrichs, Katrin

2016-10-15

Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant differences in rates of cleavage or blastocyst development (20%-31%). However, the proportion of blastocysts that developed on Day 7 for the added-zinc treatments was significantly higher than that for the control treatment (45% vs. 8%). In experiment 3, we tested whether use of high-pH medium (pH 8.0-8.4) during ICSI procedures would improve blastocyst rate when sperm with low cleavage rates after ICSI was used. When high-pH conditions were used for sperm preparation and also for the first 2 hours of incubation of injected oocytes after ICSI, the cleavage rate was unaffected but no blastocysts developed (0% vs. 24% for control). When high-pH conditions were used for sperm preparation only, the blastocyst rate was 37%. This was repeated using sperm from a second stallion; there was no significant difference in cleavage or blastocyst rates between sperm preparation in high pH vs. control medium. These findings add to our knowledge of factors affecting in vitro production of equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

PubMed Central

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

Selection of competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for patients undergoing preimplantation genetic screening: a prospective study with sibling oocytes

PubMed Central

2014-01-01

Background Recent advances in time-lapse monitoring in IVF treatment have provided new morphokinetic markers for embryonic competence. However, there is still very limited information about the relationship between morphokinetic parameters, chromosomal compositions and implantation potential. Accordingly, this study aimed at investigating the effects of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing on pregnancy and implantation outcomes for patients undergoing preimplantation genetic screening (PGS). Methods A total of 1163 metaphase II (MII) oocytes were retrieved from 138 PGS patients at a mean age of 36.6 ± 2.4 years. These sibling MII oocytes were then randomized into two groups after ICSI: 1) Group A, oocytes (n = 582) were cultured in the time-lapse system and 2) Group B, oocytes (n = 581) were cultured in the conventional incubator. For both groups, whole genomic amplification and array CGH testing were performed after trophectoderm biopsy on day 5. One to two euploid blastocysts within the most predictive morphokinetic parameters (Group A) or with the best morphological grade available (Group B) were selected for transfer to individual patients on day 6. Ongoing pregnancy and implantation rates were compared between the two groups. Results There were significant differences in clinical pregnancy rates between Group A and Group B (71.1% vs. 45.9%, respectively, p = 0.037). The observed implantation rate per embryo transfer significantly increased in Group A compared to Group B (66.2% vs. 42.4%, respectively, p = 0.011). Moreover, a significant increase in ongoing pregnancy rates was also observed in Group A compared to Group B (68.9% vs. 40.5%. respectively, p = 0.019). However, there was no significant difference in miscarriage rate between the time-lapse system and the conventional incubator (3.1% vs. 11.8%, respectively, p = 0.273). Conclusions This is the first prospective investigation using

Reliability and agreement on embryo assessment: 5 years of an external quality control programme.

PubMed

Martínez-Granados, Luis; Serrano, María; González-Utor, Antonio; Ortiz, Nereyda; Badajoz, Vicente; López-Regalado, María Luisa; Boada, Montserrat; Castilla, Jose A

2018-03-01

An external quality-control programme for morphology-based embryo quality assessment, incorporating a standardized embryo grading scheme, was evaluated over a period of 5 years to determine levels of inter-observer reliability and agreement between practising clinical embryologists at IVF centres and the opinions of a panel of experts. Following Guidelines for Reporting Reliability and Agreement Studies, the Gwet index and proportion of positive (Ppos) and negative agreement were calculated. For embryo morphology assessment, a substantial degree of reliability was measured between the centres and the panel of experts (Gwet index: 0.76; 95% CI 0.70 to 0.84). The agreement was higher for good- versus poor-quality embryos. When multinucleation or vacuoles were observed, low levels of reliability were obtained (Ppos: 0.56 and 0.43, respectively). In blastocysts, the characteristic that presented the largest discrepancy was that related to the inner cell mass. In decisions about the final disposition of the embryo, reliability between centre and the panel of experts was moderate (Gwet index: 0.51; 95% CI 0.41 to 0.60). In conclusion, the ability of clinical embryologists to evaluate the presence of multinucleation and vacuoles in the early cleavage embryo, and to determine the category of the inner cell mass in blastocysts, needs to be improved. Copyright © 2017 Reproductive Healthcare Ltd. All rights reserved.

Vitamin D deficiency and pregnancy rates in women undergoing single embryo, blastocyst stage, transfer (SET) for IVF/ICSI.

PubMed

Polyzos, Nikolaos P; Anckaert, Ellen; Guzman, Luis; Schiettecatte, Johan; Van Landuyt, Lisbet; Camus, Michel; Smitz, Johan; Tournaye, Herman

2014-09-01

What is the influence of vitamin D deficiency on pregnancy rates among women undergoing IVF/ICSI and Day 5 (blastocyst stage) single embryo transfer (SET)? Vitamin D deficiency results in significantly lower pregnancy rates in women undergoing single blastocyst transfer. Preliminary experiments have identified the presence of vitamin D receptors in the female reproductive system. However, results regarding the effect of vitamin D deficiency on clinical outcomes are conflicting. None of the previous studies adopted a SET strategy. Serum vitamin D concentration was measured retrospectively in patients who underwent SET on Day 5. Overall 368 consecutive infertile women treated within a period of 15 months were included in the study. All patients underwent ovarian stimulation for IVF/ICSI and Day 5 SET. Serum samples were obtained 7 days prior to embryo transfer and stored frozen at -20°C. Samples were collectively analyzed for their 25-OH vitamin D content. Vitamin D deficiency was defined as serum 25-OH vitamin D levels <20 ng/ml in accordance with the Institute of Medicine and the Endocrine Society clinical practice guidelines. Clinical pregnancy rates were significantly lower in women with vitamin D deficiency compared with those with higher vitamin D values (41 versus 54%, P = 0.015).Logistic regression analysis was performed to identify whether vitamin D deficiency is independently associated with clinical pregnancy rates after controlling for 16 potential confounding factors. According to our results vitamin D deficiency was independently associated with lower clinical pregnancy rates, odds ratios [ORs (95% confidence interval (CI) 0.61 (0.39-0.95)] for vitamin D deficiency (deficient versus non-deficient women), P = 0.030. Finally, even when restricting our analysis to women undergoing elective SET (274 patients), vitamin D deficiency was again independently associated with pregnancy rates [OR (95% CI) 0.56 (0.33-0.93), P = 0.024]. Our results refer only to

The impact of histones linked to sperm chromatin on embryo development and ART outcome.

PubMed

Fournier, C; Labrune, E; Lornage, J; Soignon, G; Giscard d'Estaing, S; Guérin, J-F; Benchaib, M

2018-03-02

The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone-to-protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c-IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = -0.12, p < 0.05). Regarding the type of ART procedure (c-IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR<6%; p < 0.01) and 74.6% (HPR >26%; p < 0.01). The highest delivery rate (DR; 24.5%) was obtained for HPR within the range [6%; 26%]; DR was 21.9% for HPR<6% and 18.3% for HPR>26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy. © 2018 American Society of Andrology and European Academy of Andrology.

Ovulation, fertilization and preimplantation embryonic development in raccoon dogs (Nyctereutes procyonoides).

PubMed

Xu, Baozeng; Feng, Huai L

2017-01-01

A study involving 32 sexual mature females was conducted to characterize ovulation, fertilization and early embryonic development in vivo in raccoon dogs. Oocytes and embryos were collected from the oviducts and uteri, evaluated by stereomicroscopy. Ovulation occurred 25-32h after a female first accepted mounting, regardless of copulation, when the females were paired with a male in the same cage. Ovulated oocytes were at the primary stage. The number of ovulated eggs in females with or without mating was 9.96±2.65 and 9.00±1.92, respectively. Embryos at 2-4 cell, 8-16 cell, morula, blastocyst, and hatched blastocyst stage were observed at 29-73, 48-100, 98-126, 169-198 and 217-268h after first mating, respectively. Embryos were located in the oviduct prior to 4-cell stage and moved into the uterus after 16-cell stage. Embryos at different stages were often obtained from the same female. During the zygote underwent a series of cleavage divisions, the diameter of the embryo cell mass continuously increased through the 2-cell and 4-cell stage, then started to decrease and was the minimum size at the morula stage. At the blastocyst stage, embryos increased in volume, and finally developed into a hatching blastocyst with a thinner zona pellucida. This is the first full report of preimplantation embryonic development in the raccoon dog, which will facilitate the application of advanced assisted reproductive technology in canine species. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of reactive oxygen species levels in prepared culture media on embryo development: a comparison of two media.

PubMed

Shih, Ying-Fu; Lee, Tsung-Hsien; Liu, Chung-Hsien; Tsao, Hui-Mei; Huang, Chun-Chia; Lee, Maw-Sheng

2014-12-01

This study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos. This was an autocontrolled comparison study. A total of 159 patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinn's Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test. The QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media. The elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification. Copyright © 2014. Published by Elsevier B.V.

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

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.

Embryonic development in human oocytes fertilized by split insemination

PubMed Central

Kim, Myo Sun; Kim, Jayeon; Youm, Hye Won; Park, Jung Yeon; Choi, Hwa Young

2015-01-01

Objective To compare the laboratory outcomes of intracytoplasmic sperm injection (ICSI) and conventional insemination using sibling oocytes in poor prognosis IVF cycles where ICSI is not indicated. Methods Couples undergoing IVF with following conditions were enrolled: history of more than 3 years of unexplained infertility, history of ≥3 failed intrauterine insemination, leukocytospermia or wide variation in semen analysis, poor oocyte quality, or ≥50% of embryos had poor quality in previous IVF cycle(s). Couples with severe male factor requiring ICSI were excluded. Oocytes were randomly assigned to the conventional insemination (conventional group) or ICSI (ICSI group). Fertilization rate (FR), total fertilization failure, and embryonic development at day 3 and day 5 were assessed. Results A total of 309 mature oocytes from 37 IVF cycles (32 couples) were obtained: 161 were assigned to conventional group and 148 to ICSI group. FR was significantly higher in the ICSI group compared to the conventional group (90.5% vs. 72.7%, P<0.001). Total fertilization failure occurred in only one cycle in conventional group. On day 3, the percentage of cleavage stage embryos was higher in ICSI group however the difference was marginally significant (P=0.055). In 11 cycles in which day 5 culture was attempted, the percentage of blastocyst (per cleaved embryo) was significantly higher in the ICSI group than the conventional group (55.9% vs. 25.9%, P=0.029). Conclusion Higher FR and more blastocyst could be achieved by ICSI in specific circumstances. Fertilization method can be tailored accordingly to improve IVF outcomes. PMID:26023671

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Developmental expression of the Notch signaling pathway genes during mouse preimplantation development.

PubMed

Cormier, Sarah; Vandormael-Pournin, Sandrine; Babinet, Charles; Cohen-Tannoudji, Michel

2004-10-01

Notch signaling is an evolutionary conserved pathway involved in intercellular signaling and essential for proper cell fate choices during development. Thus, it could be involved in mouse preimplantation development where intercellular signaling plays a crucial role, particularly between the inner cell mass and the trophectoderm of the blastocyst. At their face value, the phenotypes observed when disrupting each of the four Notch genes known in the mouse do not support this view as none of them involves perturbation of preimplantation development. However this could be due to functional redundancy and/or maternal expression. As a first step to address this issue, we decided to examine the expression in early development of various genes known to participate in Notch signaling. Here, we report on the expression pattern of Notch1-4, Jagged1 (Jag1), Jag2, Delta-like1 (Dll-1), Dll-3, Dll-4, Rbpsuh, Deltex1(Dtx1)and Dtx2 genes during preimplantation development from unfertilized eggs until late blastocyst stage using a RT-PCR strategy. We show that Notch1, 2, Jag1-2, Dll-3, Rbpsuh and Dtx2 transcripts are expressed at all stages. Notch4 and Dll-4 mRNAs are synthesized from the 2-cell through to the hatched blastocyst stage. Notch3, Dll-1 and Dtx1exhibit a stage dependent expression as their mRNAs are detected in 2-cell embryos and in hatched blastocysts, but are absent or weakly detected at the morula stage. Finally, we show that all the above genes are expressed both in Embryonic and Trophoblast Stem cells (ES and TS cells, respectively). Our results suggest that the Notch pathway may be active during mouse preimplantation development.

Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

PubMed Central

Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

2011-01-01

Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

PubMed

Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

2009-09-01

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P<0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development.

PubMed

Santos, Elisa Caroline da Silva; Somfai, Tamas; Appeltant, Ruth; Dang-Nguyen, Thanh Quang; Noguchi, Junko; Kaneko, Hiroyuki; Kikuchi, Kazuhiro

2017-08-01

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts. © 2016 Japanese Society of Animal Science.

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

PubMed Central

de Semir, D.; Maurisse, R.; Du, F.; Xu, J.; Yang, X.; Illek, B.; Gruenert, D. C.

2013-01-01

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology. PMID:22234514

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation.

PubMed

Leppens-Luisier, G; Sakkas, D

1997-03-01

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.

Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography.

PubMed

Ugajin, Tomohisa; Terada, Yukihiro; Hasegawa, Hisataka; Velayo, Clarissa L; Nabeshima, Hiroshi; Yaegashi, Nobuo

2010-05-15

To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. An experimental laboratory of the university. We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

PubMed

Neira, J A; Tainturier, D; Peña, M A; Martal, J

2010-03-15

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

PubMed

Wei, Ruxue; Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

2016-11-01

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10 -7  M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Role of glucose in mouse preimplantation embryo development.

PubMed

Martin, K L; Leese, H J

1995-04-01

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca x C57BL/6) were cultured from the one- and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D + L-lactate, in the presence and absence of 1 mM glucose (M16 + G and M16 - G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16 - G were transferred to M16 + G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16 + G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16 - G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 +/- 2.5 [28] vs. 105 +/- 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16 - G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16 - G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16 + G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of peri-conception nutrition on embryo quality in the superovulated ewe.

PubMed

Kakar, M A; Maddocks, S; Lorimer, M F; Kleemann, D O; Rudiger, S R; Hartwich, K M; Walker, S K

2005-09-15

Evidence indicates that oocyte/embryo quality in the sheep is affected by nutrient status during the cycle of conception. This study aimed to determine, in the superovulated ewe, if there are stages during the peri-conception period (-18 days to +6 days relative to the day of ovulation [Day 0]) when quality is more likely to be influenced by nutrition. In Experiment 1, ewes were provided with either a 0.5 x maintenance (L), 1.0 x maintenance (M) or 1.5 x maintenance (H) diet (in terms of daily energy requirements) during the peri-conception period. Diet did not affect the mean ovulation rate (range: 15.4+/-1.47 to 16.1+/-1.55) nor the mean number of embryos collected per ewe (range: 10.9+/-2.05 to 12.4+/-1.82) but there was an increase (P<0.05) in the mean number of cells per blastocyst in the L diet (74.7+/-1.45) compared with either the M (66.4+/-1.29) or H (62.0+/-0.84) diets. This increase was due to an increase in the number of trophectoderm (Tr) cells, resulting in a shift (P<0.05) in the Tr:inner cell mass (ICM) cell ratio (range 0.69+/-0.03 to 0.73+/-0.04). In Experiment 2, six diets (HHH, MHH, MHL, MLH, MLL and LLL) were imposed during three 6-day periods commencing 12 days before and continuing until 6 days after ovulation. Although diet had minimal effect on the superovulatory response, both the mean number of cells per blastocyst and the Tr:ICM ratio were increased (P<0.05) when the L diet was provided after Day 0 (diets MHL, MLL and LLL). It is concluded that the ewe is able to respond to acute changes in nutrition imposed immediately after ovulation, resulting in changes in embryo development including cell lineage differentiation. The significance of these findings, in terms of fetal development, embryo-maternal signalling and the nutritional management of the ewe is discussed.

Melatonin protect the development of preimplantation mouse embryos from sodium fluoride-induced oxidative injury.

PubMed

Zhao, Jiamin; Fu, Beibei; Peng, Wei; Mao, Tingchao; Wu, Haibo; Zhang, Yong

2017-09-01

Recently study shows that melatonin can protect embryos from the culture environment oxidative stress. However, the protective effect of melatonin on the mouse development of preimplantation embryos under sodium fluoride (NaF) induced oxidative stress is still unclear. Here, we showed that exposure to NaF significantly increased the reactive oxygen species (ROS) level, decreased the blastocyst formation rates, and increased the fragmentation, apoptosis and retardation of blastocysts in the development of mouse preimplantation embryos. However, the protective of melatonin remarkable increased the of blastocyst formation rates, maintained mitochondrial function and total antioxidant capacity by clearing ROS. Importantly the data showed that melatonin improved the activity of enzymatic antioxidants, including glutathione(GSH), superoxide dismutase(SOD), and malonaldehyde (MDA), and increased the expression levels of antioxidative genes. Taken together, our results indicate that melatonin prevent NaF-induced oxidative damage to mouse preimplantation embryo through down regulation of ROS level, stabilization of mitochondrial function and modulation of the activity of antioxidases and antioxidant genes. Copyright © 2017 Elsevier B.V. All rights reserved.

ZINC INFLUENCES THE IN VITRO DEVELOPMENT OF PERI-IMPLANTATION MOUSE EMBRYOS

EPA Science Inventory

Background: For humans, it is estimated that over 70% of concepti are lost during early development. In culture, mouse peri-implantation embryos can mimic development from the blastocyst to the egg cylinder stage of development, a period during which implantation occurs in viv...

Cryo-survival, fertilization and early embryonic development of vitrified oocytes derived from mice of different reproductive age

PubMed Central

Yan, Jie; Suzuki, Joao; Yu, Xiaomin; Kan, Frederick W. K.

2010-01-01

Purpose To evaluate the effect of female reproductive age on oocyte cryo-survival, fertilization and the subsequent embryonic development following vitrification using the mouse model in order to address the question of how maternal reproductive age is related to fertility preservation. Methods Oocytes were collected from mice of different reproductive age: (1) 8–10 weeks, (2) 16–20 weeks, (3) 32–36 weeks, and (4) 44–48 weeks. Following vitrification and warming, the oocytes in each group were assessed for cryo-survival, fertilization and embryonic development as well as for the quality of blastocysts. Fresh oocytes without undergoing vitrification were used in each age group as controls. Results The mean number of oocytes retrieved following superovulation was found to reduce significantly (P < 0.05) in mice from 32–36 weeks of age (18.1 ± 8.5) compared with 8–10 weeks of age (26.8 ± 9.8) and 16–20 weeks of age (23.9 ± 4.2) respectively. The cryo-survival rate of oocytes was reduced significantly (P < 0.05) in mice of 44–48 weeks of age (90.4% ± 7.9) compared with the other 3 groups (98.8% ± 2.1, 98.0% ± 3.3 and 98.5% ± 2.2, respectively). The cleavage rate of vitrified oocytes declined significantly following the increase in maternal age in mice of 32–36 weeks of age (69.7% ± 20.8) forward (63.6% ± 9.2). However, no significant difference in the cleavage rate was found among the control groups of different maternal ages. The rate of embryo development to the blastocyst stage in the vitrified oocytes also significantly declined following the increase in maternal age (71.8% ± 8.8, 66.4% ± 10.7, 64.2% ± 17.4 and 4.1% ± 8.3 respectively). There were no such differences in the rates of embryo development to the blastocyst stage among the control groups following the increase in maternal age (75.9% ± 12.2, 79.5% ± 28.9, 70.2% ± 17.4 and 69.3% ± 19

Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

PubMed Central

Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

2012-01-01

Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring

Evidence Supporting a Functional Requirement of SMAD4 for Bovine Preimplantation Embryonic Development: A Potential Link to Embryotrophic Actions of Follistatin1

PubMed Central

Lee, Kyung-Bon; Zhang, Kun; Folger, Joseph K.; Knott, Jason G.; Smith, George W.

2014-01-01

ABSTRACT Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent. PMID:25031360

RBP-Jkappa-dependent notch signaling is dispensable for mouse early embryonic development.

PubMed

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-07-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

PubMed Central

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

Number of blastocysts biopsied as a predictive indicator to obtain at least one normal/balanced embryo following preimplantation genetic diagnosis with single nucleotide polymorphism microarray in translocation cases.

PubMed

Wang, Yi-Zi; Ding, Chen-Hui; Wang, Jing; Zeng, Yan-Hong; Zhou, Wen; Li, Rong; Zhou, Can-Quan; Deng, Ming-Fen; Xu, Yan-Wen

2017-01-01

The aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers. This blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed. Reliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836). The minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol.

PubMed

Oikawa, Toshinori; Itahashi, Tomoko; Numabe, Takashi

2016-01-01

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

PubMed Central

OIKAWA, Toshinori; ITAHASHI, Tomoko; NUMABE, Takashi

2015-01-01

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF. PMID:26460690

Total Quality and Organization Development. Total Quality Series.

ERIC Educational Resources Information Center

Lindsay, William M.; Petrick, Joseph A.

As the global business environment becomes more turbulent, quality management seems more indispensable. This book offers strategies for integrating the theory and practice of Total Quality Management (TQM) with organizational-development (OD) theory at all organizational levels. Chapter 1 answers the question "Why Total Quality Management and…

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

PubMed

Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

2010-02-18

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Effect of the male factor on the clinical outcome of intracytoplasmic sperm injection combined with preimplantation aneuploidy testing: observational longitudinal cohort study of 1,219 consecutive cycles.

PubMed

Mazzilli, Rossella; Cimadomo, Danilo; Vaiarelli, Alberto; Capalbo, Antonio; Dovere, Lisa; Alviggi, Erminia; Dusi, Ludovica; Foresta, Carlo; Lombardo, Francesco; Lenzi, Andrea; Tournaye, Herman; Alviggi, Carlo; Rienzi, Laura; Ubaldi, Filippo Maria

2017-12-01

To evaluate the impact of the male factor on the outcomes of intracytoplasmic sperm injection (ICSI) cycles combined with preimplantation genetic testing for aneuploidies (PGT-A). Observational longitudinal cohort study. Private in vitro fertilization (IVF) center. A total of 1,219 oocyte retrievals divided into five study groups according to sperm parameters: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), obstructive azoospermia (OA), and nonobstructive azoospermia (NOA). ICSI with ejaculated/surgically retrieved sperm, blastocyst culture, trophectoderm-based quantitative polymerase chain reaction PGT-A, and frozen-warmed euploid embryo transfer (ET). The primary outcome measures were fertilization, blastocyst development, and euploidy rates; the secondary outcome measures were live birth and miscarriage rates. Perinatal and obstetrical outcomes were monitored as well. A total of 9,042 metaphase II oocytes were inseminated. The fertilization rate was significantly reduced in MMF, OAT-S, OA, and NOA compared with N (74.8%, 68.7%, 67.3%, and 53.1% vs. 77.2%). The blastocyst rate per fertilized oocyte was significantly reduced in MMF and NOA compared with N (48.6% and 40.6% vs. 49.3%). The timing of blastocyst development also was affected in OA and NOA. Logistic regression analysis adjusted for confounders highlighted NOA as a negative predictor of obtaining an euploid blastocyst per OPU (odds ratio 0.5). When the analysis was performed per obtained blastocyst, however, no correlation between male factor and euploidy rate was observed. Embryo transfers also resulted in similar live birth and miscarriage rates. No impact of sperm factor on obstetrical/perinatal outcomes was observed. Severe male factor impairs early embryonic competence in terms of fertilization rate and developmental potential. However, the euploidy rate and implantation potential of the obtained blastocysts are independent from sperm quality

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes.

PubMed

Sugawara, Atsushi; Sugimura, Satoshi; Hoshino, Yumi; Sato, Eimei

2009-08-01

Cloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

Inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst

PubMed Central

Kono, Kanako; Tamashiro, Dana Ann A.; Alarcon, Vernadeth B.

2014-01-01

Specification of the trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst correlates with cell position, as TE derives from outer cells whereas ICM from inner cells. Differences in position are reflected by cell polarization and Hippo signaling. Only in outer cells, the apical-basal cell polarity is established, and Hippo signaling is inhibited in such a manner that LATS1 and 2 (LATS1/2) kinases are prevented from phosphorylating YAP, a key transcriptional co-activator of the TE-specifying gene Cdx2. However, the molecular mechanisms that regulate these events are not fully understood. Here, we showed that inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling and disruption of apical-basal polarity. Embryos treated with ROCK inhibitor Y-27632 exhibited elevated expression of ICM marker NANOG and reduced expression of CDX2 at the blastocyst stage. Y-27632-treated embryos failed to accumulate YAP in the nucleus, although it was rescued by concomitant inhibition of LATS1/2. Segregation between apical and basal polarity regulators, namely PARD6B, PRKCZ, SCRIB, and LLGL1, was dampened by Y-27632 treatment, whereas some of the polarization events at the late 8-cell stage such as compaction and apical localization of p-ERM and tyrosinated tubulin occurred normally. Similar abnormalities of Hippo signaling and apical-basal polarization were also observed in embryos that were treated with RHO GTPases inhibitor. These results suggest that RHO-ROCK signaling plays an essential role in regulating Hippo signaling and cell polarization to enable proper specification of the ICM and TE lineages. PMID:24997360

In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

PubMed

Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

2015-01-01

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

Blastocyst transfer does not improve cycle outcome as compared to D3 transfer in antagonist cycles with an elevated progesterone level on the day of hCG.

PubMed

Demirel, Cem; Aydoğdu, Serkan; Özdemir, Arzu İlknur; Keskin, Gülşah; Baştu, Ercan; Buyru, Faruk

2017-09-01

To evaluate the association between progesterone elevation on the day of human chorionic gonadotropin (hCG) administration and clinical pregnancy rates of gonadotropin-releasing hormone (GnRH) antagonist in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles with the transfer of embryos at different developmental stages (day-3 versus day-5 ETs). This is a retrospective analysis of fresh IVF/ICSI; 194 cycles out of 2676 conducted in a single center. A total of 2676 cycles were analyzed, of which 386 had no progesterone measurements available. Two hundred eighteen cycles had progesterone elevation (p>1.5 ng/mL) giving an overall incidence of 9.5%. Twenty-four cycles were excluded from further analysis. Of the remaining 194 cycles, 151 had day-3 transfers and 43 had blastocyst transfers. There was no statistically significant difference in pregnancy and clinical pregnancy rates per transfer between the D3-ET and D5-ET groups (46% vs. 49%, and 39% vs. 35%, respectively). The results of this study suggest that blastocyst transfer does not improve cycle outcomes compared with D3 transfer in GnRH antagonist cycles with an elevated progesterone level on the day of hCG.

Measuring quality of care: considering conceptual approaches to quality indicator development and evaluation.

PubMed

Stelfox, Henry T; Straus, Sharon E

2013-12-01

In this article, we describe one approach for developing and evaluating quality indicators. We focus on describing different conceptual approaches to quality indicator development, review one approach for developing quality indicators, outline how to evaluate quality indicators once developed, and discuss quality indicator maintenance. The key steps for developing quality indicators include specifying a clear goal for the indicators; using methodologies to incorporate evidence, expertise, and patient perspectives; and considering contextual factors and logistics of implementation. The Strategic Framework Board and the National Quality Measure Clearinghouse have developed criteria for evaluating quality indicators that complement traditional psychometric evaluations. Optimal strategies for quality indicator maintenance and dissemination have not been determined, but experiences with clinical guideline maintenance may be informative. For quality indicators to effectively guide quality improvement efforts, they must be developed, evaluated, maintained, and implemented using rigorous evidence-informed practices. Copyright © 2013 Elsevier Inc. All rights reserved.

[Performance of in vitro fertilization in Germany].

PubMed

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.

Development of a generally applicable morphokinetic algorithm capable of predicting the implantation potential of embryos transferred on Day 3.

PubMed

Petersen, Bjørn Molt; Boel, Mikkel; Montag, Markus; Gardner, David K

2016-10-01

developed algorithm (KIDScore), which was based on six annotations (the number of pronuclei equals 2 at the 1-cell stage, time from insemination to pronuclei fading at the 1-cell stage, time from insemination to the 2-cell stage, time from insemination to the 3-cell stage, time from insemination to the 5-cell stage and time from insemination to the 8-cell stage) and ranking the embryos in five groups, the implantation potential of the embryos was predicted with an AUC of 0.650. On Day 3 the KIDScore algorithm was capable of predicting blastocyst development with an AUC of 0.745 and blastocyst quality with an AUC of 0.679. In a comparison of blastocyst prediction including six other published algorithms and KIDScore, only KIDScore and one more algorithm surpassed an algorithm constructed on conventional Alpha/ESHRE consensus timings in terms of predictive power. Some morphological assessments were not available and consequently three of the algorithms in the comparison were not used in full and may therefore have been put at a disadvantage. Algorithms based on implantation data from Day 3 embryo transfers require adjustments to be capable of predicting the implantation potential of Day 5 embryo transfers. The current study is restricted by its retrospective nature and absence of live birth information. Prospective Randomized Controlled Trials should be used in future studies to establish the value of time-lapse technology and morphokinetic evaluation. Algorithms applicable to different culture conditions can be developed if based on large data sets of heterogeneous origin. This study was funded by Vitrolife A/S, Denmark and Vitrolife AB, Sweden. B.M.P.'s company BMP Analytics is performing consultancy for Vitrolife A/S. M.B. is employed at Vitrolife A/S. M.M.'s company ilabcomm GmbH received honorarium for consultancy from Vitrolife AB. D.K.G. received research support from Vitrolife AB. © The Author 2016. Published by Oxford University Press on behalf of the European

Development of a generally applicable morphokinetic algorithm capable of predicting the implantation potential of embryos transferred on Day 3

PubMed Central

Petersen, Bjørn Molt; Boel, Mikkel; Montag, Markus; Gardner, David K.

2016-01-01

curve (AUC) to establish the predictive strength of the algorithm. MAIN RESULTS AND THE ROLE OF CHANCE By applying the here developed algorithm (KIDScore), which was based on six annotations (the number of pronuclei equals 2 at the 1-cell stage, time from insemination to pronuclei fading at the 1-cell stage, time from insemination to the 2-cell stage, time from insemination to the 3-cell stage, time from insemination to the 5-cell stage and time from insemination to the 8-cell stage) and ranking the embryos in five groups, the implantation potential of the embryos was predicted with an AUC of 0.650. On Day 3 the KIDScore algorithm was capable of predicting blastocyst development with an AUC of 0.745 and blastocyst quality with an AUC of 0.679. In a comparison of blastocyst prediction including six other published algorithms and KIDScore, only KIDScore and one more algorithm surpassed an algorithm constructed on conventional Alpha/ESHRE consensus timings in terms of predictive power. LIMITATIONS, REASONS FOR CAUTION Some morphological assessments were not available and consequently three of the algorithms in the comparison were not used in full and may therefore have been put at a disadvantage. Algorithms based on implantation data from Day 3 embryo transfers require adjustments to be capable of predicting the implantation potential of Day 5 embryo transfers. The current study is restricted by its retrospective nature and absence of live birth information. Prospective Randomized Controlled Trials should be used in future studies to establish the value of time-lapse technology and morphokinetic evaluation. WIDER IMPLICATIONS OF THE FINDINGS Algorithms applicable to different culture conditions can be developed if based on large data sets of heterogeneous origin. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Vitrolife A/S, Denmark and Vitrolife AB, Sweden. B.M.P.’s company BMP Analytics is performing consultancy for Vitrolife A/S. M.B. is employed at

Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

PubMed

Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

2015-05-13

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle.

PubMed

Gerger, R P C; Rossetto, Rafael; Ribeiro, E S; Ortigari, Ivens; Zago, Fabiano Carminatti; Aguiar, L H; Costa, U M; Lopes, Rui Fernando Félix; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Rodrigues, José Luiz; Forell, Fabiana; Bertolini, Luciana Relly; Bertolini, Marcelo

2017-06-01

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

Developing Quality Schools: A Handbook.

ERIC Educational Resources Information Center

Barlosky, Martin; Lawton, Stephen

This handbook combines the outcomes of the "Symposium on Quality Schools" (April 14, 15 and June 2, 3, 1994, Toronto, Canada) with a continuing presentation of the quality philosophy. It is designed to guide the development and implementation of quality-driven educational programs. Quality schools are committed to creating enhanced…

Oxidative stress differentially impacts male and female bovine embryos depending on the culture medium and the stress condition.

PubMed

Dallemagne, Matthew; Ghys, Emmanuelle; De Schrevel, Catalina; Mwema, Ariane; De Troy, Delphine; Rasse, Catherine; Donnay, Isabelle

2018-09-01

Male and female embryos are known to differ for their metabolism and response to environmental factors very early in development. The present study aimed to evaluate the response to oxidative stress of male and female bovine embryos at the morula-blastocyst stages in terms of developmental rates, total cell number and apoptotic rates in two culture conditions. Embryos where cultured in a medium supplemented with either 5% fetal calf serum (FCS) or 4 mg/mL bovine serum albumin and a mixture of insulin, transferrin and selenium (BSA-ITS). Oxidative stress was applied at Day-5 post insemination (pi) by adding either AAPH or menadione to the culture medium, and blastocysts were analyzed at Day-7pi. The impact on development and blastocyst quality was dependent on the culture medium and the stress inducer but differed between male and female embryos. Male embryos resisted better to oxidative stress in FCS supplemented medium, no matter the stress inducer. Accordingly, the impact on blastocyst cell number tended to be higher in female blastocysts after stress induction with AAPH in FCS supplemented medium. On the other hand, in BSA-ITS supplemented medium, female embryos were more resistant to AAPH induced stress, while menadione had no impact on sex ratio. The weaker resistance of males to AAPH in this medium is in accordance with their trend to show a higher increase in apoptotic rates than females in this condition. In conclusion, this study shows that oxidative stress has differential impact on male and female bovine blastocysts depending on the culture condition and on the way oxidative stress is induced. Copyright © 2018 Elsevier Inc. All rights reserved.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

PubMed

Morovic, Martin; Strejcek, Frantisek; Nakagawa, Shoma; Deshmukh, Rahul S; Murin, Matej; Benc, Michal; Fulka, Helena; Kyogoku, Hirohisa; Pendovski, Lazo; Fulka, Josef; Laurincik, Jozef

2017-12-01

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

RBP-Jκ-Dependent Notch Signaling Is Dispensable for Mouse Early Embryonic Development

PubMed Central

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-01-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jκ-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. PMID:16782866

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

PubMed Central

Kang, Jung-Taek; Kwon, Dae-Kee; Park, Sol-Ji; Kim, Su-Jin; Moon, Joon-Ho; Koo, Ok-Jae; Jang, Goo

2013-01-01

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development. PMID:23388446

Effect of embryo source and recipient progesterone environment on embryo development in cattle.

PubMed

Lonergan, P; Woods, A; Fair, T; Carter, F; Rizos, D; Ward, F; Quinn, K; Evans, A

2007-01-01

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 +/- 1.28 and 10.30 +/- 0.51 ng mL(-1), respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 +/- 0.45 mm(2) (n = 28) compared with 1.66 +/- 0.38 mm(2) (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.

Embryonic genotype and inbreeding affect preimplantation development in cattle.

PubMed

Lazzari, G; Colleoni, S; Duchi, R; Galli, A; Houghton, F D; Galli, C

2011-05-01

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

PubMed

Lucas, Caroline Gomes; Remião, Mariana Härter; Komninou, Eliza Rossi; Domingues, William Borges; Haas, Cristina; Leon, Priscila Marques Moura de; Campos, Vinicius Farias; Ourique, Aline; Guterres, Silvia S; Pohlmann, Adriana R; Basso, Andrea Cristina; Seixas, Fabiana Kömmling; Beck, Ruy Carlos Ruver; Collares, Tiago

2015-12-01

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates. Copyright © 2015 Elsevier Inc. All rights reserved.

Improving Metabolic Health in Obese Male Mice via Diet and Exercise Restores Embryo Development and Fetal Growth

PubMed Central

McPherson, Nicole O.; Bakos, Hassan W.; Owens, Julie A.; Setchell, Brian P.; Lane, Michelle

2013-01-01

Paternal obesity is now clearly associated with or causal of impaired embryo and fetal development and reduced pregnancy rates in humans and rodents. This appears to be a result of reduced blastocyst potential. Whether these adverse embryo and fetal outcomes can be ameliorated by interventions to reduce paternal obesity has not been established. Here, male mice fed a high fat diet (HFD) to induce obesity were used, to determine if early embryo and fetal development is improved by interventions of diet (CD) and/or exercise to reduce adiposity and improve metabolism. Exercise and to a lesser extent CD in obese males improved embryo development rates, with increased cell to cell contacts in the compacting embryo measured by E-cadherin in exercise interventions and subsequently, increased blastocyst trophectoderm (TE), inner cell mass (ICM) and epiblast cell numbers. Implantation rates and fetal development from resulting blastocysts were also improved by exercise in obese males. Additionally, all interventions to obese males increased fetal weight, with CD alone and exercise alone, also increasing fetal crown-rump length. Measures of embryo and fetal development correlated with paternal measures of glycaemia, insulin action and serum lipids regardless of paternal adiposity or intervention, suggesting a link between paternal metabolic health and subsequent embryo and fetal development. This is the first study to show that improvements to metabolic health of obese males through diet and exercise can improve embryo and fetal development, suggesting such interventions are likely to improve offspring health. PMID:23977045

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice.

PubMed

Paul, Catriona; Murray, Alison A; Spears, Norah; Saunders, Philippa T K

2008-07-01

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Increased risk of preterm birth in singleton pregnancies after blastocyst versus Day 3 embryo transfer: Canadian ART Register (CARTR) analysis.

PubMed

Dar, S; Librach, C L; Gunby, J; Bissonnette, F; Cowan, L

2013-04-01

Are the fetal outcomes of singleton pregnancies that result from cleavage stage embryo transfer (ET) different from the outcomes from Day 5/6 blastocyst stage ET? There was a significantly higher risk of preterm birth (<37 weeks) in singletons after extended embryo culture (Day 5/6) compared with cleavage stage (Day 3) transfer. Two recent studies, from Sweden and the USA, reported an increased risk of preterm birth in singleton pregnancies after Day 5/6 ET compared with Day 3 ET. The US study also showed increased early preterm births and the Swedish study showed increased fetal malformations in this group. A retrospective cohort study was performed. Data were collected from the Canadian ART Register database for all singleton births after fresh IVF/ICSI ET cycles (2001-2009). A total of 12 712 singleton births were included. Of these, 9506 resulted from a Day 3 ET and 3206 resulted from a blastocyst (Day 5/6) ET. Preterm birth rate <37 weeks (unadjusted by potential confounding factors) was higher with Day 5/6 versus Day 3 transfers (17.2 versus 14.1%, P < 0.001). Using logistic regression analysis to adjust for confounding factors, preterm birth rate <37 weeks was the only outcome significantly increased after Day 5/6 compared with Day 3 transfer (odds ratio 1.32, 95% confidence interval 1.17-1.49). The following confounding factors were adjusted for: year of treatment (2001-2009), maternal age (continuous), parity (0 versus ≥1 birth), diagnosis category, number of oocytes retrieved [≤20 versus >20 (high responder group)], insemination method (IVF versus ICSI), number of embryos transferred (1, 2 or ≥3) and the presence of a vanishing twin (≥1 fetal heart on the initial ultrasonographic examination). Post-natal follow-up studies will be required to determine if this difference we observed translates into adverse long-term effects on these offspring. The rate of early preterm births (<32 weeks) was higher in Day 5/6 versus Day 3, but the low number of

Moving Forward--Shaping a Career Development Culture: Quality Standards, Quality Practice, Quality Outcomes

ERIC Educational Resources Information Center

McMahon, Mary

2004-01-01

This paper represents the second of two papers written as part of the National Standards and Accreditation of Career Practitioners project. The first, a scoping paper titled Shaping a career development culture: Quality standards, quality practice, quality outcomes (McMahon, 2004), provided information for and guided discussion at the National…

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius).

PubMed

Herrid, M; Billah, M; Malo, C; Skidmore, J A

2016-03-01

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other

The effects of ethanol and strontium on growth and development of two-cell arrested mouse embryos.

PubMed

Darabi, Mohammad Reza; Shiravi, Abdolhossein; Hojati, Vida

2012-01-01

Arresting at a certain stage of development like the two-cell stage could be one of the causes of infertility. The aim of this study is to evaluate the effects of ethanol and strontium on growth and development of mice embryos arrested at the two-cell stage. In this experimental study, female mice were coupled with a male following superovulation. Positive vaginal plug mice were sacrificed 48 hours after human chorionic gonadotropin (hCG) injection. Two-cell embryos were transferred to M16 medium and divided to four groups. The first control group was incubated without any exposure to low temperatures. Groups 2, 3 and 4 were exposed to 4°C for 24 hours. The second control group was incubated immediately, while the third and fourth groups were exposed to 10 mM strontium for five minutes and 0.1% ethanol for a further five minutes. Growth rate and developmental parameters of embryos were analyzed by one- way ANOVA. The significant difference between the groups was determined by Post Hoc. The data shows that developmental rate is decreased significantly by 4°C exposure. The mean percentage of degenerated embryo was significantly different between groups but the mean cleavage rate was not significantly different. The mean percent of morula, blastocyst and hatched blastocyst formation were significantly different between groups during a 120 hours study post hCG injection. The effect of strontium and ethanol on arrested two-cell embryos had no significant effect on the mean percentage of morula, but ethanol treatment significantly increased the percentage of blastocyst and hatched blastocyst formation compared to strontium.

Removal of O-GlcNAcylation is important for pig preimplantation development

PubMed Central

SHIBUTANI, Mihiro; MORI, Takeshi; MIYANO, Takashi; MIYAKE, Masashi

2015-01-01

Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error. PMID:26004176

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

PubMed

Alomar, M; Tasiaux, H; Remacle, S; George, F; Paul, D; Donnay, I

2008-08-01

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex

[Effect of TGF-beta1 on embryo implantation and development in mice in vitro].

PubMed

Luo, Shan; Yin, Hai-ning; Li, Shang-wei

2010-03-01

To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice. Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer. TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053. TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

Grade and looseness of the inner cell mass may lead to the development of monochorionic diamniotic twins.

PubMed

Otsuki, Junko; Iwasaki, Toshiroh; Katada, Yuya; Sato, Haruka; Furuhashi, Kohyu; Tsuji, Yuta; Matsumoto, Yukiko; Shiotani, Masahide

2016-09-01

To examine the relationship between the inner cell mass (ICM) grade and its morphological configuration on the occurrence of monochorionic diamniotic (M-D) twinning. Retrospective embryo cohort study. Private IVF clinic. Evaluation of frozen-thawed single blastocyst transfers with hormone replacement treatment in 8,435. This cohort included 71 blastocysts and their ICMs observed by time-lapse photography. Any changes in configuration of the ICMs observed by time-lapse photography were analyzed retrospectively. The amount of loosening of blastomeres within the ICM was evaluated by time-lapse observations. The number of cells that were involved in the loosening process was also assessed. Both of these parameters were correlated with the type of monozygotic twinning that eventuated. The M-D twinning incidence resulting from blastocysts with a high grade ICM (grade A) were transferred was 0.38% (3/796), whereas it was significantly higher, 1.38% (34/2,463), when blastocysts with a poorer (B and C) grade ICM were transferred. Among 71 transferred frozen-thawed blastocysts that were studied with time-lapse photography, there were two dichorionic diamniotic and one M-D twins. Careful observations of the embryo that resulted in the one M-D case, revealed that the ICM acquired a looser appearance due to decompaction of at least eight cells. This type of decompaction was not observed in the ICMs of other transferred blastocysts. The occurrence of M-D twinning may be avoided by excluding blastocysts that contain decompacting ICMs. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development.

PubMed

Salviano, M B; Collares, F J F; Becker, B S; Rodrigues, B A; Rodrigues, J L

2016-04-01

Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage

Functional analysis of lysosomes during mouse preimplantation embryo development.

PubMed

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2013-01-01

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos.

PubMed

Xu, J S; Lee, Y L; Lee, K F; Kwok, K L; Lee, W M; Luk, J M; Yeung, W S B

2004-12-01

Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua

Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin Cmore » treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.« less

Developing a Quality Curriculum.

ERIC Educational Resources Information Center

Glatthorn, Allan A.

In the face of increasing demands for school reform, educational leaders are looking anew at the core elements of the instructional program, including the curriculum. This book serves as a guide to both understanding and practicing sound curriculum development. It lays out the steps of a quality curriculum-development process and emphasizes that…

Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos.

PubMed

Cheng, Shih-Lin; Lai, Yi-Ling; Lee, Ming-Che; Shen, Perng-Chih; Liu, Shyh-Shyan; Liu, Bing-Tsan

2014-07-03

The deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Mouse 4-cells embryos were divided into 16 groups for 72 h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. The 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0-90.4%, P>0.05) and normal morphological rate (84.4-85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25 μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). The FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

PubMed

Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

2011-09-15

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro porcine blastocyst development in three-dimentional alginate hydrogels

USDA-ARS?s Scientific Manuscript database

Appropriate embryonic and fetal development significantly impact pregnancy success and, therefore, the efficiency of swine production. The pre-implantation period of porcine pregnancy is characterized by several developmental hallmarks, which are initiated by the dramatic morphological change that o...

Tauroursodeoxycholic acid enhances the pre-implantation embryo development by reducing apoptosis in pigs.

PubMed

Kim, J-S; Song, B-S; Lee, K-S; Kim, D-H; Kim, S-U; Choo, Y-K; Chang, K-T; Koo, D-B

2012-10-01

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncan's multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 μm and with TUDCA at concentrations of 0, 100, 200 or 300 μm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 μm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 μm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig

Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

PubMed

Xia, P; Liu, Z; Qin, P

2011-04-01

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc. © 2010 Blackwell Verlag GmbH.

The Ornithine Decarboxylase Gene Is Essential for Cell Survival during Early Murine Development

PubMed Central

Pendeville, Hélène; Carpino, Nick; Marine, Jean-Christophe; Takahashi, Yutaka; Muller, Marc; Martial, Joseph A.; Cleveland, John L.

2001-01-01

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation. PMID:11533243

Cell fate regulation in early mammalian development

NASA Astrophysics Data System (ADS)

Oron, Efrat; Ivanova, Natalia

2012-08-01

Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos.

PubMed

Amarnath, Dasari; Wakayama, Sayaka; Zhu, Jie; Moawad, Adel R; Wakayama, Teruhiko; Campbell, Keith H S

2011-12-01

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes. Copyright © 2011 Elsevier Inc. All rights reserved.

A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos.

PubMed

Dai, Xiangpeng; Hao, Jie; Zhou, Qi

2009-08-01

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and alphaMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10-30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo.

PubMed

Tribulo, Paula; Leão, Beatriz Caetano da Silva; Lehloenya, Khoboso C; Mingoti, Gisele Zoccal; Hansen, Peter J

2017-06-01

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

Inverted light-sheet microscope for imaging mouse pre-implantation development.

PubMed

Strnad, Petr; Gunther, Stefan; Reichmann, Judith; Krzic, Uros; Balazs, Balint; de Medeiros, Gustavo; Norlin, Nils; Hiiragi, Takashi; Hufnagel, Lars; Ellenberg, Jan

2016-02-01

Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

Nepro is localized in the nucleolus and essential for preimplantation development in mice.

PubMed

Hashimoto, Masakazu; Sato, Tatsuya; Muroyama, Yuko; Fujimura, Lisa; Hatano, Masahiko; Saito, Tetsuichiro

2015-09-01

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2-cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria-associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus-associated protein, and its loss leads to the apoptosis before blastocyst formation in mice. © 2015 Japanese Society of Developmental Biologists.

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization.

PubMed

Wang, Feng; Tian, XiuZhi; Zhang, Lu; He, ChangJiu; Ji, PengYun; Li, Yu; Tan, DunXian; Liu, GuoShi

2014-02-01

To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. In vitro prospective study. University research laboratory. Animal models for human studies. In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1. Copyright © 2014 American Society for Reproductive Medicine. All rights reserved.

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

NASA Astrophysics Data System (ADS)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

PubMed

Tominaga, K; Yoneda, K; Utsumi, K

1996-03-01

The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

Effect of the nuclear-donor cell lineage, type, and cell donor on development of somatic cell nuclear transfer embryos in cattle.

PubMed

Batchelder, Cynthia A; Hoffert, Kara A; Bertolini, Marcelo; Moyer, Alice L; Mason, Jeffery B; Petkov, Stoyan G; Famula, Thomas R; Anderson, Gary B

2005-01-01

Potential applications of somatic cell nuclear transfer to agriculture and medicine are currently constrained by low efficiency and high rates of embryonic, fetal, and neonatal loss. Nuclear transfer efficiency in cattle was compared between three donor-cell treatments from a single animal, between four donor-cell treatments in sequential stages of differentiation from a single cell lineage and genotype, and between the same cell type in two donors. Cumulus and granulosa donor cells resulted in a greater proportion of viable day-7 embryos than ear-skin cells; pregnancy rate and losses were not different among treatments. The least differentiated cell type in the follicular cell lineage, preantral follicle cells, resulted in fewer cloned blastocysts (11%) than cumulus (30%), granulosa (23%), and luteal (25%) donor cells. Cloned blastocysts that did develop from preantral follicle cells (75%) were more likely to progress through implantation into later stages of pregnancy than cloned blastocysts from cumulus (10%), granulosa (9%), and luteal (11%) donor cells (p < 0.05). Day-7 embryo development from granulosa cells was similar between two donors (19 vs. 24%) and proved to be a poor indicator of further development as day-30 pregnancy rates varied threefold between donors (48 vs. 15%, p < 0.05). Results reported here emphasize the crucial role of the nuclear donor cell in the outcome of the nuclear-transfer process.

Quality risk management in pharmaceutical development.

PubMed

Charoo, Naseem Ahmad; Ali, Areeg Anwer

2013-07-01

The objective of ICH Q8, Q9 and Q10 documents is application of systemic and science based approach to formulation development for building quality into product. There is always some uncertainty in new product development. Good risk management practice is essential for success of new product development in decreasing this uncertainty. In quality by design paradigm, the product performance properties relevant to the patient are predefined in target product profile (TPP). Together with prior knowledge and experience, TPP helps in identification of critical quality attributes (CQA's). Initial risk assessment which identifies risks to these CQA's provides impetus for product development. Product and process are designed to gain knowledge about these risks, devise strategies to eliminate or mitigate these risks and meet objectives set in TPP. By laying more emphasis on high risk events the protection level of patient is increased. The process being scientifically driven improves the transparency and reliability of the manufacturer. The focus on risk to the patient together with flexible development approach saves invaluable resources, increases confidence on quality and reduces compliance risk. The knowledge acquired in analysing risks to CQA's permits construction of meaningful design space. Within the boundaries of the design space, variation in critical material characteristics and process parameters must be managed in order to yield a product having the desired characteristics. Specifications based on product and process understanding are established such that product will meet the specifications if tested. In this way, the product is amenable to real time release, since specifications only confirm quality but they do not serve as a means of effective process control.

DEVELOPMENT OF MARINE WATER QUALITY CRITERIA

EPA Science Inventory

The U.S. Environmental Protectional Agency has developed guidelines for deriving numerical national water quality criteria for the protection of aquatic organisms and their uses. These guidelines provide the method for deriving water quality criteria, including minimum data base...

Fundamentals of human embryonic growth in vitro and the selection of high-quality embryos for transfer.

PubMed

Boiso, Irene; Veiga, Anna; Edwards, Robert G

2002-01-01

Knowledge of the nature of embryo growth, and the handling and scoring of quality in human embryos are significant aspects for embryologists in IVF clinics. This review describes the formation, growth and maturation of human oocytes, many aspects of fertilization in vitro, embryonic transcription during preimplantation stages, and the formation of polarities, timing controls, role of mitochondria and functions of endocrine and paracrine systems. Modern concepts are fully discussed, together with their significance in the practice of IVF. This knowledge is essential for the correct clinical care of human embryos growing in vitro, especially in view of their uncharacteristic tendency to vary widely in implantation potential. Underlying causes of such variation have not been identified. Stringent tests must be enforced to ensure human embryos develop under optimal conditions, and are scored for quality using the most advanced techniques. Optimal methods of culture are described, including methods such as co-culture introduced to improve embryo quality but less important today. Detailed attention is given to quality as assessed from embryonic characteristics determined by timers, polarities, disturbed embryo growth and anomalous cell cycles. Methods for classification are described. Approaches to single embryo transfers are described, including the use of sequential media to produce high-quality blastocysts. These approaches, and others involved in surgical methods to remove fragments, transfer ooplasm or utilize newer approaches such as preimplantation diagnosis of chromosomal complements in embryos are covered. New outlooks in this field are summarized.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

PubMed

Muñoz, M; Martin, D; Carrocera, S; Alonso-Guervos, M; Mora, M I; Corrales, F J; Peynot, N; Giraud-Delville, C; Duranthon, V; Sandra, O; Gómez, E

2017-10-01

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Measuring the Quality of Professional Development Training

ERIC Educational Resources Information Center

Gaumer Erickson, Amy S.; Noonan, Patricia M.; Brussow, Jennifer; Supon Carter, Kayla

2017-01-01

High-quality, evidence-based professional development is essential to ensure that teachers obtain the knowledge, strategies and skills necessary to positively impact student learning. While the primary form of professional development, training has rarely been evaluated for quality beyond the satisfaction of those being trained. The Observation…

Developmental competence of bovine embryos from heat-stressed ova.

PubMed

Edwards, J L; Bogart, A N; Rispoli, L A; Saxton, A M; Schrick, F N

2009-02-01

Because multiple ovulation embryo transfer procedures are occasionally performed in cows experiencing heat stress, the goal of this study was to assess the developmental competence of otherwise morphologically normal embryos from heat-stressed ova. To this end, the ability of compact morulae from heat-stressed and non-heat-stressed bovine ova to undergo blastocyst development after culture at 38.5 or 41.0 degrees C was examined. It was hypothesized that heat-induced perturbations in the ooplasm carry over to increase the susceptibility of the preattachment embryo to heat stress. Initially, ova were matured at 38.5 or 41.0 degrees C. The consequences of heat stress did not include altered cleavage, but did reduce the proportion of 8- to 16-cell-stage embryos (55.3 vs. 50.6%; SEM +/- 1.9). Although proportionately fewer, compact morulae from heat-stressed ova were equivalent in quality to those from non-heat-stressed ova (2.1 and 2.1; SEM = 0.04). Culture of compact morulae from non-heat-stressed ova at 41.0 degrees C did not affect blastocyst development (71.9 and 71.5%; SEM = 3.0). Furthermore, the development of compact morulae from heat-stressed ova was similar to that of non-heat-stressed ova after culture at 38.5 degrees C (68.2 vs. 71.9 and 71.5%; SEM = 3.0). However, blastocyst development was reduced when compact morulae from heat-stressed ova were cultured at 41.0 degrees C (62.3 vs. 71.9, 71.5 and 68.2; SEM = 3.1). In summary, reduced compaction rates of heat-stressed ova explained in part why fewer develop to the blastocyst stage after fertilization. The thermolability of the few embryos that develop from otherwise developmentally challenged ova emphasizes the importance of minimizing exposure to stressor(s) during oocyte maturation.

Professional Development for Water Quality Control Personnel.

ERIC Educational Resources Information Center

Shepard, Clinton Lewis

This study investigated the availability of professional development opportunities for water quality control personnel in the midwest. The major objective of the study was to establish a listing of educational opportunities for the professional development of water quality control personnel and to compare these with the opportunities technicians…

Pragmatic quality metrics for evolutionary software development models

NASA Technical Reports Server (NTRS)

Royce, Walker

1990-01-01

Due to the large number of product, project, and people parameters which impact large custom software development efforts, measurement of software product quality is a complex undertaking. Furthermore, the absolute perspective from which quality is measured (customer satisfaction) is intangible. While we probably can't say what the absolute quality of a software product is, we can determine the relative quality, the adequacy of this quality with respect to pragmatic considerations, and identify good and bad trends during development. While no two software engineers will ever agree on an optimum definition of software quality, they will agree that the most important perspective of software quality is its ease of change. We can call this flexibility, adaptability, or some other vague term, but the critical characteristic of software is that it is soft. The easier the product is to modify, the easier it is to achieve any other software quality perspective. This paper presents objective quality metrics derived from consistent lifecycle perspectives of rework which, when used in concert with an evolutionary development approach, can provide useful insight to produce better quality per unit cost/schedule or to achieve adequate quality more efficiently. The usefulness of these metrics is evaluated by applying them to a large, real world, Ada project.

Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.

PubMed

Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha

2010-10-01

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle.

PubMed

Wongsrikeao, Pimprapar; Nagai, Takashi; Agung, Budiyanto; Taniguchi, Masayasu; Kunishi, Miho; Suto, Shizuyo; Otoi, Takeshige

2007-06-01

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos. (c) 2006 Wiley-Liss, Inc.

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

PubMed Central

Canovas, Sebastian; Ivanova, Elena; Romar, Raquel; García-Martínez, Soledad; Soriano-Úbeda, Cristina; García-Vázquez, Francisco A; Saadeh, Heba; Andrews, Simon; Kelsey, Gavin; Coy, Pilar

2017-01-01

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT. DOI: http://dx.doi.org/10.7554/eLife.23670.001 PMID:28134613

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production.

PubMed

Baltazar, A L; de Mattos, G M; Ropelli, B M; Firetti, Smg; Castilho, C; Pugliesi, G; Maldonado, Mbc; Binelli, M; Silva, Jof; Lupatini, G C; Lafuente, B S; Membrive, Cmb

2018-06-01

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro. © 2018 Blackwell Verlag GmbH.

Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

PubMed

Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

2016-03-24

Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

Relating Preschool Quality to Children's Literacy Development

ERIC Educational Resources Information Center

Cunningham, Denise D.

2010-01-01

Preschool classrooms were investigated to determine the extent to which quality is related to children's literacy development. The study included 24 classrooms of 428 prekindergarten children in a large, urban Midwestern school district. Results suggest that global classroom quality and literacy environment quality are strongly related. Literacy…

Rho differentially regulates the Hippo pathway by modulating the interaction between Amot and Nf2 in the blastocyst.

PubMed

Shi, Xianle; Yin, Zixi; Ling, Bin; Wang, Lingling; Liu, Chang; Ruan, Xianhui; Zhang, Weiyu; Chen, Lingyi

2017-11-01

The Hippo pathway modulates the transcriptional activity of Yap to regulate the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) in blastocysts. Yet how Hippo signaling is differentially regulated in ICM and TE cells is poorly understood. Through an inhibitor/activator screen, we have identified Rho as a negative regulator of Hippo in TE cells, and PKA as a positive regulator of Hippo in ICM cells. We further elucidated a novel mechanism by which Rho suppresses Hippo, distinct from the prevailing view that Rho inhibits Hippo signaling through modulating cytoskeleton remodeling and/or cell polarity. Active Rho prevents the phosphorylation of Amot Ser176, thus stabilizing the interaction between Amot and F-actin, and restricting the binding between Amot and Nf2. Moreover, Rho attenuates the interaction between Amot and Nf2 by binding to the coiled-coil domain of Amot. By blocking the association of Nf2 and Amot, Rho suppresses Hippo in TE cells. © 2017. Published by The Company of Biologists Ltd.

Future Development of Nursing Home Quality Indicators

ERIC Educational Resources Information Center

Arling, Greg; Kane, Robert L.; Lewis, Teresa; Mueller, Christine

2005-01-01

Nursing home quality indicators have been developed over the past 10 years to quantify nursing home quality and to draw systematic comparisons between facilities. Although these indicators have been applied widely for nursing home regulation, quality improvement, and public reporting, researchers and stakeholders have raised concerns about their…

Rotorcraft handling-qualities design criteria development

NASA Technical Reports Server (NTRS)

Aiken, Edwin W.; Lebacqz, J. Victor; Chen, Robert T. N.; Key, David L.

1988-01-01

Joint NASA/Army efforts at the Ames Research Center to develop rotorcraft handling-qualities design criteria began in earnest in 1975. Notable results were the UH-1H VSTOLAND variable stability helicopter, the VFA-2 camera-and-terrain-board simulator visual system, and the generic helicopter real-time mathematical model, ARMCOP. An initial series of handling-qualities studies was conducted to assess the effects of rotor design parameters, interaxis coupling, and various levels of stability and control augmentation. The ability to conduct in-flight handling-qualities research was enhanced by the development of the NASA/Army CH-47 variable-stability helicopter. Research programs conducted using this vehicle include vertical-response investigations, hover augmentation systems, and the effects of control-force characteristics. The handling-qualities data base was judged to be sufficient to allow an update of the military helicopter handling-qualities specification, MIL-H-8501. These efforts, including not only the in-house experimental work but also contracted research and collaborative programs performed under the auspices of various international agreements. The report concludes by reviewing the topics that are currently most in need of work, and the plans for addressing these topics.

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

PubMed

Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

2010-12-01

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

PubMed

Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

2009-09-01

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

PubMed

Takai, Y; Tsutsumi, O; Ikezuki, Y; Hiroi, H; Osuga, Y; Momoeda, M; Yano, T; Taketani, Y

2000-04-21

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos. Copyright 2000 Academic Press.

Handmade Cloned Buffalo (Bubalus bubalis) Embryos Produced from Somatic Cells Isolated from Milk and Ear Skin Differ in Their Developmental Competence, Epigenetic Status, and Gene Expression.

PubMed

Jyotsana, Basanti; Sahare, Amol A; Raja, Anuj K; Singh, Karn P; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

2015-10-01

We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p < 0.05) for milk-derived than that for skin-derived embryos, whereas the total cell number and apoptotic index were similar. The global level of H3K9ac was higher (p < 0.05) in skin- than in milk-derived cells, whereas the level of H3K27me3 was similar in the two groups. The global level of H3K9ac was similar between milk-derived and in vitro-fertilized (IVF) blastocysts, which was higher (p < 0.05) than that in skin-derived blastocysts. The level of H3K27me3 was similar among the three groups. The expression level of IGF-1R and G6PD was higher (p < 0.05) in skin- than in milk-derived cells, whereas DNMT1, DNMT3a, and HDAC1 expression level was similar. In the blastocysts, the expression level of DNMT1, HDAC1, OCT4, and CDX2 was higher (p < 0.05) in skin-derived than that in IVF blastocysts. The expression level of DNMT3a and IGF-1R, was in the order (p < 0.05) skin-derived and IVF > milk-derived blastocysts and that of NANOG was (p < 0.05) IVF-> milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.

Liposome-encapsulated diacyl glycerol and inositol triphosphate-induced delayed oocyte activation and poor development of parthenotes.

PubMed

Nair, Ramya; Manikkath, Jyothsna; Hegde, Aswathi R; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

2017-09-01

To explore the ability of diacyl glycerol (DAG) and inositol triphosphate (IP3), two major secondary messengers in the calcium signaling pathway, in activating oocytes. Oocyte cumulus complex obtained from superovulated Swiss albino mice were incubated in M16 medium with liposome-encapsulated 1,2-Dipalmitoyl-sn-glycerol (LEDAG) and/or IP3 for 3 h. Strontium chloride was used as positive control. The activation potential, ploidy status, and blastocyst rate was calculated. Both DAG and IP3, individually, induced activation in ~98% of oocytes, which was significantly higher (p<0.01) than activation induced by strontium chloride (60%). Delayed pronucleus formation and a higher percentage of diploid parthenotes was observed in oocytes activated with LEDAG and/or IP3. However, these embryos failed to progress beyond the 6-8-cell stage. Only when the medium was supplemented with LEDAG (5 μg/mL) and IP3 (10 μg/mL) could activated oocytes progress till the blastocyst stage (5.26%), which was lower than the blastocyst rate in the positive controls (13.91%). The results of the present study indicate that DAG and IP3 can induce delayed oocyte activation and poor development of parthenotes in vitro.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

PubMed

Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

2016-04-01

Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos.

PubMed

Pan, Yangyang; He, Honghong; Cui, Yan; Baloch, Abdul Rasheed; Li, Qin; Fan, Jiangfeng; He, Junfeng; Yu, Sijiu

2015-12-01

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

PubMed Central

Pan, Yangyang; He, Honghong; Cui, Yan; Baloch, Abdul Rasheed; Li, Qin; Fan, Jiangfeng; He, Junfeng

2015-01-01

Abstract This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus–oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian

C-Phycocyanin protects against mitochondrial dysfunction and oxidative stress in parthenogenetic porcine embryos.

PubMed

Niu, Ying-Jie; Zhou, Wenjun; Guo, Jing; Nie, Zheng-Wen; Shin, Kyung-Tae; Kim, Nam-Hyung; Lv, Wen-Fa; Cui, Xiang-Shun

2017-12-05

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of early porcine embryos as well as its underlying mechanisms. Different concentrations of CP (2, 5, 8, 10 μg/mL) were added to porcine zygote medium 5 during in vitro culture. The results showed that 5 μg/mL CP significantly increased blastocyst formation and hatching rate. Blastocyst formation and quality were significantly increased in the 50 μM H 2 O 2 treatment group following 5 μg/mL CP addition. CP prevented the H 2 O 2 -induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H 2 O 2 -induced oxidative injury group compared to in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

PubMed

Moro, L N; Jarazo, J; Buemo, C; Hiriart, M I; Sestelo, A; Salamone, D F

2015-10-01

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. © 2015 Blackwell Verlag GmbH.

Histone Deacetylase Inhibitor Improves the Development and Acetylation Levels of Cat–Cow Interspecies Cloned Embryos

PubMed Central

Wittayarat, Manita; Sato, Yoko; Do, Lanh Thi Kim; Morita, Yasuhiro; Chatdarong, Kaywalee; Techakumphu, Mongkol; Taniguchi, Masayasu

2013-01-01

Abstract Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA–treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro–fertilized embryos and significantly higher (p<0.05) than those of non-TSA–treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9. PMID:23790014

Staff Development and Total Quality Management.

ERIC Educational Resources Information Center

Norris, Gerald L.; Norris, Joye H.

Professional development is an emerging view of faculty development that places teachers in charge of their own professional growth. The emergence of Total Quality Management (TQM) provides a vehicle for designing professional development to meet the needs of individuals and the organizations that employ them. The eight tenets of Deming's theory…

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

PubMed

Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

2018-01-01

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Several fibroblast growth factors are expressed during pre-attachment bovine conceptus development and regulate interferon-tau expression from trophectoderm.

PubMed

Cooke, Flavia N T; Pennington, Kathleen A; Yang, Qien; Ealy, Alan D

2009-02-01

The trophectoderm-derived factor interferon tau (IFNT) maintains the uterus in a pregnancy-receptive state in cattle and sheep. Fibroblast growth factors (FGFs) are implicated in regulating IFNT expression and potentially other critical events associated with early conceptus development in cattle. The overall objectives of this work were to identify the various FGFs and FGF receptors (FGFRs) expressed in elongating pre-attachment bovine conceptuses and determine if these FGFs regulate conceptus development and/or mediate IFNT production. In vitro-derived bovine blastocysts and in vivo-derived elongated conceptuses collected at day 17 of pregnancy express at least four FGFR subtypes (R1c, R2b, R3c, R4). In addition, transcripts for FGF1, 2, and 10 but not FGF7 are present in elongated bovine conceptuses. The expression pattern of FGF10 most closely resembled that of IFNT, with both transcripts remaining low in day 8 and day 11 conceptuses and increasing substantially in day 14 and day 17 conceptuses. Supplementation with recombinant FGF1, 2 or 10 increased IFNT mRNA levels in bovine trophectoderm cells and bovine blastocysts and increased IFNT protein concentrations in trophectoderm-conditioned medium. Blastocyst development was not affected by any of the FGFs. In summary, at least four FGFRs reside in pre- and peri-attachment bovine conceptuses. Moreover, conceptuses express at least three candidate FGFs during elongation, the time of peak IFNT expression. These findings provide new insight for how conceptus-derived factors such as FGF1, 2, and 10 may control IFNT expression during early pregnancy in cattle.

Developing Interoperable Air Quality Community Portals

NASA Astrophysics Data System (ADS)

Falke, S. R.; Husar, R. B.; Yang, C. P.; Robinson, E. M.; Fialkowski, W. E.

2009-04-01

Web portals are intended to provide consolidated discovery, filtering and aggregation of content from multiple, distributed web sources targeted at particular user communities. This paper presents a standards-based information architectural approach to developing portals aimed at air quality community collaboration in data access and analysis. An important characteristic of the approach is to advance beyond the present stand-alone design of most portals to achieve interoperability with other portals and information sources. We show how using metadata standards, web services, RSS feeds and other Web 2.0 technologies, such as Yahoo! Pipes and del.icio.us, helps increase interoperability among portals. The approach is illustrated within the context of the GEOSS Architecture Implementation Pilot where an air quality community portal is being developed to provide a user interface between the portals and clearinghouse of the GEOSS Common Infrastructure and the air quality community catalog of metadata and data services.

Developing a multidisciplinary robotic surgery quality assessment program.

PubMed

Gonsenhauser, Iahn; Abaza, Ronney; Mekhjian, Hagop; Moffatt-Bruce, Susan D

2012-01-01

The objective of this study was to test the feasibility of a novel quality-improvement (QI) program designed to incorporate multiple robotic surgical sub-specialties in one health care system. A robotic surgery quality assessment program was developed by The Ohio State University College of Medicine (OSUMC) in conjunction with The Ohio State University Medical Center Quality Improvement and Operations Department. A retrospective review of cases was performed using data interrogated from the OSUMC Information Warehouse from January 2007 through August 2009. Robotic surgery cases (n=2200) were assessed for operative times, length of stay (LOS), conversions, returns to surgery, readmissions and cancellations as potential quality indicators. An actionable and reproducible framework for the quality measurement and assessment of a multidisciplinary and interdepartmental robotic surgery program was successfully completed demonstrating areas for improvement opportunities. This report supports that standard quality indicators can be applied to multiple specialties within a health care system to develop a useful quality tracking and assessment tool in the highly specialized area of robotic surgery. © 2012 National Association for Healthcare Quality.

Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain

PubMed Central

Sugawara, Atsushi; Ward, Monika A.

2012-01-01

Preimplantation genetic diagnosis (PGD) is considered highly successful in respect to its accuracy in detecting genetic anomalies but the effects of embryo biopsy on embryonic/fetal growth and development are less known, particularly in conjunction with in vitro fertilization (IVF). Here, we compared biopsied (B) and non-biopsied (NB) mouse embryos for their developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the 4-cell stage, and were either cultured for 120 h and subjected to differential inner cell mass (ICM) and trophoblast (T) staining, or were transferred into the uterine tubes of surrogate mothers after 72 h of culture, to examine their pre- and post-implantation development, respectively. Non-biopsied embryos from the same IVF cohorts served as controls. Embryo biopsy negatively affected preimplantation development to blastocyst in C57BL/6 (69 vs 79%, P<0.01) but not in B6D2F1 mice (89 vs 91%, P=NS). Although B6 embryos had lower total cell number than F2 (B6: 47 and 61 vs. F1: 53 and 70; B and NB, respectively, P<0.05) there were no differences between B and NB blastocysts in %ICM (B6: 19.8 vs 19.8; F2: 20.9 vs 20.4, P=NS) and ICM:T ratio (B6: 4.7 vs 4.7; F2: 4.4 vs. 4.7) in both mouse strains. Post-implantation development to live fetuses of B embryos as compared to NB counterparts was impaired in C57BL/6 (6 vs 18%, P<0.001) but not in B6D2F1 mice (26 vs 35%, P=NS). We conclude that blastomere biopsy impairs embryonic/fetal development in mice known to be sensitive to in vitro culture and manipulations. Such mice model infertile couples with poor quality gametes seeking help in assisted reproduction technologies (ART) clinics. PMID:23174776

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

PubMed

Aberkane, A; Essahib, W; Spits, C; De Paepe, C; Sermon, K; Adriaenssens, T; Mackens, S; Tournaye, H; Brosens, J J; Van de, Velde H

2018-05-26

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in-vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six-days post fertilisation (6dpf) human embryos were co-cultured with Ishikawa cells for 12 h, 24 h (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 115 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV, and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expression was validated at the protein

Effect of micro-vibration culture system on embryo development.

PubMed

Hur, Yong Soo; Park, Jeong Hyun; Ryu, Eun Kyung; Park, Sung Jin; Lee, Jun Ho; Lee, Soo Hee; Yoon, Jung; Yoon, San Hyun; Hur, Chang Young; Lee, Won Don; Lim, Jin Ho

2013-06-01

Micro-vibration culture system was examined to determine the effects on mouse and human embryo development and possible improvement of clinical outcomes in poor responders. The embryonic development rates and cell numbers of blastocysts were compared between a static culture group (n = 178) and a micro-vibration culture group (n = 181) in mice. The embryonic development rates and clinical results were compared between a static culture group (n = 159 cycles) and a micro-vibration culture group (n = 166 cycles) in poor responders. A micro-vibrator was set at a frequency of 42 Hz, 5 s/60 min duration for mouse and human embryo development. The embryonic development rate was significantly improved in the micro-vibration culture group in mice (p < 0.05). The cell numbers of mouse blastocysts were significantly higher in the micro-vibration group than in the static culture group (p < 0.05). In the poor responders, the rate of high grade embryos was not significantly improved in the micro-vibration culture group on day 3. However, the optimal embryonic development rate on day 5 was improved in the micro-vibration group, and the total pregnancy rate and implantation rate were significantly higher in the micro-vibration group than in the static culture group (p < 0.05). Micro-vibration culture methods have a beneficial effect on embryonic development in mouse embryos. In poor responders, the embryo development rate was improved to a limited extent under the micro-vibration culture conditions, but the clinical results were significantly improved.

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

PubMed

Oliveira, C S; de Souza, M M; Saraiva, N Z; Tetzner, T A D; Lima, M R; Lopes, F L; Garcia, J M

2012-06-01

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. © 2011 Blackwell Verlag GmbH.

The quality and availability of hardwood logging residue based on developed quality levels

Treesearch

Floyd G. Timson

1980-01-01

Hardwood logging residue was examined for salvageable quality material. Four quality levels (QL 1 to QL 4), based on four sets of specifications, were developed. The specifications used surface indicators, sweep, center decay, and piece size to determine quality. Twenty-six percent of the total logging residue (residue ≥ 4 inches in diameter outside bark at...

Emblems of Quality in Higher Education. Developing and Sustaining High-Quality Programs.

ERIC Educational Resources Information Center

Haworth, Jennifer Grant; Conrad, Clifton F.

This book proposes an "engagement" theory of program quality to evaluate and improve higher education programs at all degree levels. Based on interviews with 781 participants in a national study of Masters degree programs, it focuses on the interactive roles of students, faculty, and administrators in developing high-quality programs…

Development of an Instructional Quality Assurance Model in Nursing Science

ERIC Educational Resources Information Center

Ajpru, Haruthai; Pasiphol, Shotiga; Wongwanich, Suwimon

2011-01-01

The purpose of this study was to develop an instructional quality assurance model in nursing science. The study was divided into 3 phases; (1) to study the information for instructional quality assurance model development (2) to develop an instructional quality assurance model in nursing science and (3) to audit and the assessment of the developed…

Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development

PubMed Central

Yoisungnern, Ton; Choi, Yun-Jung; Woong Han, Jae; Kang, Min-Hee; Das, Joydeep; Gurunathan, Sangiliyandi; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Kyung Chang, Won; Chang, Byung-Soo; Parnpai, Rangsun; Kim, Jin-Hoi

2015-01-01

Silver nanoparticles (AgNPs) have many features that make them attractive as medical devices, especially in therapeutic agents and drug delivery systems. Here we have introduced AgNPs into mouse spermatozoa and then determined the cytotoxic effects of AgNPs on sperm function and subsequent embryo development. Scanning electron microscopy and transmission electron microscopy analyses showed that AgNPs could be internalized into sperm cells. Furthermore, exposure to AgNPs inhibited sperm viability and the acrosome reaction in a dose-dependent manner, whereas sperm mitochondrial copy numbers, morphological abnormalities, and mortality due to reactive oxygen species were significantly increased. Likewise, sperm abnormalities due to AgNPs internalization significantly decreased the rate of oocyte fertilization and blastocyst formation. Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes. Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development. Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs. PMID:26054035

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

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

The Effects of Development Team Skill on Software Product Quality

NASA Technical Reports Server (NTRS)

Beaver, Justin M.; Schiavone, Guy A.

2006-01-01

This paper provides an analysis of the effect of the skill/experience of the software development team on the quality of the final software product. A method for the assessment of software development team skill and experience is proposed, and was derived from a workforce management tool currently in use by the National Aeronautics and Space Administration. Using data from 26 smallscale software development projects, the team skill measures are correlated to 5 software product quality metrics from the ISO/IEC 9126 Software Engineering Product Quality standard. in the analysis of the results, development team skill is found to be a significant factor in the adequacy of the design and implementation. In addition, the results imply that inexperienced software developers are tasked with responsibilities ill-suited to their skill level, and thus have a significant adverse effect on the quality of the software product. Keywords: software quality, development skill, software metrics

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

PubMed

Chen, Paula R; Redel, Bethany K; Spate, Lee D; Ji, Tieming; Salazar, Shirley Rojas; Prather, Randall S

2018-05-31

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism were corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs.

PubMed

Lee, Joohyeong; Park, Jong-Im; Yun, Jung Im; Lee, Yongjin; Yong, Hwanyul; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Geun-Shik; Lee, Eunsong

2015-01-01

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.

Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs

PubMed Central

Lee, Joohyeong; Park, Jong-Im; Yun, Jung Im; Lee, Yongjin; Yong, Hwanyul; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Geun-Shik

2015-01-01

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs. PMID:25797293

The ovine uterus as a host for in vitro-produced bovine embryos.

PubMed

Rexroad, C E; Powell, A M

1999-07-15

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.

Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

PubMed

Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

2016-10-01

Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1-/- female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1-/- mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

DEVELOPING WATER QUALITY CRITERIA FOR SUSPENDED AND BEDDED SEDIMENTS

EPA Science Inventory

The U.S. EPA’s Framework for Developing Suspended and Bedded Sediments (SABS) Water Quality Criteria (SABS Framework) is a nationally-consistent process for developing ambient sediment quality criteria for surface waters. The SABS Framework accommodates natural variation among wa...

Day 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles.

PubMed

Li, Ryh-Sheng; Hwu, Yuh-Ming; Lee, Robert Kuo-Kuang; Li, Sheng-Hsiang; Lin, Ming-Huei

2018-02-01

Embryo transfers during cleavage stage (day 2 or day 3) and blastocyst stages (day 5 or day 6) are common in current daily practice in fresh IVF/ET cycles. Data regarding transferring day 4 embryos, morula/compact stage, is still restricted and the grading system is also inconsistent, as between IVF clinics. This study provided a new detailed classification system for morula/compact stage embryos and compared successes rates between day 4 and day 5 ET. This was a retrospective study. A review of medical records from January 1st, 2013, to December 31st 2015, performed for all conventional insemination and ICSI cycles with a GnRH-antagonist protocol at the Infertility Division of MacKay Memorial Hospital in Taipei City, Taiwan. There were 427 cycles included in our study, 107 in study group (day 4 MET) and 320 in control group (day 5 BET). Pregnancy rates and live birth rate were compatible, as between morula embryo transfer (MET) and blastocyst embryo transfer (BET). The implantation rate (36.3% vs. 39.6%, respectively, p = 0.500), clinical pregnancy rate (49.5% vs. 51.9%, respectively, p = 0.737), and live birth rate (42.1% vs. 45.6%, respectively, p = 0.574) were statistically insignificant between groups. The term birth rate was statistically higher in the MET group than in the BET group (95.7% vs. 79.5%, respectively, p = 0.006). When the clinical outcomes between day 4 good MET and day 5 good BET were compared, the results were compatible. The implantation rate (48.8% vs. 41.1%, respectively, p = 0.335), clinical pregnancy rate (55.0% vs. 53.2%, respectively, p = 0.867), and live birth rate (47.5% vs. 47.1%, respectively, p = 1.000) showed no significant difference. The term birth rate was also higher in day 4 good MET group than in day 5 good BET group (100% vs. 78.3%, respectively, p = 0.025). In this study, we performed day 4 MET avoid BET on Sunday. The grading system we provided was more detailed for embryo selection and it was easier to

Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

PubMed

Bodri, Daniel; Kato, Ryutaro; Kondo, Masae; Hosomi, Naoko; Katsumata, Yoshinari; Kawachiya, Satoshi; Matsumoto, Tsunekazu

2015-05-01

To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. Retrospective case series. Private infertility clinic. Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

PubMed

Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

In vitro development and cytological quality of inter-species (porcine→bovine) cloned embryos are affected by trichostatin A-dependent epigenomic modulation of adult mesenchymal stem cells.

PubMed

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.

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

PubMed

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

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

PubMed Central

Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Somfai, Tamás; Inaba, Yasushi; Hirayama, Muneyuki; Yamanouchi, Tadayuki; Matsuda, Hideo; Kobayashi, Shuji; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

2012-01-01

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos. PMID:22590579

Pharmaceutical product development: A quality by design approach

PubMed Central

Pramod, Kannissery; Tahir, M. Abu; Charoo, Naseem A.; Ansari, Shahid H.; Ali, Javed

2016-01-01

The application of quality by design (QbD) in pharmaceutical product development is now a thrust area for the regulatory authorities and the pharmaceutical industry. International Conference on Harmonization and United States Food and Drug Administration (USFDA) emphasized the principles and applications of QbD in pharmaceutical development in their guidance for the industry. QbD attributes are addressed in question-based review, developed by USFDA for chemistry, manufacturing, and controls section of abbreviated new drug applications. QbD principles, when implemented, lead to a successful product development, subsequent prompt regulatory approval, reduce exhaustive validation burden, and significantly reduce post-approval changes. The key elements of QbD viz., target product quality profile, critical quality attributes, risk assessments, design space, control strategy, product lifecycle management, and continual improvement are discussed to understand the performance of dosage forms within design space. Design of experiments, risk assessment tools, and process analytical technology are also discussed for their role in QbD. This review underlines the importance of QbD in inculcating science-based approach in pharmaceutical product development. PMID:27606256

Pharmaceutical product development: A quality by design approach.

PubMed

Pramod, Kannissery; Tahir, M Abu; Charoo, Naseem A; Ansari, Shahid H; Ali, Javed

2016-01-01

The application of quality by design (QbD) in pharmaceutical product development is now a thrust area for the regulatory authorities and the pharmaceutical industry. International Conference on Harmonization and United States Food and Drug Administration (USFDA) emphasized the principles and applications of QbD in pharmaceutical development in their guidance for the industry. QbD attributes are addressed in question-based review, developed by USFDA for chemistry, manufacturing, and controls section of abbreviated new drug applications. QbD principles, when implemented, lead to a successful product development, subsequent prompt regulatory approval, reduce exhaustive validation burden, and significantly reduce post-approval changes. The key elements of QbD viz., target product quality profile, critical quality attributes, risk assessments, design space, control strategy, product lifecycle management, and continual improvement are discussed to understand the performance of dosage forms within design space. Design of experiments, risk assessment tools, and process analytical technology are also discussed for their role in QbD. This review underlines the importance of QbD in inculcating science-based approach in pharmaceutical product development.

Leading quality through the development of a multi-year corporate quality plan: sharing The Ottawa Hospital experience.

PubMed

Hunter, Linda; Myles, Joanne; Worthington, James R; Lebrun, Monique

2011-01-01

This article discusses the background and process for developing a multi-year corporate quality plan. The Ottawa Hospital's goal is to be a top 10% performer in quality and patient safety in North America. In order to create long-term measurable and sustainable changes in the quality of patient care, The Ottawa Hospital embarked on the development of a three-year strategic corporate quality plan. This was accomplished by engaging the organization at all levels and defining quality frameworks, aligning with internal and external expectations, prioritizing strategic goals, articulating performance measurements and reporting to stakeholders while maintaining a transparent communication process. The plan was developed through an iterative process that engaged a broad base of health professionals, physicians, support staff, administration and senior management. A literature review of quality frameworks was undertaken, a Quality Plan Working Group was established, 25 key stakeholder interviews were conducted and 48 clinical and support staff consultations were held. The intent was to gather information on current quality initiatives and challenges encountered and to prioritize corporate goals and then create the quality plan. Goals were created and then prioritized through an affinity exercise. Action plans were developed for each goal and included objectives, tasks and activities, performance measures (structure, process and outcome), accountabilities and timelines. This collaborative methodology resulted in the development of a three-year quality plan. Six corporate goals were outlined by the tenets of the quality framework for The Ottawa Hospital: access to care, appropriate care (effective and efficient), safe care and satisfaction with care. Each of the six corporate goals identified objectives and supporting action plans with accountabilities outlining what would be accomplished in years one, two and three. The three-year quality plan was approved by senior

Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

PubMed

Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

2014-10-13

Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

PubMed

Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T

2016-12-01

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.

Quality Assurance Project Plan Development Tool

EPA Pesticide Factsheets

This tool contains information designed to assist in developing a Quality Assurance (QA) Project Plan that meets EPA requirements for projects that involve surface or groundwater monitoring and/or the collection and analysis of water samples.

Gene expression of porcine blastocysts from gilts fed organic or inorganic selenium and pyridoxine.

PubMed

Dalto, B D; Tsoi, S; Audet, I; Dyck, M K; Foxcroft, G R; Matte, J J

2015-01-01

In this study, we determined how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of porcine expanded blastocysts (PEB) during pregnancy. Eighteen gilts were randomly assigned to one of the three experimental diets (n=6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT+0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT+0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and killed 5 days later for embryo harvesting. A porcine embryo-specific microarray was used to detect differentially gene expression between MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610. CONT gilts had lower whole blood Se and erythrocyte pyridoxal-5-P concentrations than supplemented gilts (P<0.05). No treatment effect was observed on blood plasma Se-glutathione peroxidase activity (P=0.57). There were 10, 247, and 96 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610 respectively. No specific biological process was associated with MSeB610 vs CONT. However, for OSeB610 vs CONT, upregulated genes were related with global protein synthesis but not to selenoproteins. The stimulation of some genes related with monooxygenase and thioredoxin families was confirmed by quantitative real-time RT-PCR. In conclusion, OSeB610 affects PEB metabolism more markedly than MSeB610. Neither Se sources with pyridoxine influenced the Se-glutathione peroxidase metabolic pathway in the PEB, but OSeB610 selectively stimulated genes involved with antioxidant defense. © 2015 Society for Reproduction and Fertility.

Quality Assurance Through Quality Improvement and Professional Development in the National Breast and Cervical Cancer Early Detection Program

PubMed Central

Siegl, Elvira J.; Miller, Jacqueline W.; Khan, Kris; Harris, Susan E.

2015-01-01

Quality assurance (QA) is the process of providing evidence that the outcome meets the established standards. Quality improvement (QI), by contrast, is the act of methodically developing ways to meet acceptable quality standards and evaluating current processes to improve overall performance. In the case of the National Breast and Cervical Cancer Early Detection Program (NBCCEDP), the desired outcome is the delivery of quality health care services to program clients. The NBCCEDP provides professional development to ensure that participating providers have current knowledge of evidence-based clinical standards regarding breast and cervical cancer screening and diagnosis and are monitoring women with abnormal screening results for timely follow-up. To assess the quality of clinical care provided to NBCCEDP clients, performance data are collected by NBCCEDP grantees and compared against predetermined Centers for Disease Control and Prevention (CDC) benchmarks known as Data Quality Indicator Guides. In this article, the authors describe 1) the development and use of indicators for QI in the NBCCEDP and 2) the professional development activities implemented to improve clinical outcomes. QA identifies problems, whereas QI systematically corrects them. The quality of service delivery and improved patient outcomes among NBCCEDP grantees has enhanced significantly because of continuous monitoring of performance and professional development. By using QA, NBCCEDP grantees can maximize the quality of patient screening, diagnostic services, and follow-up. Examples of grantee activities to maintain quality of care are also described in this report. PMID:25099901

Embryo-endometrial interactions during early development after embryonic diapause in the marsupial tammar wallaby.

PubMed

Renfree, Marilyn B; Shaw, Geoff

2014-01-01

The marsupial tammar wallaby has the longest period of embryonic diapause of any mammal. Reproduction in the tammar is seasonal, regulated by photoperiod and also lactation. Reactivation is triggered by falling daylength after the austral summer solstice in December. Young are born late January and commence a 9-10-month lactation. Females mate immediately after birth. The resulting conceptus develops over 6- 7 days to form a unilaminar blastocyst of 80-100 cells and enters lactationally, and later seasonally, controlled diapause. The proximate endocrine signal for reactivation is an increase in progesterone which alters uterine secretions. Since the diapausing blastocyst is surrounded by the zona and 2 other acellular coats, the mucoid layer and shell coat, the uterine signals that maintain or terminate diapause must involve soluble factors in the secretions rather than any direct cellular interaction between uterus and embryo. Our studies suggest involvement of a number of cytokines in the regulation of diapause in tammars. The endometrium secretes platelet activating factor (PAF) and leukaemia inhibitory factor, which increase after reactivation. Receptors for PAF are low on the blastocyst during diapause but are upregulated at reactivation. Conversely, there is endometrial expression of the muscle segment homeobox gene MSX2 throughout diapause, but it is rapidly downregulated at reactivation. These patterns are consistent with those observed in diapausing mice and mink after reactivation, despite the very different patterns of endocrine control of diapause in these 3 divergent species. These common patterns suggest a similar underlying mechanism for diapause, perhaps common to all mammals, but which is activated in only a few.

Effect of Sex of Embryo on Developmental Competence, Epigenetic Status, and Gene Expression in Buffalo (Bubalus bubalis) Embryos Produced by Hand-Made Cloning.

PubMed

Sandhu, Anjit; Mohapatra, Sushil K; Agrawal, Himanshu; Singh, Manoj K; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S

2016-10-01

Buffalo embryos were produced by hand-made cloning using skin fibroblasts from male and female buffaloes (n = 4 each) as donor cells for examining the effect of sex. Although the rate of blastocyst formation (43.8% ± 1.31% vs. 42.2% ± 1.22%) was similar, the total cell number (333 ± 10.4 vs. 270 ± 10.9) was higher (p < 0.05) whereas the apoptotic index (6.39 ± 0.25 vs. 8.52 ± 0.38) was lower (p < 0.05) for male than for female blastocysts. In the blastocysts, the global level of H3K18ac was found to be in the following order: male>female>IVF (in vitro fertilization) blastocysts (p < 0.05). The global level of H3K9me2 was not significantly different between male and female blastocysts and was higher (p < 0.05) compared with that in their IVF counterparts. The relative mRNA abundance of X-chromosome-linked (XIST, HPRT, PGK, and G6PD), apoptosis- (CASPASE3) and pregnancy-related genes (IFN-τ) was significantly higher (p < 0.05) whereas that of DNMT1 was significantly lower (p < 0.05) in female than in male blastocysts; however, in the case of apoptosis- (BCL-XL) and developmental competence-related genes (IGF1R and OCT4), the expression level was similar between the two groups. The gene expression level of OCT4 and IFN-τ but not of IGF1R was significantly lower (p < 0.05) in cloned than in IVF blastocysts. This study demonstrates that the epigenetic status, quality, and expression level of several genes but not the developmental competence are affected by the sex of cloned embryos.

Improving in vitro maturation and pregnancy outcome in cattle using a novel oocyte shipping and maturation system not requiring a CO₂ gas phase.

PubMed

Barceló-Fimbres, M; Campos-Chillón, L F; Mtango, N R; Altermatt, J; Bonilla, L; Koppang, R; Verstegen, J P

2015-07-01

The present work evaluated the benefit of a novel shipping and maturation medium (SMM) not requiring a CO2 gas for maturation and subsequent embryonic development of slaughterhouse and ovum pickup (OPU) bovine cumulus-oocyte complexes (COCs). Four experiments were conducted. In experiment 1, COCs were maturated for 18 hours in SMM and then incubated for 6 hours in, or 24 hours in a conventional system (control). Experiment 2 compared maturation for 24 hours in SMM versus 24 hours in the control. Experiment 3 compared three different incubation temperatures (37 °C, 38 °C, and 38.5 °C) for COCs maturation in SMM. In experiment 4, COCs obtained from 166 OPU sessions (representing two dairy and two beef breeds) in two locations (Wisconsin and California) were matured in SMM or control and evaluated relative to embryo production and pregnancy rates. Frozen semen was used for all experiments. The results for experiment 1 showed that the blastocyst rate and total embryo production rate (TE, Day-7 morulae plus all blastocysts) were higher for SMM than those in the control. However, no differences were observed for cleavage rate or blastocyst stage. In experiment 2, the blastocyst rate and TE were higher for SMM than those in the control; however, there was no difference for cleavage rate, total cell number, blastocyst stage. In experiment 3, the cleavage rate was similar, but the blastocyst rate and TE were greater for 38.5 °C than those for 38.0 °C and 37.5 °C. For experiment 4, Wisconsin OPU-derived COCs had a greater cleavage rate, blastocyst rate, TE, and blastocyst stage for SMM versus control. There were no breed effects. For the California trial, OPU-derived COCs matured in SMM had similar cleavage and pregnancy rates at Day 35 but greater blastocyst rates and transferred embryos per session than the control, which resulted in 2.2 more pregnancies per OPU session. Holstein COCs had superior embryonic development but similar pregnancy compared with Jersey. We

Estimation of the optimal timing of fertilization for embryo development of in vitro-matured bovine oocytes based on the times of nuclear maturation and sperm penetration.

PubMed

Koyama, Keisuke; Kang, Sung-Sik; Huang, Weiping; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

2014-05-01

The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14-22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12-30 hr, sperm penetration was examined after 4-18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12-30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x - 0.297x(2), P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr.

Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type.

PubMed

Lagutina, Irina; Lazzari, Giovanna; Duchi, Roberto; Colleoni, Silvia; Ponderato, Nunzia; Turini, Paola; Crotti, Gabriella; Galli, Cesare

2005-10-01

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

The development of the Pictorial Thai Quality of Life.

PubMed

Phattharayuttawat, Sucheera; Ngamthipwatthana, Thienchai; Pitiyawaranun, Buncha

2005-11-01

"Quality of life" has become a main focus of interest in medicine. The Pictorial Thai Quality of Life (PTQL) was developed in order to measure the Thai mental illness both in a clinical setting and community. The purpose of this study was to develop the Pictorial Thai Quality of Life (PTQL), having adequate and sufficient construct validity, discriminant power, concurrent validity, and reliability. To develop the Pictorial Thai Quality of Life Test, two samples groups were used in the present study: (1) pilot study samples: 30 samples and (2) survey samples were 672 samples consisting of normal, and psychiatric patients. The developing tests items were collected from a review of the literature in which all the items were based on the WHO definition of Quality of Life. Then, experts judgment by the Delphi technique was used in the first stage. After that a pilot study was used to evaluate the testing administration, and wording of the tests items. The final stage was collected data from the survey samples. The results of the present study showed that the final test was composed 25 items. The construct validity of this test consists of six domains: Physical, Cognitive, Affective, Social Function, Economic and Self-Esteem. All the PTQL items have sufficient discriminant power It was found to be statistically significant different at the. 001 level between those people with mental disorders and normal people. There was a high level of concurrent validity association with WHOQOL-BREF, Pearson correlation coefficient and Area under ROC curve were 0.92 and 0.97 respectively. The reliability coefficients for the Alpha coefficients of the PTQL total test was 0.88. The values of the six scales were from 0.81 to 0:91. The present study was directed at developing an effective psychometric properties pictorial quality of life questionnaire. The result will be a more direct and meaningful application of an instrument to detect the mental health illness poor quality of life in

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

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.

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

PubMed

Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

2016-06-01

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

Develop to Term Rat Oocytes Injected with Heat-Dried Sperm Heads

PubMed Central

Lee, Kyung-Bon; Park, Ki-Eun; Kwon, In-Kiu; Tripurani, Swamy K.; Kim, Keun Jung; Lee, Ji Hye; Niwa, Koji; Kim, Min Kyu

2013-01-01

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term. PMID:24223784

Effect of triiodothyronine on developmental competence of bovine oocytes.

PubMed

Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

2013-09-01

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

Developing and Implementing a Quality Assurance Strategy for Electroconvulsive Therapy.

PubMed

Hollingsworth, Jessa; Baliko, Beverly; McKinney, Selina; Rosenquist, Peter

2018-04-17

The literature provides scant guidance in effective quality assurance strategies concerning the use of electroconvulsive therapy (ECT) for the treatment of psychiatric conditions. Numerous guidelines are published that provide guidance in the delivery of care; however, little has been done to determine how a program or facility might ensure compliance to best practice for safety, tolerability, and efficacy in performing ECT. The objective of this project was to create a quality assurance strategy specific to ECT. Determining standards for quality care and clarifying facility policy were key outcomes in establishing an effective quality assurance strategy. An audit tool was developed utilizing quality criteria derived from a systematic review of ECT practice guidelines, peer review, and facility policy. All ECT procedures occurring over a 2-month period of May to June 2017 were retrospectively audited and compared against target compliance rates set for the facility's ECT program. Facility policy was adapted to reflect quality standards, and audit findings were used to inform possible practice change initiatives, were used to create benchmarks for continuous quality monitoring, and were integrated into regular hospital quality meetings. Clarification on standards of care and the use of clinical auditing in ECT was an effective starting point in the development of a quality assurance strategy. Audit findings were successfully integrated into the hospital's overall quality program, and recognition of practice compliance informed areas for future quality development and policy revision in this small community-based hospital in the southeastern United States. This project sets the foundation for a quality assurance strategy that can be used to help monitor procedural safety and guide future improvement efforts in delivering ECT. Although it is just the first step in creating meaningful quality improvement, setting clear standards and identifying areas of greatest

Friendship Quality Scale: Conceptualization, Development and Validation

ERIC Educational Resources Information Center

Thien, Lei Mee; Razak, Nordin Abd; Jamil, Hazri