Search

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 10 5  cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content., Competing Interests: Declaration of competing interest Tokunori Yamamoto is President of Meis Technology, Inc., which possesses a patent related to this work (Japan Patent No. 6970853). The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Research Question: Can a deep learning image analysis model be developed to assess oocyte quality by predicting blastocyst development from images of denuded mature oocytes?, Design: A deep learning model was developed utilizing 37,133 static oocyte images with associated laboratory outcomes from eight fertility clinics (six countries). A subset of data (n = 7807) was allocated to test model performance. External model validation was conducted to assess generalizability and robustness on new data (n = 12,357) from two fertility clinics (two countries). Performance was assessed by calculating area under the curve (AUC), balanced accuracy, specificity and sensitivity. Subgroup analyses were performed on the test dataset for age group, male factor and geographical location of the clinic. Model probabilities of the external dataset were converted to a 0-10 scoring scale to facilitate analysis of correlation with blastocyst development and quality., Results: The deep learning model demonstrated AUC of 0.64, balanced accuracy of 0.60, specificity of 0.55 and sensitivity of 0.65 on the test dataset. Subgroup analyses displayed the highest performance for age group 38-39 years (AUC 0.68), a negligible impact of male factor, and good model generalizability across geographical locations. Model performance was confirmed on external data: AUC of 0.63, balanced accuracy of 0.58, specificity of 0.57 and sensitivity of 0.59. Analysis of the scoring scale revealed that higher scoring oocytes correlated with higher likelihood of blastocyst development and good-quality blastocyst formation., Conclusion: The deep learning model showed a favourable performance for the evaluation of oocytes in terms of competence to develop into a blastocyst, and when the predictions were converted into scores, they correlated with blastocyst quality. This represents a significant first step in oocyte evaluation for scientific and clinical applications., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Embryonic diapause in mammals is a period of developmental pause of the embryo at the blastocyst stage. During diapause, the blastocyst has minimal cell proliferation, metabolic activity and gene expression. At reactivation, blastocyst development resumes, characterised by increases in cell number, biosynthesis and metabolism. Until recently, it has been unknown how diapause is maintained without any loss of blastocyst viability. This review focuses on recent progress in the identification of molecular pathways occurring in the blastocyst that can both cause and maintain the diapause state. A switch to lipid metabolism now appears essential to maintaining the diapause state and is induced by forkhead box protein O1. The forkhead box protein O transcription family is important for diapause in insects, nematodes and fish, but this is the first time a conclusive role has been established in mammals. Multiple epigenetic modifications are also essential to inducing and maintaining the diapause state, including both DNA and RNA methylation mechanisms. Finally, it now appears that diapause embryos, dormant stem cells and chemotherapeutic-resistant cancer cells may all share a universal system of quiescence., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jane Fenelon reports a relationship with Colossal Biosciences that includes: employment., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g -1 (PFOS-02) representing average human exposure and 53 ng g -1 (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic changes seen in the blastocysts. The gene expression changes were more pronounced after exposure to PFOS-53 compared to PFOS-02. DNA-methylation changes were associated with similar biological functions as the transcriptomic data, with the most significantly associated pathway being TP53. Collectively, these results reveal that brief PFOS exposure during oocyte maturation alters the early embryo development at concentrations relevant to humans. This study adds to the evidence that PFOS has the potential to affect female fertility., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: An increased incidence of monozygotic twinning after a blastocyst transfer has been previously reported in assisted reproductive technology treatment. It is uncertain whether this phenomenon is due to the extended culture time, culture medium, or inherent blastocyst parameters., Objective: This study aimed to investigate the association between blastocyst parameters (in vitro culture time, blastocyst stage, and inner cell mass and trophectoderm grading) and the incidence of monozygotic twinning after assisted reproductive technology., Study Design: This was a retrospective cohort study employing data from a multicenter, large, electronic database from 4 academic hospitals. All clinical pregnancies after a single blastocyst transfer between January 2014 and February 2020 were included. Blastocyst morphology was evaluated based on the Gardner grading system, considering the blastocyst stage, and inner cell mass and trophectoderm grading (grades A, B, and C). Monozygotic twinning was defined as ≥2 fetal heartbeats in a single gestational sac or 2 gestational sacs with sex concordance at birth. The multivariable predicted marginal proportions from logistic regression models were used to compute adjusted relative risks for the association between blastocyst parameters and the incidence of monozygotic twinning., Results: The overall monozygotic twinning rate was 1.53% (402 of 26,254 cases). The monozygotic twinning was not associated with the culture time in vitro (day 5 vs day 6) or blastocyst stage (early, blastocyst, expanded, hatching, and hatched). Alternatively, monozygotic twinning was associated with lower inner cell mass grading (B vs A: adjusted relative risk, 1.67 [95 % confidence interval, 1.28-2.25]; C vs A: adjusted relative risk, 1.98 [95% confidence interval, 1.18-3.11]) and higher trophectoderm grading (B vs C: adjusted relative risk, 1.38 [95% confidence interval, 1.03-1.92]; A vs C: adjusted relative risk, 2.14 [95% confidence interval, 1.45-3.20]). The incidence of monozygotic twinning was the lowest in blastocysts with grade A inner cell mass and grade B or C trophectoderm (0.82%, as the reference) and the highest in blastocysts with grade B or C inner cell mass and grade A trophectoderm (2.40%; adjusted relative risk, 2.62; 95% confidence interval, 1.60-4.43). The incidence of monozygotic twinning in blastocysts with consistent inner cell mass or trophectoderm grading was somewhere in between (both A: 1.58%; adjusted relative risk, 1.86 [95% confidence interval, 1.23-3.04]; both B or C: 1.59%; adjusted relative risk, 1.84 [95% confidence interval, 1.29-2.90])., Conclusion: Higher risk of monozygotic twinning was associated with blastocyst morphology specific to those blastocysts with loosely arranged inner cell mass cells combined with tightly packed trophectoderm cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Research Question: What impact does maternal age and embryo morphology have on sustained implantation rates of euploid blastocysts?, Design: This was a retrospective analysis of sustained implantation rates of euploid blastocysts stratified by maternal age and morphology. The primary analysis included 208 embryo transfers with a total of 229 embryos transferred from January 2017 through August 2020., Results: For all ages the sustained implantation rates for day 5 good quality blastocysts were higher than for day 5 fair, day 5 poor and day 6 blastocysts. At a maternal age of 36 years the best-fit sustained implantation rates were 86% for day 5 good quality blastocysts, 64% for day 5 fair, 63% for day 5 poor, and 51% for all day 6 blastocysts analysed as one group. When controlling for morphology and day of biopsy, there were higher sustained implantation rates for euploid embryos of younger patients compared with older patients. The best-fit sustained implantation rates for age 33 compared to age 39 years were 86% versus 80% for day 5 good, 71% versus 62% for day 5 fair, 59% versus 55% for day 5 poor, and 81% versus 46% for all day 6., Conclusions: There was a clinically significant higher sustained implantation rate at all ages for euploid day 5 good quality embryos compared with day 5 fair, day 5 poor and day 6 embryos., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

WW domain-containing transcription regulator 1 (WWTR1) is one of the primary effectors in the Hippo pathway, which plays essential roles in cell differentiation into trophectoderm (TE) and inner cell mass cell lineages at the blastocyst stage. However, little is known about the roles of WWTR1 in preimplantation development. The present study aimed to explore the significance of WWTR1 expression in preimplantation development using an mRNA knockdown (KD) system in bovine embryos. We first quantitated WWTR1 expression at protein and mRNA levels from fertilization to blastocyst stage. WWTR1 proteins gradually shifted from extranuclear localization during the 16-cell stage to nuclear localization by morula stage. WWTR1 mRNA expression was also transiently upregulated at the 16-cell stage. WWTR1 KD efficiently repressed WWTR1 expression at protein and mRNA levels. The WWTR1 KD embryos developed to the blastocyst stage at rates equivalent to those of controls, but TE cell numbers were significantly decreased. Representative TE-expressed genes, including CDX2 and IFNT were also significantly decreased in WWTR1 KD blastocysts. These results provide the first demonstration that WWTR1 expression is responsible for normal TE cell development in preimplantation embryos., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Research Question: The study aimed to develop an artificial intelligence model based on artificial neural networks (ANNs) to predict the likelihood of achieving a live birth using the proteomic profile of spent culture media and blastocyst morphology., Design: This retrospective cohort study included 212 patients who underwent single blastocyst transfer at IVI Valencia. A single image of each of 186 embryos was studied, and the protein profile was analysed in 81 samples of spent embryo culture medium from patients included in the preimplantation genetic testing programme. The information extracted from the analyses was used as input data for the ANN. The multilayer perceptron and the back-propagation learning method were used to train the ANN. Finally, predictive power was measured using the area under the curve (AUC) of the receiver operating characteristic curve., Results: Three ANN architectures classified most of the embryos correctly as leading (LB+) or not leading (LB-) to a live birth: 100.0% for ANN1 (morphological variables and two proteins), 85.7% for ANN2 (morphological variables and seven proteins), and 83.3% for ANN3 (morphological variables and 25 proteins). The artificial intelligence model using information extracted from blastocyst image analysis and concentrations of interleukin-6 and matrix metalloproteinase-1 was able to predict live birth with an AUC of 1.0., Conclusions: The model proposed in this preliminary report may provide a promising tool to select the embryo most likely to lead to a live birth in a euploid cohort. The accuracy of prediction demonstrated by this software may improve the efficacy of an assisted reproduction treatment by reducing the number of transfers per patient. Prospective studies are, however, needed., (Copyright © 2020 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 μg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 μg/mL and ETU at 300 μg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 μg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor β and integrin β3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor β and integrin β3 expression of endometrial epithelial cells., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Research Question: Day of cryopreservation, inner cell mass (ICM) grade, trophectoderm grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. This study sought to examine the likelihood of live birth and whether the morphological grade of the blastocyst is more or equally useful in FET cycles among preimplantation genetic testing for aneuploidies (PGT-A) tested and untested blastocysts., Design: This was a retrospective cohort study of 6271 vitrified-warmed, autologous, single-embryo transfer cycles among patients undergoing IVF from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equations., Results: Among PGT-A tested embryos, inferior ICM grade was associated with a lower chance of live birth (ICM grade B versus A: adjusted risk ratio [aRR] 0.91, 95% confidence interval [CI] 0.84-0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved on day 6 versus day 5 (aRR 0.87, 95% CI 0.78-0.96), and those with inferior pre-vitrification trophectoderm grade (trophectoderm grade B versus A: aRR 0.86, 95% CI 0.79-0.94). Blastocysts with a higher pre-vitrification expansion grade (pre-vitrification expansion grade 5 versus 4: aRR 1.1, 95% CI 1.01-1.2) were associated, but ICM grade was not associated (ICM grade B versus A: aRR 0.93, 95% CI 0.86-1.02), with chance of live birth., Conclusions: Among PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, trophectoderm grade and expansion grade may help to identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphological grade and day of cryopreservation less important., (Copyright © 2020 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

During preimplantation development, the mouse embryo forms the blastocyst, which consists of a squamous epithelium enveloping a fluid-filled lumen and a cluster of pluripotent cells. The shaping of the blastocyst into its specific architecture is a prerequisite to implantation and further development of the embryo. Recent studies identified the central role of the actomyosin cortex in generating the forces driving the successive steps of blastocyst morphogenesis. As seen in other developing animals, actomyosin functions across spatial scales from the subcellular to the tissue levels. In addition, the slow development of the mouse embryo reveals that actomyosin contractility operates at multiple timescales with periodic cortical waves of contraction every ∼80 s and tissue remodeling over hours., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020. Published by Elsevier Ltd.)

After fertilization, mouse embryos go through preimplantation development to give rise to blastocyst. Two key molecular events, zygotic genome activation (ZGA) and the first cell lineage specification, are essential for the process. Recent advances in low-input epigenomics profiling techniques allow the analysis of these events at a molecular level, which revealed a critical role of epigenetic and chromatin reprogramming in ZGA and the first cell lineage specification. Additionally, the establishment of an in vitro embryonic stem cell (ESC) to two-cell embryo-like conversion system have also contributed to the molecular understanding of preimplantation development. In this review, we summarize recent advances in epigenetic regulation of mouse preimplantation development, point out the remaining questions, and propose strategies to tackle these questions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Study of early human embryo development is essential for advancing reproductive and regenerative medicine. Traditional human embryological studies rely on embryonic tissue specimens, which are difficult to acquire due to technical challenges and ethical restrictions. The availability of human stem cells with developmental potentials comparable to pre-implantation and peri-implantation human embryonic and extraembryonic cells, together with properly engineered in vitro culture environments, allow for the first time researchers to generate self-organized multicellular structures in vitro that mimic the structural and molecular features of their in vivo counterparts. The development of these stem cell-based, synthetic human embryo models offers a paradigm-shifting experimental system for quantitative measurements and perturbations of multicellular development, critical for advancing human embryology and reproductive and regenerative medicine without using intact human embryos., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Research Question: Is the spatiotemporal phenomenology of the cytoplasmic halo during fertilization related to embryonic competence?, Design: Time-lapse images from 1009 zygotes were retrospectively analysed from 560 patients who underwent IVF with minimal stimulation and single vitrified-warmed blastocyst transfer between April 2017 and March 2018. Halo presence and morphokinetics were monitored and compared relative to embryo quality, blastocyst expansion and ongoing pregnancy., Results: Halo was observed in 88% of fertilized oocytes. Embryos derived from zygotes without halo had significantly higher rates of rapid cleavage (P = 0.0004), cell fusion (P = 0.0028) and asymmetrical division (P = 0.0002) compared with those derived from zygotes with halo. Multivariate logistic regression analysis had significantly higher developmental rates compared with the expanded blastocyst stage in embryos displaying a halo, regardless of its distribution (adjusted odds ratio 0.435; P = 0.0004). Prolonged halo time intervals were significantly correlated with increased asymmetrical division at first cell division (P = 0.0412, P = 0.0088, respectively) and decreased developmental rates to expanded blastocyst stage (P = 0.0062, P = 0.0020, respectively). Additionally, prolonged presence of the cytoplasmic halo was associated with a decreased ongoing pregnancy rate (adjusted odds ratio 0.871; P = 0.006). Poor sperm quality and decreased oocyte diameter were correlated with absence of the cytoplasmic halo (P = 0.0477, P < 0.0001, respectively) or prolonged halo presence (P = 0.0139, P = 0.0002, respectively)., Conclusions: Halo presence and morphokinetics are associated with cleavage patterns, development to blastocyst stage and ongoing pregnancy rate after single blastocyst transfer. Halo morphokinetics seems to reflect sperm and oocyte quality. Cytoplasmic halo might be valuable predictor for refining selection of more developmentally competent blastocysts., (Copyright © 2020. Published by Elsevier Ltd.)

Objective: To determine whether preimplantation genetic testing for aneuploidy (PGT-A) could pick up true abnormalities that have clinical relevance., Materials and Methods: This was a retrospective cohort study of patients who underwent in vitro fertilization with PGT-A from 2015 to 2017. We evaluated the associations of aneuploidy and mosaicism with maternal age, the chromosome abnormalities present in individual chromosomes, and the effect of embryo sex on the proportion of each type of error in the four chromosomes most frequently affected., Result(s): A total of 1043 embryos from 255 patients (mean maternal age = 39 ± 4 years) were included in the initial analysis. Of these, 36% (377/1043) were euploid, 47% (487/1043) were aneuploid, 13% (140/1043) contained mosaicism, and 4% (39/1043) gave no result. We excluded the 39 embryos with no result; thus, 1004 embryos were included in the analysis. Increased aneuploidy was associated with increased maternal age, but the rate of embryo mosaicism was not. A combined analysis of aneuploidy with noncomplex abnormalities and mosaicism showed that chromosomes 22, 21, 16, and 15 were the most frequently involved. Chromosome 22 showed the highest proportion of mosaicism and chromosome 15 showed the highest proportion of aneuploidy. When we included embryo sex in the analysis, embryo sex was associated with these chromosome errors in the most susceptible chromosome, 22., Conclusion(s): PGT-A showed that chromosomes 22, 21, 16, and 15 were the most frequently involved among common chromosome abnormalities, comparable with those of published data analyzed from spontaneous abortion. This result suggested that PGT-A could pick up abnormalities that have clinical relevance to spontaneous abortion. Moreover, we identified a role of embryo sex in these chromosomal errors on chromosome 22., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Somatic cell nuclear transfer (SCNT) has potential applications in agriculture and biomedicine, but the efficiency of cloning is still low. In this study, the transcriptional profiles in cloned and fertilized embryos were measured and compared by RNA sequencing. The 2-cell embryos were detected to identify the earliest transcriptional differences between embryos derived through IVF and SCNT. As a result, 364 genes showed decreased expression in cloned 2-cell embryos and were enriched in "intracellular protein transport" and "ubiquitin mediated proteolysis". In blastocysts, 593 genes showed decreased expression in cloned blastocysts and were enriched in "RNA binding", "nucleotide binding", "embryo development", and "adherens junction". We identified 14 development related genes that were not activated in the cloned embryos. Then, 68 and 245 long non-coding RNAs were recognized abnormally expressed in cloned 2-cell embryos and cloned blastocysts, respectively. Furthermore, we found that incomplete RNA-editing occurred in cloned embryos and might be caused by decreased ADAR expression. In conclusion, our study revealed the abnormal transcripts and deficient RNA-editing sites in cloned embryos and provided new data for further mechanistic studies of somatic nuclear reprogramming., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The clinical application of a PGT-A program implementing single euploid embryo transfer is evaluated over a 6.5 year period, beginning with its early validation phases. Euploidy embryo status is inversely correlated to oocyte source age and positively correlated to blastocyst quality grades. However, once a single euploid embryo is transferred, high levels of implantation and live birth success are attained independent of patient age and embryo quality, with only AA blastocysts exhibiting improved implantation. Factors influencing successful outcomes are discussed, including the management of mosaic NGS profiles. Overall, distinct advantages to a dedicated PGT-A/single euploid embryo transfer program are clearly evident in per cycle start comparisons to control cycles and national average statistics by age groups., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO 2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO 2 - than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO 2 (20.3 ± 4.6%). Cleavage for MAT CR- CO 2 and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO 2 ), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte., (Copyright © 2020 Elsevier Inc. All rights reserved.)

At least two distinct pluripotent states, referred to as naïve and primed, define the early mammalian embryo. In the mouse, the pluripotent epiblast cells in the pre/peri-implantation embryo are the source of naïve embryonic stem cells (ESCs). After the embryo implants, the epiblast lineage generates a restricted or primed population of stem cells, referred to as epiblast stem cells (EpiSCs). ESCs can be cultured in EpiSC media to generate epiblast-like cells (EpiLCs). The differentiation of naive ESCs into primed EpiLCs permits insights into the development and differentiation of the pluripotent epiblast lineage. This chapter describes the generation and characterization of EpiSCs as well as EpiLCs., (© 2020 Elsevier Inc. All rights reserved.)

Development of the mammalian embryo begins with formation of the totipotent zygote during fertilization. This initial cell is able to give rise to every embryonic tissue of the developing organism as well as all extra-embryonic lineages, such as the placenta and the yolk sac, which are essential for the initial patterning and support growth of the fetus until birth. As the embryo transits from pre- to post-implantation, major structural and transcriptional changes occur within the embryonic lineage to set up the basis for the subsequent phase of gastrulation. Fine-tuned coordination of cell division, morphogenesis and differentiation is essential to ultimately promote assembly of the future fetus. Here, we review the current knowledge of mammalian development of both mouse and human focusing on morphogenetic processes leading to the onset of gastrulation, when the embryonic anterior-posterior axis becomes established and the three germ layers start to be specified., (© 2020 Elsevier Inc. All rights reserved.)

Background: Blastocyst morphology is a predictive marker for implantation success of in vitro fertilized human embryos. Morphology grading is therefore commonly used to select the embryo with the highest implantation potential. One of the challenges, however, is that morphology grading can be highly subjective when performed manually by embryologists. Grading systems generally discretize a continuous scale of low to high score, resulting in floating and unclear boundaries between grading categories. Manual annotations therefore suffer from large inter-and intra-observer variances., Method: In this paper, we propose a method based on deep learning to automatically grade the morphological appearance of human blastocysts from time-lapse imaging. A convolutional neural network is trained to jointly predict inner cell mass (ICM) and trophectoderm (TE) grades from a single image frame, and a recurrent neural network is applied on top to incorporate temporal information of the expanding blastocysts from multiple frames., Results: Results showed that the method achieved above human-level accuracies when evaluated on majority votes from an independent test set labeled by multiple embryologists. Furthermore, when evaluating implantation rates for embryos grouped by morphology grades, human embryologists and our method had a similar correlation between predicted embryo quality and pregnancy outcome., Conclusions: The proposed method has shown improved performance of predicting ICM and TE grades on human blastocysts when utilizing temporal information available with time-lapse imaging. The algorithm is considered at least on par with human embryologists on quality estimation, as it performed better than the average human embryologist at ICM and TE prediction and provided a slightly better correlation between predicted embryo quality and implantability than human embryologists., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Research Question: Does complete mechanical removal of the zona pellucida modify the outcome of transfer of vitrified-warmed human blastocysts?, Design: In a prospective randomized controlled study, 419 couples were allocated to either zona pellucida-free (n = 209) or zona intact (n = 210) vitrified-warmed embryo transfer. Main outcome measures included clinical pregnancy, implantation and ongoing pregnancy rates., Results: Transfer of zona pellucida-free blastocysts resulted in clinical pregnancy, implantation and ongoing pregnancy rates (35,9%, 33,9% and 32,1%, respectively), similar to those achieved with zona intact control embryos (39%, 36,4% and 33,1%, respectively)., Conclusion: Total mechanical removal of the zona pellucida did not affect the tested parameters of clinical outcomes., (Copyright © 2019. Published by Elsevier Ltd.)

This systematic review and meta-analysis determined the association between aspirated after ovarian stimulation and top/good quality embryos obtained in women undergoing ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI). MEDLINE, EMBASE, Scopus, CINAHL and Web of Science were searched for English-language publications on top/good-quality embryos at cleavage (day 2/3) and/or blastocyst (day 5/6) developmental stages, up to 18 November 2017. Twenty-eight studies (three prospective and 25 retrospective) reporting data on 291,752 assisted reproductive technology (ART) cycles were considered eligible. We confirmed a strong positive association between oocytes retrieved and top/good-quality day 2/3 embryos (weighted correlation coefficient [r w ] = 0.791), day 5/6 embryos (r w  = 0.901), metaphase II oocytes (r w  = 0.988), oocytes exhibiting two pronuclei (r w  = 0.987) and euploid embryos (r w  = 0.851); P < 0.001 for all correlations (evaluated in subsets of the 17 studies). Data from 5657 cycles showed that the group with the most oocytes aspirated had the most top/good-quality day 2/3 embryos (pooled standardized mean differences (high [>15] versus low [<4] 1.91, 95% confidence interval [CI] 1.05-2.77, P < 0.0001; high versus medium [4-15] 1.15, 95% CI 0.74-1.55, P < 0.0001; medium versus low 1.41, 95% CI 0.79-2.03, P < 0.0001). Individual participant meta-analysis would enable accurate determination of these associations and other outcomes., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-μm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-μm group (39%) was higher than that in the 77-μm group (28%), and the yield in the 57-μm group (31%) was the intermediate. The 37-μm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-μm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Research Question: Which blastocyst morphology parameter is associated with live birth after controlling for female age and endometrial receptivity?, Design: Retrospective study including fresh single blastocyst transfers (n = 2461) where the value of serum progesterone on day of human chorionic gonadotrophin trigger (PdHCG) was available. Generalized estimating equation regression models evaluated the independent effects of developmental stage (DevSt), inner cell mass (ICM) and trophectoderm grade on live birth rates while controlling for the confounding effects of female age and PdHCG., Results: DevSt was strongly associated with the probability of live birth (P < 0.0001) independently of female age (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.87-0.91) and PdHCG (OR 0.80, 95% CI 0.74-0.87). For full blastocysts, expanded blastocysts and hatching blastocysts, addition of ICM and trophectoderm grading in the multivariable analysis suggested that besides female age (OR 0.92, 95% CI 0.90-0.94) and PdHCG (OR 0.80, 95% CI 0.73-0.87), only DevSt (P = 0.001) and trophectoderm quality (P = 0.004) were independent predictors of live birth, while the predictive capacity of ICM was no longer significant. The mean probability of live birth was highest for AA blastocysts (35.0%), followed by BA blastocysts (31.2%) and AB blastocysts (27.7%)., Conclusion: This large study analyses for the first time the independent role of blastocyst morphology in predicting live birth while controlling for female age and PdHCG. Its findings suggest that DevSt and then trophectoderm grade are stronger predictors of live birth over ICM grade when selecting a single blastocyst for transfer., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research Question: Are the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm associated with the clinical outcomes of single-embryo transfer (SET)?, Design: Time-lapse microscopy was used to compare morphokinetic variables between expanded blastocysts derived from preimplantation genetic testing for aneuploidy cycles using high-resolution next-generation sequencing (hr-NGS). The clinical efficacy of the morphokinetic algorithm KIDScore D5 was evaluated after euploid SET., Results: Compared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), P = 0.034) (where t is the time for the embryo to reach the specific stage in hours after ICSI or conventional IVF) and t8 (median 58.6 hpi, P = 0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, P = 0.012). A significantly higher incidence (P = 0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, P = 0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (P = 0.0171 to <0.0001)., Conclusions: Although morphokinetic features appear dissimilar in embryos with different diploid-aneuploid mosaic levels, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. When combined with hr-NGS, use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of a 4 month-old boy with catecholaminergic polymorphic ventricular tachycardia carrying the double heterozygous mutations RyR2-A1855D and SCN10A-Q1362H. PBMCs were reprogrammed using non-integrative Sendai viral vectors containing reprogramming factors OCT4, SOX2, KLF4 and C-MYC. The iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry RyR2-A1855D and SCN10A-Q1362H mutations and have a normal karyotype. They will be useful for studying the pathogenesis of CPVT patients with ≥2 variants., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Monosomy of chromosome X is associated with high prenatal mortality of female embryos and severe developmental abnormalities of patients born with Turner's syndrome (45,XO). The CDMLe012-A-1 human embryonic stem cell (hESC) line, derived from a day six blastocyst with a normal 46,XX female karyotype spontaneously lost an X-chromosome during cell culture. This 45,XO karyotype was stably maintained for more than 55 passages. Since the CDMLe012-A-1 cells express pluripotent stem cell markers and differentiate into cells derived from the three germ layers, the cell line represents a stable, pluripotent stem cell model of Turner's syndrome., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Time-lapse monitoring (TLM) has emerged as a novel technology for the continuous and noninvasive evaluation of embryos. TLM has revealed the prevalence of specific dysmorphisms such as abnormal development during the early-cleavage stage of embryos. However, little information is available on the prevalence and consequences of abnormal cleavage in bovine embryos. Hence, this study aimed to investigate growth potential of bovine embryos presenting abnormal cleavage, such as reverse cleavage (RC), direct cleavage (DC), and irregular and unsmooth ruffling of the oolema membrane (ruffling). Bovine embryos derived through in vitro fertilization (IVF) were cultured in the microwell culture dishes, and the kinetics of in vitro development were observed through TLM at 20-min intervals for 10 d. Approximately 36% of embryos that developed into a blastocyst presented abnormal cleavage. Morphokinetic evaluations revealed that RC, DC, and ruffling embryos showed slower development compared to embryos with normal cleavage (P < 0.01). Embryos with RC and DC, but not ruffling, revealed impaired hatchability (P < 0.05) with increased collapses of the blastocyst cavity until hatching (P < 0.0001). Moreover, the RC and DC embryos presented increased chromosomal aneuploidy (P < 0.05). These results suggest a compromised viability of embryos with RC and DC. This is the first report that clarified the effect of abnormal cleavage on the morphokinetics and growth potential of bovine IVF embryos. Results indicate that the kinetic evaluation of bovine embryos using the time-lapse imaging system will be beneficial for selecting embryos with a high viability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

With new time-lapse incubators, IVF laboratories have increased use of single step media, often used in an uninterrupted approach. This simplifies the culture process for embryologists, may help reduce costs, offers the potential to reduce cell handling and associated harmful environmental stressors, and improves embryo quality and outcomes. One could argue, however, that optimized quality control and culture conditions are required to implement uninterrupted culture successfully. Without impeccable laboratory conditions and oversight, while trying to reduce harmful environmental stress, the laboratory could be imparting stress. Factors such as medium evaporation and associated osmolality and pH increases, as well as volatile organic compound accumulation, could offset any advantage of reduced dish or embryo handling. When implementing uninterrupted embryo culture, attention must be paid to incubator humidity, amount, quality and type of oil used, medium formulation and protein quality, as well as laboratory air and gas supply quality, and volatile organic compound content., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research Question: How can the kinetics of human blastocyst expansion be used to evaluate an embryo's ploidy identified using preimplantation genetic testing for aneuploidy (PGT-A)?, Design: This was a retrospective observational study of 188 autologous blastocysts from 34 sequential treatment cycles using PGT-A and blastocyst biopsy. Using time-lapse imaging, blastocyst expansion was evaluated using a quantitative standardized expansion assay (qSEA). Trophectoderm cell division was examined in selected, unbiopsied embryos (n = 7) to evaluate the contribution of mitosis to the expansion rate., Results: The averaged euploid blastocyst expansion rate was significantly (52.8%) faster than in aneuploid blastocysts (P = 0.0041). Scatterplots, representing 'expansion maps', revealed that both populations showed a similarly overlapping distribution of blastocyst formation times at 80-140 h from fertilization. Euploidy and aneuploidy were better distinguished in regions of higher and lower expansion, respectively, in expansion maps. Based upon the expansion slopes, rank-ordering of individual embryos within cohorts resulted in more than 90% euploid embryos in the first two ranks in patients less than 35 years of age. Additional detailed time-lapse image analysis provided evidence that rapid expansion was associated with robust, integrative cellular mitosis in trophectoderm cells., Conclusions: The kinetics of human blastocyst expansion are related to an embryo's ploidy. These preliminary observations describe a new quantitative, non-invasive approach to embryo assessment that may be useful to identify single blastocysts for transfer, particularly in younger patient groups. However, this approach may also be useful for euploid embryo selection after PGT-A. The results support the hypothesis that aneuploidy universally impairs general cellular processes, including cell division, in differentiated cells., (Copyright © 2019. Published by Elsevier Ltd.)

Aminopeptidase N (ANPEP) is a membrane-bound zinc-dependent peptidase. Although it is widely believed that ANPEP acts as an important angiogenesis regulatory factor, there are few studies about its function in the female reproductive system. In our previous research, we applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to analyze the influence of different controlled superovulation treatments for Assisted Reproductive Technology, In Vitro Fertilization and Embryo Transfer (IVF-ET)) patients from a global proteomic perspective to search for potential biomarkers associated with endometrium receptivity and embryo implantation. ANPEP is one of the proteins that demonstrated differential expression between different treatment groups and may be closely associated with endometrial receptivity. In this study, we assessed the expression of ANPEP in the endometrium of mice at different ages and found it to be highest in the mature period. We also detected ANPEP expression in the endometrium of IVF-ET patients in the proliferative, preimplantation and implantation stages, and the highest expression level of ANPEP was found in the last group. Human primary endometrial stromal cells were infected with an adenovirus expression vector containing the ANPEP gene and a green fluorescent protein (GFP) fusion protein; the mRNA levels of HOXA-10, LIF, and integrin β3 were found to be increased. Therefore, we conclude that ANPEP could be involved in the regulation of endometrial receptivity., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Migration of Anterior Visceral Endoderm (AVE) is a critical symmetry breaking event in the early post-implantation embryo development and is essential for establishing the correct body plan. Despite much effort, cellular and molecular events influencing AVE migration are only partially understood. Here, using time-lapse live imaging of mouse embryos, we demonstrate that cell division in the embryonic visceral endoderm is coordinated with AVE migration. Moreover, we demonstrate that temporal inhibition of FGF signalling during the pre-implantation specification of embryonic visceral endoderm perturbs cell cycle progression, thus affecting AVE migration. These findings demonstrate that coordinated cell cycle progression during the implantation stages of development is important for post-implantation morphogenesis in the mouse embryo., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Research Question: What are the live birth rates and neonatal outcomes following cleavage-stage embryo transfer and blastocyst transfer in a freeze-all treatment scenario?, Design: This was a retrospective cohort study. All good-quality embryos were frozen on the third day; the remaining embryos were grown on until they reached blastocyst stage and then frozen. Between 2007 and 2016, 11,801 patients underwent cleavage-stage embryo transfer and 1009 patients underwent blastocyst transfer in the first treatment cycle using the freeze-all strategy. The live birth rate and neonatal outcomes were evaluated., Results: The live birth rate in the first frozen embryo transfer cycle was higher following blastocyst transfer than following cleavage-stage transfer (69.1% versus 55.5%, P < 0.01), but there was no difference in live birth rate in the second frozen embryo transfer cycle between blastocyst transfer and cleavage-stage transfer (45.2% versus 52.7%, P > 0.05). Similarly, no difference was found in the cumulative live birth rate for the first complete IVF cycle (71.1% versus 69.2%, P > 0.05). Blastocyst transfer gave a higher risk of preterm singleton delivery than did cleavage-stage transfer. However, there was no difference in the risk of early preterm delivery, low birth weight, very low birth weight, high birth weight and very high birth weight between the two groups., Conclusions: There is no evidence to support the superiority of blastocyst transfer compared with cleavage-stage transfer in a freeze-all treatment scenario. There may be a higher risk of preterm singleton delivery following blastocyst transfer than following cleavage-stage transfer but further studies are needed to verify this., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Assisted reproductive technologies (ART) are widely used for both humans and domestic animals. In bovine species, in vitro embryo production is increasingly used and significant efforts are being made to optimize media and culture conditions. Phospholipase A2 (PLA2) are lipolytic enzymes that hydrolyze glycerophospholipids to produce free fatty acids and lysophospholipids that have been found to be critical for many biological processes. Mouse group X secreted PLA 2 (mGX) is abundant in the male reproductive tract and its use during sperm capacitation has been shown to improve in vitro production of viable embryos in a mouse model. Here, we examined its effect in the bovine species, testing the impact of mGX on the three steps involved in vitro production of preimplantation embryos: oocyte maturation, fertilization and preimplantation development. We found that incubating cumulus oocyte complexes (COC) or gametes with mGX resulted in increased blastocyst hatching and blastocyst production, respectively. The increases of embryo production induced by the phospholipase mGX were not observed for the catalytically inactive mutant H48Q-mGX, suggesting that these effects require the enzymatic activity of mGX. We also tested bGIB, a bovine homolog of mGX. bGIB failed to improve blastocyst production, underlining the high specificity of mGX. In conclusion, the results presented show that the effects of mGX are not restricted to the mouse model and that it is potent in the bovine species as well. This result strengthens the potential of mGX as a "pro-fertility drug" for mammalian reproduction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Research Question: What pregnancy rates are achieved after transfer of cryopreserved double slow-frozen embryos in IVF cycles? Patients in whom surplus thawed cleaved embryos (day 2 or 3) were grown to the blastocyst stage, re-frozen and then re-thawed for transfer (double freezing) were included., Design: Data were collected on all patients who had undergone the above procedure at the IVF unit of Assuta Ramat Hachayal Hospital, Tel Aviv, during a 7-year period. For each patient in the study group, the two-consecutive, matched-by-age patients treated with frozen-thawed single blastocyst transfer were selected to form a 2:1 ratio control group. All embryos were frozen using the slow freeze protocol., Results: A total of 54 patients had 70 embryos that were re-frozen at the blastocyst stage. Twenty-eight of these blastocysts were thawed and 27 underwent transfer to 25 patients. A single embryo was transferred to 23 patients and two embryos were transferred to two patients. The survival rate of the second thawing was 96.4% (27/28). Clinical pregnancy rate was 16% (4/25) and implantation rate was 14.8% (4/27). In the study group, pregnancies were achieved in 22 out of the 25 patients using IVF treatment, indicating good receptivity of the uterus. In the control group, the implantation/pregnancy rates were significantly higher (44.2% [23/52]; P < 0.01)., Conclusion: The transfer of twice slow-frozen and thawed embryos does not seem to be a beneficial approach in the planned management of cryopreserved surplus embryos owing to the low pregnancy rate achieved after transfer of the re-frozen blastocyst embryos., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The most important factor for a successful pregnancy after in vitro fertilization is embryo quality. The aim of this study was to explore the possibility that using the immunomagnetic reduction (IMR) assay to quantitatively measure β-subunit of human chorionic gonadotropin (β-hCG) in blastocyst culture media to differentiate embryo quality., Methods: This was a prospective case-control study including 28 samples of blastocyst culture media. We used single-step blastocyst culture and IMR assay to analyze β-hCG concentrations in culture media. We also explored the relationship between IMR signals of β-hCG and morphological grading of blastocysts., Results: β-hCG concentration-dependent IMR signals were highly correlated with blastocyst morphological quality (Spearman correlation coefficient: 0.731). Receiver-operating characteristic curve analysis showed a cut-off IMR value to differentiate embryo quality of 0.873%, with an area under the curve of 0.947, sensitivity of 0.882 and specificity of 0.818. Furthermore, subanalysis also revealed a positive correlation between β-hCG concentration-dependent IMR signals and trophectoderm grading, with a Spearman correlation coefficient of 0.576., Conclusions: An IMR assay can quantitatively measure β-hCG in blastocyst culture media, and may be a potential clinical tool to assist in the assessment of good blastocyst quality before embryo transfer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C. After 2 months' storage, some of the 4°C-stored spermatozoa were exposed to 40 °C for 1 week or 1 month, then again stored at 4 °C for the remaining storage period. Following storage, rehydrated spermatozoa were injected into mouse oocytes. The resulting zygotes were assessed for chromosome damage, in vitro development up to the blastocyst stage, and post-implantation development to normal fetuses on day 18 of gestation. In storage at 4 °C, one-week exposure to 40 °C had no adverse effect on the chromosome integrity and developmental competence compared to non-exposure to 40 °C (continuous storage at 4 °C). In contrast, one-month exposure to 40 °C caused an increasing level of chromosome damage (36%, P < 0.05) and reduced frequencies of blastocysts (54%, P < 0.05) and normal fetuses (36%, P < 0.05) compared to the frequencies obtained by continuous storage at 4 °C (15%, 82% and 52%, respectively). Storage at 25 °C resulted in accumulation of chromosome damage (27%, P < 0.05), leading to decreased blastocyst formation (63%, P < 0.05). But, the frequency of normal fetus (44%) was not significantly different from that obtained by continuous storage at 4 °C. Consequently, mouse spermatozoa freeze-dried in K-ETBS withstood temporary exposure to 40 °C for 1 week. Chromosome damage accumulated in spermatozoa during storage at 25 °C., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Research Question: Can detailed scrutiny of time-lapse imaging (TLI) of inner cell mass (ICM) splitting help to reduce the frequency of multiple pregnancies following elective single embryo transfer (eSET)?, Design: Retrospective analysis of time-lapse images of an embryo in vitro, which resulted in a monochorionic triamniotic pregnancy following eSET on Day 5 of development., Results: A 37-year-old female patient underwent a frozen embryo transfer cycle whereby a single vitrified/warmed embryo was transferred at the hatching blastocyst stage. The subsequent pregnancy scan revealed a monochorionic triamniotic pregnancy. Because the blastocyst was cultured in an incubator incorporating TLI, retrospective scrutiny of the digital recordings demonstrated two distinct ICM structures splitting apart, which formed during the '8-shaped hatching'., Conclusions: Assisted reproductive techniques and in-vitro culture have been associated with an increased frequency of embryo splitting. This has been postulated to be linked to the in-vitro hatching method observed at the blastocyst stage of development. This case report highlights the need to objectively assess any splitting of the ICM, beyond standard grading of the quality of the ICM and the trophectoderm. Such assessments of ICM splitting should be routine practice in clinical embryology when selecting embryos for transfer., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Culture conditions determine embryo quality, which may be affected on many levels (timing of development, blastomere count, transcripts, metabolite content, apoptosis). Molecular interactions of signalling pathways like MEK/ERK and WNT/β-catenin are critical for cell-to-cell communication and cellular differentiation. Both pathways are important regulators of apoptosis. We have aimed to verify the prolonged effect of MEK/ERK silencing and WNT activation by chemical inhibitors (2i or 3i systems) on bovine IVP embryos. Apoptotic index, total cell count and transcription of embryo quality markers were evaluated. A higher rate of apoptosis was observed in 2i blastocysts, but was not accompanied by changes in transcript content of genes controlling apoptosis (BAX, BCL2, BAK, BAX/BCL2 ratio). Therefore, alternative pathways of apoptotic activation cannot be ruled out. The expression of genes related to embryo quality (HSPA1A, SLC2A1) was not affected. GJA1 transcripts were significantly higher in 3i blastocysts, what indicates a stimulatory effect of the applied inhibitors on cell-to-cell interactions. The lowest mRNA level of the IFNT2 gene was found in 2i embryos. A variation in the SDHA gene transcript was observed (with the highest content in the 3i blastocysts), what may suggest their reduced quality. It may be concluded that the modifications of culture conditions (activation of the WNT and silencing of the MEK/ERK signalling) might alter pathways crucial for embryo development without causing embryonic death., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Research Question: Does autologous endometrial cell co-culture (AECC) improve the number of good-quality blastocysts obtained by IVF/intracytoplasmic sperm injection (ICSI), compared with conventional embryo culture medium in a broad group of patients referred to assisted reproductive technology (ART)?, Design: This interventional, randomized, double-blind study took place at Clinique Ovo from March 2013 to October 2015 and included 207 healthy patients undergoing an IVF or ICSI protocol, of which 71 were excluded before randomization. On the previous cycle, all participants underwent an endometrial biopsy at D5 to D7 post-ovulation, following which the endometrial cells were prepared for AECC., Results: The data demonstrated that AECC significantly increased the incidence of good-quality blastocysts compared with culture in conventional media (42.6% vs 28.4%, P < 0.001). No significant differences were found in pregnancy and live birth rates., Conclusion: This study demonstrated the benefits of AECC on blastocyst quality compared with conventional embryo culture medium, in a broader category of patients referred to ART as opposed to other studies that concentrated on specific causes of infertility only. However, limitations of the study design should be taken into consideration; the analysis was performed using embryos rather than patients and a follow-up of children born following the treatments could not be conducted., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research Question: Does the composite morphology score or a particular developmental component (expansion stage, inner cell mass [ICM] or trophectoderm [TE]) of euploid blastocysts undergoing single frozen embryo transfer (FET) impact ongoing pregnancy/live birth (OP/LB) rates?, Design: Retrospective cohort study including a total of 2236 embryos from 1629 patients who underwent single euploid FET between 2012 and 2017., Results: Embryos with an ICM grade of A compared with C had a higher OP/LB rate (55.6% versus 32.3%, P < 0.001). Blastocysts with a TE grade of A or B compared with C had a higher likelihood of OP/LB (A versus C: odds ratio [OR] 1.6, 99% confidence interval [CI] 1.1-2.3, B versus C: OR 1.5, 99% CI 1.1-2.1), and blastocysts with a developmental stage of 4 or 5 compared with 6 had higher odds of OP/LB (4 versus 6: OR 1.6, 99% CI 1.2-2.2, 5 versus 6: OR 1.6, 99% CI 1.2-2.3)., Conclusions: Among euploid embryos, ICM morphology is the best predictor of sustained implantation; however, a composite score may provide additional guidance. While there is a known benefit in genomic screening prior to embryo selection, morphology provides individualized, prognostic information about implantation potential., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)