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What is the incidence of chromosomal mosaicism in human blastocysts and can a single trophectoderm (TE) biopsy accurately predict the chromosomal constitution of the inner cell mass (ICM)? Observational study in 46 surplus cryopreserved preimplantation embryos of unknown chromosomal constitution. For each embryo, a TE biopsy was performed and the ICM was collected separately. Both samples underwent next-generation sequencing (NGS) for cytogenetic analysis and were classified as chromosomally normal, abnormal or mosaic. Mosaic samples were classified as low or high mosaic, based on the majority dominance of either normal or abnormal cells in the biopsied sample. Findings within each embryo were compared. Chromosomal mosaicism was detected in 59% (n = 27/46) of the embryos, with a cytogenetic concordance rate between TE and corresponding ICM of 48% (n = 22/46). Concordance was higher from a clinical perspective: in 86% of embryos with a high-mosaic or abnormal TE, the ICM was also high-mosaic or abnormal. In 88% of the blastocysts with a normal or low-mosaic TE biopsy, a normal or low-mosaic ICM was observed. Despite the low cytogenetic concordance rate due to chromosomal mosaicism present in blastocysts, it was found that a single TE biopsy could correctly predict whether the ICM consists of mostly normal or abnormal cells in the majority of cases. [ABSTRACT FROM AUTHOR]

The emergence of the earliest cell lineages in mammalian embryos is a complex process that utilizes an extensive network of chromatin regulators, transcription factors, cell polarity regulators, and cellular signaling pathways. These factors and pathways operate over a protracted period of time as embryos cleave, undergo compaction, and form blastocysts. The first cell fate specification event separates the pluripotent inner cell mass from the trophectoderm lineage. The second event separates pluripotent epiblast from hypoblast. This review summarizes over 50 years of study of these early lineage forming events, addressing the complexity of the network of interacting molecules, cellular functions and pathways that drive them, interspecies differences, and aspects of these mechanisms that likely underlie their high susceptibility to disruption by numerous environmental factors that can compromise embryo viability, such as maternal health and diet, environmental toxins, and other stressors., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Abstract Purpose This study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells. Methods TE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next‐generation sequencing. Results Discordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality. Conclusion Chromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection.

Objective: To analyze the correlation between TE grading and initial β-hCG serum level after single euploid embryo transfer. Secondarily, to explore the association between TE grading with subsequent IVF outcomes. Design: Retrospective cohort analysis. Setting: Single, academic, private infertility and assisted reproductive care institute. Patients or other participants: Infertility patients who underwent a single euploid embryo transfer that resulted in a positive pregnancy test. Intervention(s): β-hCG measurements. Main outcome measure(s): Correlation between TE grade with first β-hCG measurement. Second outcome measurements included ongoing pregnancy, biochemical pregnancy loss, and clinical pregnancy loss rates. Results: 2,798 cases were analyzed. A significant difference in initial β-hCG measurement among groups (TE A: median 143.4 mIU/mL IQR 79.2–211.2; TE B: 119 mIU/mL IQR 57.1–177.8; TE C: 82.4 mIU/mL IQR 36.3–136.4, p ≤ 0.0001) was observed. There was a significant correlation found between the TE grade and β-hCG measurements (p ≤ 0.0001, r2 = 0.10). TE grade was not associated with higher odds of biochemical pregnancy loss (TE A vs. TE B: aOR 1.01 CI95% 0.97–1.05; TE A vs. TE C: aOR 1.03 CI95% 0.98–1.08), or higher odds of clinical pregnancy loss (TE A vs. TE B: aOR 1.02 CI95% 0.98–1.05; TE A vs. TE C: aOR 1.03 CI95% 0.98–1.07). Conclusions: In patients with euploid embryos, TE grade correlates with the first pregnancy test measurement of β-hCG. We propose this finding helps to appoint a relevant link between morphology assessment and early embryo development in vivo. [ABSTRACT FROM AUTHOR]

Objective: Trophectoderm (TE) cells are the first differentiating cells in embryo development and have epithelial features. TE cells, which associate with implantation of the blastocyst into the uterine endometrium, contribute to the formation of the placenta. Inner cells mass (ICM) together with TE cells are used for determining embryo quality. The aim of this study was to investigate the role of TE and ICM cells on pregnancy outcome in 5th day blastocyst transferred in-vitro-fertilization (IVF) pregnancy. Material and Methods: This was a retrospective study using data from all patients who applied for blastocyst transfer IVF between January 2015 and March 2019 at the Reproductive Endocrinology and Infertility Center of Akdeniz University Faculty of Medicine, Department of Obstetrics and Gynecology. ALPHA İstanbul consensus evaluation system was used for grading of the blastocyst. The embryo quality, expansion, ICM and TE morphology of the 5th day transferred blastocyst was assessed, together with abortion rate, live birth rate, pregnancy complications, and pregnancy outcomes. Results: There was a significantly increased risk of preeclampsia (PE) (7.8% vs 1.1%; p=0.041), preterm delivery (PD) (36% vs 17.7%; p=0.037), and antenatal bleeding rates (13.6% vs 5%; p=0.021) in TE-C compared to the TE-A + TE-B blastocysts. Furthermore, a higher rate of obstetric complications was observed in ICM-C compared to ICM-A and B (p=0.003). There was a significant correlation between TE morphology and implantation success, ongoing pregnancy rate, and abortion incidence. Conclusion: These results suggest that TE cell morphology is related to implantation success and pregnancy outcomes, especially in terms of the risk of abortion, PE, PD, and antenatal bleeding. It may be advisable to counsel women concerning possible poor obstetric outcome due to poor ICM quality. Future prospective and controlled studies are needed to clarify this association. [ABSTRACT FROM AUTHOR]

Background. Transfer of embryos at the blastocyst stage is one of the best approaches for achieving a higher success rate in In vitro fertilization (IVF) treatment as it demonstrates an improved uterine and embryonic synchrony at implantation. Despite novel biochemical and genetic markers proposed for the prediction of embryo viability in recent years, the conventional morphological grading of blastocysts remains the classical way of selection in routine practice. This study aims to investigate the association between the morphological features of blastocysts and pregnancy outcomes. Methods. This prospective study included women undergoing single or double frozen blastocyst transfers following their autologous cycles in a period between October 2020 and September 2021. The morphological grades (A--good, B--average, and C--poor) of inner cell mass (ICM) and trophectoderm (TE) of blastocysts with known implantation were compared to assess their predictive potential of pregnancy outcome. It was further explored by measuring the relationship between the two variables using logistic regression and receiver operating characteristic (ROC) analysis. Results. A total of 1,972 women underwent frozen embryo transfer (FET) cycles with a total of 3,786 blastocysts. Known implantation data (KID) from 2,060 blastocysts of 1,153 patients were subjected to statistical analysis, the rest were excluded. Implantation rates (IR) from transfer of ICM/TE grades AA, AB, BA, BB were observed as 48.5%, 39.4%, 23.4% and 25% respectively. There was a significantly higher IR observed in blastocysts with ICM grade A (p < 0:001) than those with B irrespective of their TE scores. The analysis of the interaction between the two characteristics confirmed the superiority of ICM over TE as a predictor of the outcome. The rank biserial correlation value for ICM was also greater compared to that of TE (0.11 vs 0.05). Conclusion. This study confirms that the morphology of ICM of the blastocyst is a stronger predictor of implantation and clinical pregnancy than that of TE and can be utilized as a biomarker of viability. [ABSTRACT FROM AUTHOR]

BackgroundElective single blastocyst transfer (eSBT) is considered to reduce the incidence of multiple pregnancy compared to double embryo transfer. Blastocyst selection is the key to achieving pregnancy. In the past, morphological assessment was the main criterion used to select blastocyst. Some important morphological parameters are considered to be clinically valuable, such as cytoplasmic strings traversing from the inner cell mass (ICM) and mural trophectoderm (mTE).MethodsIn this study, 1,267 elective frozen-thawed eSBT cycles cultured in a time-lapse culture system from January 2018 to May 2019 were included. Blastocysts were grouped into “present” and “absent” according to the appearance of cytoplasmic strings between ICM and mTE cells. The “present” group was further categorized according to the quantity of cytoplasmic strings between the ICM and mTE cells.ResultsA time-lapse analysis indicated that cytoplasmic strings between ICM and mTE were more visible among good quality blastocysts. Furthermore, blastocysts with cytoplasmic strings showed higher clinical pregnancy and live birth rates (P = 0.011 and 0.003), while no significant differences were observed in abortion rate and birth weight (P = 0.466 and 0.556).ConclusionsIn conclusion, although the results of previous studies about cytoplasmic strings have been controversial, the present time-lapse analysis provides evidence for the first time that cytoplasmic strings between ICM and mTE cells are a positive predictor of clinical pregnancy and live birth outcomes in elective frozen-thawed single blastocyst transfer cycles.

Background and objective: Day-5 blastocyst embryos are usually chosen for assisted reproductive therapy. We compared the reproductive outcomes of the euploid blastocysts developed on Day 5 versus Day 6. Methods: This single-center, retrospective observational study analyzed patients aged 25–45 years, who underwent intracytoplasmic sperm injection from December 2014 to November 2018. Depending on the day of trophectoderm biopsy, patients were categorized into Day-5 and Day-6 groups. Percentages of euploid embryos were calculated for both groups, and elective single euploid blastocysts were transferred in a frozen embryo transfer (FET) cycles. The study endpoints were the comparisons of the reproductive outcomes including clinical pregnancy rate (CPR), implantation rate (IR), miscarriage rate (MR), and live birth rate (LBR) between Day-5 and Day-6 euploid FET groups. Results: A total of 801 embryos from 184 patients were evaluated [Day 5 (n=769); Day 6 (n=32); 42.45% were euploid] with the rate of euploidy in Day-5 and Day-6 groups at 42.52% and 40.62%, respectively. A total of 126 patients underwent FET with 126 elective single euploid embryos (Day 5: 117; Day 6: 9). For Day-5 versus Day-6 groups, a significantly higher IR (61.54% vs. 44.44%; p = 0.0531), CPR (61.54% vs. 44.44%; p = 0.0531), and LBR (61.54% vs. 33.33%; p = 0.0014) were reported. Multivariate analysis on ANOVA suggested, comparable pregnancy rates at Day 5 and Day 6 (p = 0.728). Conclusions: Day-5 euploid blastocysts seem to offer better reproductive outcomes than Day-6 euploid blastocysts. Further research is recommended to evaluate the reproductive outcomes of Day-6 blastocysts.

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo. [Display omitted] • Internal GATA3+ cells appear in blastocysts from around 5 days after fertilization • Polar trophectoderm thickens substantially before the expected time of implantation • Some late blastocysts exhibit reduced numbers of cells in the mural trophectoderm • A reservoir of polar trophectoderm cells may be necessary for successful implantation Corujo-Simon et al. present evidence suggesting that, in preparation for implantation in the uterus, polar trophectoderm of the human blastocyst becomes multi-layered. Using sequential fluorescent labeling they show that at least the majority of the extra cells originate from the trophectoderm, not the underlying inner cell mass. [ABSTRACT FROM AUTHOR]

Human pregnancy depends on the proper development of the embryo prior to implantation and the implantation of the embryo into the uterine wall. During the pre-implantation phase, formation of the morula is followed by internalization of blastomeres that differentiate into the pluripotent inner cell mass lineage, while the cells on the surface undergo polarization and differentiate into the trophectoderm of the blastocyst. The trophectoderm mediates apposition and adhesion of the blastocyst to the uterine epithelium. These processes lead to a stable contact between embryonic and maternal tissues, resulting in the formation of a new organ, the placenta. During implantation, the trophectoderm cells start to differentiate and form the basis for multiple specialized trophoblast subpopulations, all of which fulfilling specific key functions in placentation. They either differentiate into polar cells serving typical epithelial functions, or into apolar invasive cells that adapt the uterine wall to progressing pregnancy. The composition of these trophoblast subpopulations is crucial for human placenta development and alterations are suggested to result in placenta-associated pregnancy pathologies. This review article focuses on what is known about very early processes in human reproduction and emphasizes on morphological and functional aspects of early trophoblast differentiation and subpopulations. [ABSTRACT FROM AUTHOR]

Bovine mesenchymal stem cells are a relevant cell population found in the maternal reproductive tract that exhibits the immunomodulation capacity required to prevent embryo rejection. The phenotypic plasticity showed by both endometrial mesenchymal stem cells (eMSC) and embryonic trophoblast through mesenchymal to epithelial transition and epithelial to mesenchymal transition, respectively, is essential for embryo implantation. Embryonic trophoblast maintains active crosstalk via EVs and soluble proteins with eMSC and peripheral blood MSC (pbMSC) to ensure the retention of eMSC in case of pregnancy and induce the chemotaxis of pbMSC, critical for successful implantation. Early pregnancy-related proteins and angiogenic markers are detected as cargo in EVs and the soluble fraction of the embryonic trophectoderm secretome. The pattern of protein secretion in trophectoderm-EVs changes depending on their epithelial or mesenchymal phenotype and due to the uptake of MSC EVs. However, the changes in this EV-mediated communication between maternal and embryonic MSC populations infected by viruses that cause abortions in cattle are poorly understood. They are critical in the investigation of reproductive viral pathologies. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst. Methods: Fifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS). Results: Fifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23). Conclusion: Blastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised. [ABSTRACT FROM AUTHOR]

The mechanisms regulating early stages of placentation and trophectoderm differentiation in the ruminant conceptus remain poorly understood. Here we present a model of trophectoderm (TE) differentiation in vitro from outgrowths of individual in vitro derived embryos. Cell outgrowths expressed markers of mononucleate (MNC) and binucleate (BNC) TE cells. The percentage of BNC ranged from 14 to 39% in individual outgrowths as determined by flow cytometry. Pregnancy-associated glycoproteins (PAGs), produced by BNC, were measured in culture media on days 35 to 54. Continuous secretion of PAGs was observed and indicative of BNC functionality. Gene expression was evaluated in 20 embryo cell outgrowths derived from two different sires. Expression of HAND1, which is involved in TE differentiation, and CSH2, a BNC-specific gene, was altered in cell outgrowths between the two sires tested. Single-cell RNA-seq analysis of day 40 TE cell outgrowths revealed 11 distinct cell populations, with specific clusters genes involved in TE lineage specification, proliferation, and differentiation. In addition, whole -RNAseq analysis was performed in day 35 and 40 TE cell outgrowths and confirmed sustained expression of genes expressed by BNC, such as CSH2 and some PAGs. The developed in vitro bovine embryo outgrowth culture found evidence for MNC and BNC differentiation and continuous production of PAGs, recapitulating key features of early bovine placenta development. This model can be used to understand the developmental biology of TE cells, provide insights into paternal influences on TE differentiation, and impact our understanding of early pregnancy loss in cattle.

ObjectiveThe aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst.MethodsFifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS).ResultsFifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23).ConclusionBlastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised.

There has been recent renewed interest in studying human early embryonic development. The advent of improved culture conditions to maintain blastocysts in vitro for an extended period and the emerging stem-cell-based models of the blastocyst and peri-implantation embryos have provided new information that is relevant to early human embryogenesis. However, the mechanism of lineage development and embryonic patterning, and the molecular pathways involved in their regulation, are still not well understood. Interest in human embryonic development has been reinvigorated recently given numerous technical advances. In this review, Rossant and Tam discuss new insights into human embryogenesis gathered from successes in culturing human embryos in vitro and stem-cell-based embryo models. Then they outline what questions still need answering. There has been recent renewed interest in studying human early embryonic development. The advent of improved culture conditions to maintain blastocysts in vitro for an extended period and the emerging stem-cell-based models of the blastocyst and peri-implantation embryos have provided new information that is relevant to early human embryogenesis. However, the mechanism of lineage development and embryonic patterning, and the molecular pathways involved in their regulation, are still not well understood. Interest in human embryonic development has been reinvigorated recently given numerous technical advances. In this review, Rossant and Tam discuss new insights into human embryogenesis gathered from successes in culturing post-implantation human embryos in vitro and stem-cell-based embryo models. Then they outline what questions still need answering. [ABSTRACT FROM AUTHOR]

Study Question: Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child?Summary Answer: A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed.What Is Known Already: The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting.Study Design, Size, Duration: Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth.Participants/materials, Setting, Methods: Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833).Main Results and the Role Of Chance: No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)).Limitations, Reasons For Caution: It is possible that some residual confounding remains.Wider Implications Of the Findings: Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth.Study Funding/competing Interest(s): Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Abstract Purpose To investigate the associations between blastocyst development and the sex ratio (male:female) among singleton live births resulting from single-blastocyst frozen embryo transfer (FET) cycles. Methods Patients with singleton live births following the first autologous single FET of non- preimplantation genetic testing (PGT) blastocysts in a single reproductive medicine department between January 2015 and February 2019 were included in this retrospective study. The primary outcome measure was the singleton sex ratio. Multivariable logistic regression models were used to estimate the associations between blastocyst quality and singleton sex ratio after adjustment for some potential confounders. Results There were 638 high-quality and 572 poor-quality single blastocyst FETs, and the blastocysts were conceived via 855 IVF and 355 ICSI treatments. A total of 1210 singleton live births were assessed. High-quality single blastocyst FET resulted in a significantly higher sex ratio than did poor-quality single blastocyst FET (60% vs. 49.7%, P

Epigenetic modifications, including DNA methylation and histone modifications, are reprogrammed considerably following fertilization during mammalian early embryonic development. Incomplete epigenetic reprogramming is a major factor leading to poor developmental outcome in embryos generated by assisted reproductive technologies, such as somatic cell nuclear transfer. However, the role of histone modifications in preimplantation development is poorly understood. Here, we show that co-knockdown (cKD) of Hdac1 and 2 (but not individually) resulted in developmental failure during the morula to blastocyst transition. This outcome was also confirmed with the use of small-molecule HDAC1/2-specific inhibitor FK228. We observed reduced cell proliferation and increased incidence of apoptosis in cKD embryos, which were likely caused by increased acetylation of TRP53. Importantly, both RNA-seq and immunostaining analysis revealed a failure of lineage specification to generate trophectoderm and pluripotent cells. Among many gene expression changes, a substantial decrease of Cdx2 may be partly accounted for by the aberrant Hippo pathway occurring in cKD embryos. In addition, we observed an increase in global DNA methylation, consistent with increased DNA methyltransferases and UHRF1. Interestingly, deficiency of RBBP4 and 7 (both are core components of several HDAC1/2-containing epigenetic complexes) results in similar phenotypes as those of cKD embryos. Overall, HDAC1 and 2 play redundant functions required for lineage specification, cell viability and accurate global DNA methylation, each contributing to critical developmental programmes safeguarding a successful preimplantation development.

Introduction: This study was conducted to compare hatching rates after assisted hatching, re-expansion rates after trophectoderm biopsy, and survival rates after cryopreservation using different methods of assisted hatching and biopsy in mouse embryo. Materials and Methods: Five-week-old female mice (C57BL/CBA) were superovulated, and two-cell embryos were collected. All embryos were cultured to blastocyst stage. For assisted hatching and separating trophectoderm from blastocyst, laser device and hand-made pipette were used respectively. Hatching rates after assisted hatching, re-expansion rates after trophectoderm biopsy, and survival rates after cryopreservation were calculated. Results: Hatching rate was 92% in mechanically assisted hatching group and 90% in laser group, respectively. After mechanically assisted hatching, re-expansion rate was 91.3% and survival rate was 87% in biopsy by pipette and laser group, respectively. In laser hatching group, re-expansion rate was 88.9% with biopsy by pipette and survival rate was 84.4% with biopsy by laser. Conclusion: Throughout the study, mechanical technique and laser technique showed no differences in the safety profiles in trophectoderm biopsy procedure.

Introduction: In human placental development, the trophectoderm (TE) appears in blastocysts on day 5 post-fertilization and develops after implantation into three types of trophoblast lineages: cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT). CDX2/Cdx2 is expressed in the TE, and Cdx2 expression is upregulated by knockdown of Foxo1 in mouse ESCs. However, the significance of FOXO1 in trophoblast lineage differentiation during the early developmental period remains unclear. In this study, we examined the effect of FOXO1 inhibition on the differentiation of naive human induced pluripotent stem cells (iPSCs) into TE and trophoblast lineages., Methods: We induced TE differentiation from naive iPSCs in the presence or absence of a FOXO1 inhibitor, and the resulting cells were subjected to trophoblast differentiation procedures without the FOXO1 inhibitor. The cells obtained in these processes were assessed for morphology, gene expression, and hCG secretion using phase-contrast microscopy, reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), RNA-seq, immunochromatography, and a chemiluminescent enzyme immunoassay., Results: In the induction of trophoblast differentiation from naive iPSCs, treatment with a FOXO1 inhibitor resulted in the enhanced expression of TE markers, CDX2 and HAND1, but conversely decreased the expression of ST markers, such as ERVW1 (Syncytin-1) and GCM1, and an EVT marker, HLA-G. The proportion of cells positive for an early TE marker TACSTD2 and negative for a late TE marker ENPEP was higher in FOXO1 inhibitor-treated cells than in non-treated cells. The expressions of ERVW1 (Syncytin-1), ERVFRD-1 (Syncytin-2), and other endogenous retrovirus (ERV)-associated genes that have been reported to be expressed in trophoblasts were suppressed in the cells obtained by differentiating the TE cells treated with FOXO1 inhibitor., Conclusions: Treatment with a FOXO1 inhibitor during TE induction from naive iPSCs promotes early TE differentiation but hinders the progression of differentiation into ST and EVT. The suppression of ERV-associated genes may be involved in this process., Competing Interests: The authors have no competing financial interests to declare., (© 2024 The Author(s).)

Trophectoderm-based preimplantation genetic testing for aneuploidies (PGT-A) is used worldwide as a means of selecting embryos with high potential for achieving a live birth. However, trophectoderm analysis may be impaired through embryonic mosaicism, leading to genetically healthy embryos being falsely discarded, and thus even reducing cumulative live birth rates. Polar body biopsy, a technique applied since the early days of preimplantation testing, has been abandoned by most IVF centres. In comparison to trophectoderm analysis, however, polar body biopsy might even have certain advantages over trophectoderm PGT-A. This Countercurrent contribution discusses the newest clinical evidence, as well as ethical and cost-efficiency considerations, and argue that polar body analysis should be reconsidered., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

In many mammals, including ruminants, pregnancy requires pregnancy recognition signaling molecules secreted by the conceptus; however, the mechanism underlying pregnancy establishment in cattle remains unknown. Trophoblastic vesicles (TVs) are artificially produced from the extraembryonic tissues of the elongating conceptus and may be useful tools for understanding conception. This study investigated the morphological and functional properties of TVs in comparison to those of intact conceptuses. TVs were prepared from the extraembryonic tissues of conceptuses collected 14 days after artificial insemination (AI), cryopreserved immediately after dissection, and cultured after thawing for subsequent transplantation into the uterus. The transferred TVs were collected 7 days after transplantation and compared with extraembryonic tissue samples collected from conceptuses at 21 days post-AI. The recovered TVs were 40 times longer than those of their pre-transplant counterparts. Microscopic evaluation revealed that their membrane structures consisted of trophoblast and hypoblast layers. The expression patterns of the cell differentiation markers, CDX2, SOX2, and GATA6, and interferon tau (IFNT) protein expression levels in the TVs were similar to those in control extraembryonic tissue samples. These findings suggest that TVs are capable of morphological elongation and maintain IFNT production in a similar way as original trophoblasts., (© 2024 Wiley Periodicals LLC.)

Interferon-gamma (IFNG) is a pro-inflammatory cytokine secreted by the porcine conceptus (embryo and extra-embryonic membranes) during the peri-implantation period of pregnancy. IFNG modifies the endometrial inflammatory immune response and is required for the implantation and survival of the conceptus. It is not known how IFNG from the conceptus trophectoderm is transported across the endometrial luminal epithelium (LE). In the present study, immunofluorescence analyses detected immunoreactive IFNG protein in both the trophectoderm and endometrial LE on Day 15 of pregnancy, while our previous research localized IFNG mRNA only to conceptus trophectoderm. Using minced endometrial explants to disrupt the barrier posed by the intact endometrial LE, treatment with recombinant IFNG induced the expression of genes that were not induced when IFNG was infused into the uterine lumen in vivo by McLendon et al. (Biology of Reproduction. 2020;103(5):1018-1029). We hypothesized that during pregnancy extracellular vesicles (EVs) serve as intercellular signaling vehicles to transport conceptus-derived IFNG across the intact endometrial LE and into the stromal compartment of the uterus. Western blotting detected the presence of IFNG in EVs isolated from the uterine fluid of pregnant gilts, but not nonpregnant gilts. Real-time PCR demonstrated increased expression of IFNG-stimulated genes in EV-treated endometrial explants and EV-mediated IFNG transport was confirmed in whole uterine sections cultured with EVs from Day 15 of pregnancy. These results suggest that EVs are involved in IFNG transport across the endometrial LE to enable paracrine communication between the conceptus and cells within the endometrial stroma., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The morula-to-blastocyst transition (MBT) culminates with formation of inner cell mass (ICM) and trophectoderm (TE) lineages. Recent studies identified signaling pathways driving lineage specification, but some features of these pathways display significant species divergence. To better understand evolutionary conservation of the MBT, we completed a meta-analysis of RNA sequencing data from five model species and ICMTE differences from four species. Although many genes change in expression during the MBT within any given species, the number of shared differentially expressed genes (DEGs) is comparatively small, and the number of shared ICMTE DEGs is even smaller. DEGs related to known lineage determining pathways (e.g., POU5F1) are seen, but the most prominent pathways and functions associated with shared DEGs or shared across individual species DEG lists impact basic physiological and metabolic activities, such as TCA cycle, unfolded protein response, oxidative phosphorylation, sirtuin signaling, mitotic roles of polo-like kinases, NRF2-mediated oxidative stress, estrogen receptor signaling, apoptosis, necrosis, lipid and fatty acid metabolism, cholesterol biosynthesis, endocytosis, AMPK signaling, homeostasis, transcription, and cell death. We also observed prominent differences in transcriptome regulation between ungulates and nonungulates, particularly for ICM- and TE-enhanced mRNAs. These results extend our understanding of shared mechanisms of the MBT and formation of the ICM and TE and should better inform the selection of model species for particular applications. [ABSTRACT FROM AUTHOR]

Assisted reproduction is presumed to increase monozygotic twin rates, with the possible contribution of laboratory and medical interventions. Monozygotic dichorionic gestations are supposed to originate from the splitting of an embryo during the first four days of development, before blastocyst formation. Single embryo transfers could result in dichorionic pregnancies, currently explained by embryo splitting as described in the worldwide used medical textbooks, or concomitant conception. However, such splitting has never been observed in human in vitro fertilization, and downregulated frozen cycles could also produce multiple gestations. Several models of the possible origins of dichorionicity have been suggested. However, some possible underlying mechanisms observed from assisted reproduction seem to have been overlooked. In this review, we aimed to document the current knowledge, criticize the accepted dogma, and propose new insights into the origin of zygosity and chorionicity. [ABSTRACT FROM AUTHOR]

Cellular differentiation induces various morphological changes, including elongation, in mitochondria. Preimplantation embryos have round-shaped mitochondria, characteristic of undifferentiated cells. However, there is controversy regarding the precise mitochondrial morphology in blastocyst embryos, which are generated from two cell lineages: undifferentiated inner cell mass (ICM) and differentiated trophectoderm (TE). This study attempted to precisely determine mitochondrial morphology in these two blastocyst regions. Transmission electron microscopy analyses were conducted using more than 1000 mitochondria from blastocyst embryos. No significant differences were observed in the configuration of mitochondrial cristae and frequencies of hooded mitochondria, which are specific to embryos of livestock animals, between the ICM and TE. To accurately compare mitochondrial roundness between the ICM and TE, oblateness was calculated based on both the major and minor axes. Average oblateness was significantly greater in the TE than in the ICM (P < 0.01). These results indicate tissue-specific mitochondrial maturation with complete elongation in the TE at the blastocyst stage. Since mitochondrial elongation is closely associated with cellular metabolism and differentiation, the present study provides new insights for better understanding of early embryonic development in cattle. • Mitochondrial ultrastructure of bovine blastocysts was determined by TEM analysis. • Evaluation of mitochondrial morphology by TEM was based on the oblateness value. • Mitochondria in the trophectoderm were more elongated than those in the inner cell mass. [ABSTRACT FROM AUTHOR]

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two‐round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient‐rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this “on‐gel” culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on‐gel‐cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA‐seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency‐related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways‐related genes to facilitate the functional maturation required for feto‐maternal interaction. Quantitative PCR analysis of TE cells derived from on‐gel culture further confirmed time‐dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species‐specific strategies for mammalian preimplantation development. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells. Methods: TE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next‐generation sequencing. Results: Discordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality. Conclusion: Chromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection. [ABSTRACT FROM AUTHOR]

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Summary: Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7−/− embryos are able to hatch from the zona pellucida, no expansion of Trpm7−/− trophoblast cells can be observed, and Trpm7−/− embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7−/− trophoblast stem cells and the ability of Trpm7−/− ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.

Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos ( Xiang et al., 2020). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state. This article has an associated First Person interview with the first author of the paper.

The tyrosine kinase Src is highly expressed in embryonic stem cells (ESCs) and ESC-differentiated cells, however, its functional role remains obscured. Here, we constitutively expressed Src in mouse ESCs and found these cells retained comparable levels of the core pluripotent factors, such as Oct4 and Sox2, while promoted the expression of epiblast lineage markers and restrained trophoblast lineage markers compared to the control ESCs. Knockdown of Src in mouse ESCs showed the opposite effect. Directly differentiation of these ESCs to epiblast and trophoblast lineage cells revealed that Src activation dramatically accelerated the production of epiblast-like cells and inhibited the induction of trophoblast-like cells in vitro. Mechanistically, we found Src activation enhanced the Yap1-Tead interaction and their transcriptional output in mouse ESCs through specially upregulating Yap1 tyrosine phosphorylation. Subsequently, we found that overexpression of Yap1 in mouse ESCs phenocopied the differentiation patterns of Src overexpressing cells in vitro. Moreover, inhibition of Src kinase activity by Dasatinib or Yap1/Tead-mediated transcription with Verteporfin reversed the differentiation patterns of Src overexpressing ESCs. Taken together, our results unravel a novel Src-Yap1 regulatory axis during mouse ESC differentiation to trophectoderm and epiblast lineage cells in vitro.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine preimplantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-toblastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation. [ABSTRACT FROM AUTHOR]

Study Question: Does trophectoderm (TE) quality affect birthweight after single frozen-thawed blastocyst transfer?Summary Answer: Transfer of single blastocyst with advanced TE quality was associated with higher birthweight and increased risk of a large for gestational age (LGA) baby.What Is Known Already: Transfer of blastocysts with advanced TE quality results in higher ongoing pregnancy rates and a lower miscarriage risk. However, data on the relationship between TE quality and birthweight are still lacking.Study Design, Size, Duration: This retrospective cohort study at a tertiary-care academic medical center included 1548 singleton babies born from single frozen-thawed blastocyst transfer from January 2011 to June 2019.Participants/materials, Setting, Methods: Babies were grouped into four groups according to embryo expansion (Stages 3, 4, 5 and 6), three groups according to inner cell mass (ICM) quality (A, B and C), and three groups according to TE quality (A, B and C). Main outcomes included absolute birthweight, Z-scores adjusted for gestational age and gender, and adverse neonatal outcomes. Multivariable linear and logistic regression analyses were performed to investigate the association of neonatal outcomes with expansion stage, ICM quality and TE quality.Main Results and the Role Of Chance: As TE quality decreased, birthweight (3468.10 ± 471.52, 3357.69 ± 522.06, and 3288.79 ± 501.90 for A, B and C, respectively, P = 0.002), Z-scores (0.59 ± 1.07, 0.42 ± 1.04, and 0.27 ± 1.06 for A, B and C, respectively, P = 0.002) and incidence of LGA (28.9%, 19.7% and 17.4% for A, B and C, respectively, P = 0.027) decreased correspondingly. After adjusting for confounders, compared with the Grade A group, blastocysts with TE Grade B (standardized coefficients (β): -127.97 g, 95% CI: -234.46 to -21.47, P = 0.019) and blastocysts with TE grade C (β: -200.27 g, 95% CI: -320.69 to -79.86, P = 0.001) resulted in offspring with lower birthweight. Blastocysts with TE grade C brought babies with lower Z-scores than TE Grade A (β: -0.35, 95% CI: -0.59 to -0.10, P = 0.005). Also, embryos with TE Grade B (adjusted odds ratio (aOR):0.91, 95% CI: 0.84 to 0.99, P = 0.033) and embryos with TE Grade C (aOR : 0.89, 95% CI: 0.81 to 0.98, P = 0.016) had lower chance of leading to a LGA baby than those with TE Grade A. No association between neonatal outcomes with embryo expansion stage and ICM was observed (all P > 0.05).Limitations, Reasons For Caution: The retrospective design, lack of controlling for several unknown confounders, and inter-observer variation limited this study.Wider Implications Of the Findings: The study extends our knowledge of the down-stream effect of TE quality on newborn birthweight and the risk of LGA.Study Funding/competing Interest(s): This study was funded by National Key R&D Program of China (2018YFC1003000), National Natural Science Foundation of China (81771533 to Y.P.K. and 31200825 to L.S.) and Innovative Research Team of High-level Local Universities in Shanghai (SSMU-ZLCX20180401), Shanghai Sailing Program(21YF1423200) and the Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (JYZZ117). The authors declare no conflict of interest in this present study.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to explore specific factors that predispose to monozygotic twinning (MZT) at the blastocyst stage. Methods: This was a retrospective observational study of a cohort of 2863 pregnancies after single blastocyst transfer (SBT) between January 2011 and June 2019 in our hospital. MZT pregnancy was identified as the number of fetuses exceeded the number of gestational sacs (GSs) by transvaginal ultrasound at 6–7 gestational weeks. The incidences of MZT regarding the maternal age at oocyte retrieval, paternal age, ovarian stimulation protocol, fertilization method, endometrium preparation protocol, vitrified day, and the Gardner grading of the blastocyst were calculated. The serum estrogen (E2), progesterone (P) levels, endometrium thickness and serum hCG levels on day 11 after embryo transfer (ET) were compared between the MZT and singleton pregnancies. Statistical analyses were used appropriately. Results: Fifty-one MZT pregnancies (1.78%) were identified. The only significant differences observed between MZT and singleton pregnancies were the proportion of TE grade (P = 0.022) and the hCG levels on day 11 after ET (P = 0.003). Multivariate logistic regression revealed that trophectoderm (TE) grade was an independent factor affecting MZT, the adjusted odds ratios (aORs) of grade A and B TE were 5.46 [95% confidential interval (CI) 1.48–20.16, P = 0.011) and 3.96 (95% CI 1.17–13.40, P = 0.027) compared to grade C respectively. There were no significant associations between the parental age, fertilization method, ovarian stimulation protocol, endometrium preparation protocol, vitrified day, expansion stage, inner cell mass (ICM) grade and MZT. Conclusions: TE grade is associated with MZT at the blastocyst stage, potentially mediated via increased secretion of hCG from more well developed TE. Increased hCG secretion in turn may prolong the implantation window to support the embryo splitting. [ABSTRACT FROM AUTHOR]

The first event of cellular differentiation consists of the segregation of the trophectoderm and the inner cell mass. Studies inmice suggest that cell contractility and the formation of an apical domain play important roles in this event; however, this remains unknown in the bovine. We tested the hypothesis that blocking apical domain formation would halt subsequent trophectoderm differentiation in bovine embryos. We first assessed the formation of an apical domain by the presence of Par-6 Family Cell Polarity Regulator Beta (PARD6B) and Ezrin (EZR), which appeared after the 8-cell stage. We inhibited apical domain formation by blocking cell contractility with 25 mM(-)-blebbistatin. Treatment from 90 to 186 h after insemination did not reduce blastocyst development compared with the untreated control group or the group treated with inactive (þ)-blebbistatin. Immunofluorescence staining after blebbistatin treatment revealed the absence of EZRand the trophectoderm marker Caudal Type Homeobox 2 (CDX2). Following blebbistatin treatment, Yes1 Associated Transcriptional Regulator (YAP), which is involved in the Hippo signalling pathway, exhibited cytoplasmic staining instead of nuclear localisation. Despite changes in protein expression and localisation, no difference in trophectoderm or total cell numbers was observed. In conclusion, inhibition of cell contractility inhibited apical domain formationwithout impairing blastocyst formation, suggesting that a different biological mechanism is involved in trophectoderm and inner cell mass differentiation in bovine embryos. [ABSTRACT FROM AUTHOR]

Background: Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single‐cell RNA‐seq data derived from human and mouse embryos. Results: By weighted gene‐coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage‐segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre‐EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single‐cell RNA‐seq. We found that SIN3A knockdown disrupts the gene‐silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos. Conclusion: Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development. Key Findings: A dynamic transcriptome for pre‐implantation embryos in cynomolgus monkey.SIN3A is a critical regulatory gene for pre‐implantation development in among three mammalian species.SIN3A is important for gene silencing during the EGA.New knowledge of evolutionarily conserved and divergent transcriptome dynamics during mammalian pre‐implantation development. [ABSTRACT FROM AUTHOR]

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias. [Display omitted] • Comparison of hTSCs, hTELCs, primed hPSCs, naive hPSCs, and hEPSCs • hEPSCs have DNAme levels and X activity similar to primed hPSCs • hEPSCs have a higher metabolic activity than hPSCs • hTSCs have the highest metabolic activity of all cell types measured Onfray et al. report the comparison of hTSCs, hTELCs, and primed, extended, and naive hPSCs by transcriptomics, proteomics, DNAme quantification, X, and metabolic activity. TELCs have features of trophectoderms, while TSCs represent post-implantation trophoblasts. hEPSCs are clearly primed cells but display a higher metabolic and clonogenic potential than regular primed PSCs. [ABSTRACT FROM AUTHOR]

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and ‘EOMES transcription factor mRNAs’ were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

Abstract Objective To assess the predictive value of each individual morphological parameter: blastocoele expansion degree, inner cell mass (ICM), and trophectoderm (TE) grades on the clinical pregnancy outcome in frozen–warmed embryo transfer (FET) cycles. Methods This is a retrospective cohort study, including 1154 FET cycles receiving vitrified-warmed one or two blastocysts transfer from August 2011 through to May 2018. The correlation between blastocyst morphology parameters and clinical outcome after FET was assessed. Results In the subgroup analysis based on clinical pregnancy, the patients who achieved clinical pregnancy had a significantly higher degree of blastocyst expansion (3.69 ± 0.68 vs. 3.53 ± 0.78, P = 0.000) and had a thicker endometrium (9.65 ± 1.63 vs. 9.28 ± 1.64) compared with those with non-clinical pregnancy. The logistic regression analysis showed that among the three blastocyst morphology parameters, only the blastocoele expansion degree was significantly correlated with the clinical pregnancy outcome and had ability to predict the outcome after FET cycles with one or two vitrified-warmed blastocysts transferred. Both ICM and TE stages were not associated with pregnancy outcomes. Conclusions The blastocoele expansion degree may be essential for successful pregnancy and should be given priority when selecting frozen blastocyst for transfer.

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. • WWTR1 proteins localized within nuclei of trophectoderm cells in bovine blastocysts. • WWTR1 mRNA knockdown induced reduction of trophectoderm cells. • WWTR1 mRNA knockdown significantly reduced expression of trophectoderm-specific genes. • WWTR1 mRNA knockdown specifically disrupted trophectoderm development in bovine embryos. [ABSTRACT FROM AUTHOR]

Which pre-vitrification parameters are the most predictive of survival and live birth in vitrified–warmed blastocyst transfer cycles? A retrospective study including 11,936 warmed blastocysts. Pre-vitrification morphological parameters analysed for blastocysts included day of vitrification; blastocyst expansion degree; trophoectoderm grade (A, B and C); and inner cell mass grade (A, B and C). Univariate and multivariate generalized estimating equations models were used to analyse survival, clinical pregnancy and live birth rate. A stepwise regression analysis was conducted to select and classify by order which outcomes were the most predictive. The odds of survival increased almost twice for blastocysts with lower expansion degree (OR 1.92; 95% CI 1.37 to 2.69; P < 0.001) and by about 50% for blastocysts vitrified on day 5 (OR 1.56; 95% CI 1.27 to 1.89; P < 0.001). Multivariate generalized estimating equations model showed that trophectoderm grade followed by the day of vitrification were the most significant predictors of live birth. The odds of live birth increased nearly three times for blastocysts with trophectoderm graded as A compared with those with trophectoderm graded as C (OR 2.85; 95% CI 2.48 to 3.27; P < 0.001), and double for blastocysts vitrified on day 5 compared with those vitrified on day 6 (OR 2.22; 95% CI 1.97 to 2.49; P < 0.001). The odds of live birth also increased in higher expansion degree blastocysts. Blastocysts vitrified on day 5 and those with higher trophoectoderm grade should be given priority when warming. [ABSTRACT FROM AUTHOR]

Bovine mesenchymal stem cells are a relevant cell population found in the maternal reproductive tract that exhibits the immunomodulation capacity required to prevent embryo rejection. The phenotypic plasticity showed by both endometrial mesenchymal stem cells (eMSC) and embryonic trophoblast through mesenchymal to epithelial transition and epithelial to mesenchymal transition, respectively, is essential for embryo implantation. Embryonic trophoblast maintains active crosstalk via EVs and soluble proteins with eMSC and peripheral blood MSC (pbMSC) to ensure the retention of eMSC in case of pregnancy and induce the chemotaxis of pbMSC, critical for successful implantation. Early pregnancy-related proteins and angiogenic markers are detected as cargo in EVs and the soluble fraction of the embryonic trophectoderm secretome. The pattern of protein secretion in trophectoderm-EVs changes depending on their epithelial or mesenchymal phenotype and due to the uptake of MSC EVs. However, the changes in this EV-mediated communication between maternal and embryonic MSC populations infected by viruses that cause abortions in cattle are poorly understood. They are critical in the investigation of reproductive viral pathologies.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, in vitro -produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n = 80) and vitrified (n =80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell–sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation. Global gene expression of the bovine whole embryo or isolated trophectoderm from fresh or vitrified day-14 embryos was profiled by RNA sequencing, which identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation. [ABSTRACT FROM AUTHOR]

The dysfunction of placenta development is correlated to the defects of pregnancy and fetal growth. The detailed molecular mechanism of placenta development is not identified in humans due to the lack of material in vivo. Trophoblast (TB) lineage derived from human embryonic stem cells (hESCs) induced by bone morphogenetic protein 4 (BMP4) has been applied as a model for studying TB lineage specification in vitro. With the development of single-cell sequencing technology, it became possible to detect the transcriptome of the post-implantation embryo at unprecedented precision. In this study, we reanalyzed single-cell RNA-seq of post-implantation embryos derived from two separate groups and identified different subtypes of trophoblast cells and their marker, respectively. At the same time, we focused on the gene expression patterns of trophoblast-specific transcription factors in different models. Our analysis sheds new light on the transcription regulation mechanism of trophoblast differentiation at the early stage of pregnancy establishment in human. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) are pluripotent stem cell populations derived from the preimplantation embryo and are used to study the differentiation of many types of somatic and germ cells in developing embryos. They are also used to study cell lineages of extraembryonic tissues, such as the trophectoderm (TE) and the primitive endoderm (PrE). mESC cultures are suitable systems for reproducing cellular and molecular events occurring during the differentiation of these cell types, such as changes in gene expression patterns, signaling events, and genome rearrangements although the consistency between the results obtained using mESCs and those of in vivo studies on embryos should be carefully taken into account. Since TE and PrE cells can be induced from mESCs in vitro, mESC cultures are useful systems to study differentiation of these cell lineages during development, if used appropriately. In addition, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human‐induced pluripotent stem cells (hiPSCs), are capable of generating extraembryonic lineages in vitro and are promising tools to study the differentiation of these lineages in the human embryo. [ABSTRACT FROM AUTHOR]

Objectives: To examine the characteristics and distribution of possible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target cells in the human trophectoderm (TE) and placenta.Methods: Bioinformatics analysis was performed based on published single-cell transcriptomic datasets of early TE and first- and second-trimester human placentae. We conducted the transcriptomic analysis of 4198 early TE cells, 1260 first-trimester placental cells and 189 extravillous trophoblast cells (EVTs) from 24-week placentae (EVT_24W) using the SMART-Seq2 method. In addition, to confirm the bioinformatic results, we performed immunohistochemical staining of three first-trimester, three second-trimester and three third-trimester placentae from nine women recruited prospectively to this study. We evaluated the expression of the SARS-CoV-2-related molecules angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2).Results: Via bioinformatic analysis, we identified the existence of ACE2 and TMPRSS2 expression in human TE as well as in first- and second-trimester placentae. In the human TE, 54.4% of TE1 cells, 9.0% of cytotrophoblasts (CTBs), 3.2% of EVTs and 29.5% of syncytiotrophoblasts (STBs) were ACE2-positive. In addition, 90.7% of TE1 cells, 31.5% of CTBs, 22.1% of EVTs and 70.8% of STBs were TMPRSS2-positive. In placental cells, 20.4% of CTBs, 44.1% of STBs, 3.4% of EVTs from 8-week placentae (EVT_8W) and 63% of EVT_24W were ACE2-positive, while 1.6% of CTBs, 26.5% of STBs, 1.9% of EVT_8W and 20.1% of EVT_24W were TMPRSS2-positive. Pathway analysis revealed that EVT_24W cells that were positive for both ACE2 and TMPRSS2 (ACE2 + TMPRSS2-positive) were associated with morphogenesis of branching structure, extracellular matrix interaction, oxygen binding and antioxidant activity. The ACE2 + TMPRSS2-positive TE1 cells were correlated with an increased capacity for viral invasion, epithelial-cell proliferation and cell adhesion. Expression of ACE2 and TMPRSS2 was observed on immunohistochemical staining in first-, second- and third-trimester placentae.Conclusions: ACE2- and TMPRSS2-positive cells are present in the human TE and placenta in all three trimesters of pregnancy, which indicates the possibility that SARS-CoV-2 could spread via the placenta and cause intrauterine fetal infection. © 2020 International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDK) are key regulatory enzymes that regulate proliferation dynamics and cell fate in response to extracellular inputs. It remains largely unknown how CDK activity fluctuates and influences cell commitment in vivo during early mammalian development. Here, we generated a transgenic mouse model expressing a CDK kinase translocation reporter (KTR) that enabled quantification of CDK activity in live single cells. By examining pre- and post-implantation mouse embryos at different stages, we observed a progressive decrease in CDK activity in cells from the trophectoderm (TE) prior to implantation. This drop correlated with the establishment of an FGF4-dependent signaling gradient through the embryonic-abembryonic axis. Furthermore, we showed that CDK activity levels do not determine cell fate decisions during pre-implantation development. Finally, we uncovered the existence of conserved regulatory mechanisms in mammals by revealing lineage-specific regulation of CDK activity in TE-like human cells., Competing Interests: COMPETING INTERESTS The authors declare no competing interests.