Blastocyst Trophectoderm Research Articles

Genome-wide epigenetic changes in compartments of genetically unbalanced human blastocysts

BACKGROUND: Epigenetic genome reprogramming is an important determinant of human embryo development. However, its mechanisms remain poorly elucidated, especially in genetically unbalanced embryos. AIM: The aim of this study is the analysis of DNA methylation and hydroxymethylation levels in trophectoderm and inner cell mass of genetically balanced and unbalanced human blastocysts. MATERIALS AND METHODS: Twenty-two IVF-derived human blastocysts were enrolled in the study; of these blastocysts, 15 were genetically unbalanced and 7 — genetically balanced. Detection of 5-methylcytosine and 5-hydroxymethylcytosine was performed on trophectoderm and inner cell mass nuclei by indirect immunofluorescence. RESULTS: In genetically unbalanced blastocysts, the DNA methylation level was elevated in both compartments. The DNA hydroxymethylation level, in contrast, was elevated only in inner cell mass, whereas trophectoderm cells retained the same level as in genetically balanced embryos. These changes equalized the inner cell mass and trophectoderm DNA hydroxymethylation levels in genetically unbalanced blastocysts, while in genetically balanced ones the 5-hydroxymethylcytosine content in inner cell mass lagged behind that in trophectoderm. CONCLUSIONS: Genetic imbalance is associated with differential epigenetic changes in trophectoderm and inner cell mass cells of human blastocysts: DNA methylation level increases in both compartments while DNA hydroxymethylation level increases only in inner cell mass. The trophectoderm cells in genetically unbalanced blastocysts retain the same hydroxymethylation level as in genetically balanced ones, suggesting a possible explanation of the ability of karyotypically abnormal embryos to implant.

Chromatin landscape instructs precise transcription factor regulome during embryonic lineage specification

Embryos, originating from fertilized eggs, undergo continuous cell division and differentiation, accompanied by dramatic changes in transcription, translation, and metabolism. Chromatin regulators, including transcription factors (TFs), play indispensable roles in regulating these processes. Recently, the trophoblast regulator TFAP2C was identified as crucial in initiating early cell fate decisions. However, Tfap2c transcripts persist in both the inner cell mass and trophectoderm of blastocysts, prompting inquiry into Tfap2c's function in post-lineage establishment. In this study, we delineate the dynamics of TFAP2C during the mouse peri-implantation stage and elucidate its collaboration with the key lineage regulators CDX2 and NANOG. Importantly, we propose that de novo formation of H3K9me3 in the extraembryonic ectoderm during implantation antagonizes TFAP2C binding to crucial developmental genes, thereby maintaining its lineage identity. Together, these results highlight the plasticity of the chromatin environment in designating the genomic binding of highly adaptable lineage-specific TFs and regulating embryonic cell fates.

The utility of human two plus one small pronucleated zygotes (2.1PN) based on clinical outcomes and the focused ploidy analysis

PurposeAre human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful?MethodsIn a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity.ResultsThe average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid.ConclusionsIt was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis.

Increasing maternal glucose concentrations is insufficient to restore placental glucose transfer in chorionic somatomammotropin RNA interference pregnancies.

We previously demonstrated impaired placental nutrient transfer in chorionic somatomammotropin (CSH) RNA interference (RNAi) pregnancies, with glucose transfer being the most impacted. Thus, we hypothesized that despite experimentally elevating maternal glucose, diminished umbilical glucose uptake would persist in CSH RNAi pregnancies, demonstrating the necessity of CSH for adequate placental glucose transfer. Trophectoderm of sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either nontargeting control (CON RNAi; n = 5) or CSH-specific shRNA (CSH RNAi; n = 7) before transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters and underwent steady-state metabolic studies (3H2O transplacental diffusion) at 137 ± 0 dGA, before and during a maternal hyperglycemic clamp. Umbilical glucose and oxygen uptakes, as well as insulin and IGF1 concentrations, were impaired (P ≤ 0.01) in CSH RNAi fetuses and were not rescued by elevated maternal glucose. This is partially due to impaired uterine and umbilical blood flow (P ≤ 0.01). However, uteroplacental oxygen utilization was greater (P ≤ 0.05) during the maternal hyperglycemic clamp, consistent with greater placental oxidation of substrates. The relationship between umbilical glucose uptake and the maternal-fetal glucose gradient was analyzed, and while the slope (CON RNAi, Y = 29.54X +74.15; CSH RNAi, Y = 19.05X + 52.40) was not different, the y-intercepts and elevation were (P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. Together, these data suggested that CSH plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer.NEW & NOTEWORTHY The current study demonstrated a novel, critical autocrine role for chorionic somatomammotropin in augmenting placental glucose transfer and maintaining placental oxidative metabolism. In pregnancies with CSH deficiency, excess glucose in maternal circulation is insufficient to overcome fetal hypoglycemia due to impaired placental glucose transfer and elevated placental metabolic demands. This suggests that perturbations in glucose transfer in CSH RNAi pregnancies are due to compromised metabolic efficiency along with reduced placental mass.

Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Live birth after single euploid frozen embryo transfer in a 39-year-old woman with high-grade mosaic Turner syndrome

Objective To describe the reproductive and obstetric outcomes of an intracytoplasmic sperm injection cycle with preimplantation genetic testing for aneuploidy in an advanced reproductive-age woman with high-grade mosaic Turner syndrome. Methods Case report of a 39-year-old woman diagnosed with mosaic Turner Syndrome 45,X[90]/46,XX[10] karyotype who underwent in vitro fertilization treatment with blastocyst trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing. Result(s) Two of the four blastocysts biopsied were euploid. The patient achieved ongoing pregnancy after the first single euploid frozen embryo transfer, followed by the birth of a healthy child. Conclusion Autologous intracytoplasmic sperm injection cycles can be considered in a select group of advanced reproductive-age women diagnosed with high-grade mosaic Turner syndrome.

O-093 Endometrium-blastocyst cross talk: how far are we from understanding?

Abstract The early events characterising implantation of the human embryo represent a critical step in which proper activation of orchestrated molecular pathways regulate the success of pregnancy. Understanding this complex network is considered by many the last frontier of human reproduction. Although outstanding research advancements have provided clinicians tools to select the best quality embryo, identification of the optimal timing in which the endometrium is ready to accommodate it remains a challenge. Over the last decade several studies have identified many key players involved in the preparation of the endometrium to implantation and several diffusible signals guiding the embryo in its journey toward the maternal endometrial tissue have been uncovered (Massimiani et al. 2020). However, pieces of the puzzle are still missing. For convenience, the process of implantation is subdivided into several sequential processes, the completion of one being propaedeutic to the following. Once the blastocyst has arrived in close proximity to the uterine epithelium, the first event occurring is its escape from the zona pellucida. The molecular basis of blastocyst hatching remains poorly understood, and species-specific differences have been reported. Using the mouse model, we recently demonstrated that thyroid hormone up-regulates the expression of specific lytic enzymes (Isp1 and Isp2) known mediators of mouse embryo zona lysis. Interestingly, we observed that TH-mediated upregulation of Isp1 and Isp2 is strictly dependent on the presence of endometrial stromal cell feeder layer, clearly indicating that blastocyst–endometrium interaction is indispensable for TH-mediated increased expression of these proteases. So far, no molecular signals regulating hatching in humans have been identified, and only mechanical forces have been proposed to induce zona opening; however very recently the possibility that proteases may be involved also in humans has been suggested (Almagor et al., 2020), although further studies are needed to support this hypothesis. Once free from the zona, the embryo contacts the endometrial epithelium and starts its journey to further proceed in the consolidation of pregnancy. Hormones, pro- and anti-inflammatory mediators, growth factors and their receptors, adhesion molecules, and proteases and protease inhibitors have been reported in the regulation of the process of embryo apposition, adhesion, and invasion (Massimiani et al., 2020). Among the many molecular signals, the Notch pathway has been recognized key in embryo-endometrium cross talk (Cuman et al., 2014). Transient activation of the Notch1 receptor has been demonstrated in the process of decidualization (Afshar et al., 2012), a limiting step to implantation, and blastocyst-conditioned medium has been shown to induce the expression of Notch family members in decidual cells (Hess et al., 2007). Expression of notch receptors and ligands has been reported in the blastocyst trophectoderm as well (Cuman et al., 2014). We previously demonstrated that the secreted factor epidermal growth factor-like domain 7 (EGFL7) is a novel modulator of the Notch pathway, it is expressed by trophoblast cells of the mouse blastocyst (Fitch et al., 2004; Lacko et al., 2014) and it regulates human trophoblast migration and invasion ability by activating the Notch pathway (Massimiani et al., 2015). Data will be presented demonstrating that EGFL7 is expressed in the human endometrium and its expression is mainly localized in the glandular epithelium throughout the menstrual cycle. EGFL7 is significantly upregulated in whole endometrial tissue during the secretory phase, particularly in the stromal compartment; this observation is further supported by in vitro studies showing its upregulation in decidualized endometrial stromal cells, suggesting a role for EGFL7 in implantation. Further support is provided by the evidence that its levels are strongly downregulated in the endometrium of women with impaired fertility. We propose EGFL7 as a novel player involved in the embryo-endometrium cross talk.

P-121 Identification of differentially expressed genes common to the developmental potential, maternal age, and morphology in human blastocysts

Abstract Study question Is it possible to apply RNA-sequencing to assisted reproductive technology (ART) to predict blastocyst quality? Summary answer We identified 14 commonly identified differentially expressed genes by grouping blastocysts according to three distinct parameters such as developmental potential, maternal age, and Gardner score What is known already In ART, while the selection of a suitable embryo for transfer is critical for a successful pregnancy, neither preimplantation genetic testing nor morphological and chronological evaluation of the embryo can fully guarantee a successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. We have reported that 96 differentially expressed genes (DEGs) were identified using RNA-seq of each inner cell mass (ICM) and trophectoderm (TE) in blastocysts classified according to the developmental potential which correlates with pregnancy rate at the ESHRE 37th annual meeting. Study design, size, duration After retrospectively analyzing 1,890 cases undergoing freeze-thaw blastocyst transfer from March 2018 to December 2020 to examine the correlation between blastocyst developmental potential and pregnancy rate, a total 13 blastocysts cryopreserved between February 2011 and September 2018, then scheduled for disposal and with consented, were subjected to RNA-seq to identify genes associated with pregnancy expectation. RNA-seq data were then examined whether common DEGs could be found when classified by maternal age and Gardner score, respectively. Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE cells and ICM cells were collected from blastocysts classified by developmental potential and subjected to RNA-seq to identify DEGs. In addition, RNA-seq data were regrouped by maternal age and Gardner score to find common DEGs. DEGs (q-value < 0.01) were identified using the R package “DESeq2” (version 1.32.0). Main results and the role of chance When the RNA-seq data obtained from blastocysts classified according to pregnancy expectation were re-grouped by maternal age and re-analyzed, we identified 7 and 17 genes that were down- and up-regulated in the elder group, respectively, in ICM. In TE, 2 and 12 genes were down- and up-regulated in the elder group, respectively. On the other hand, when re-grouped by Gardner score (<BB versus <BB), we identified 26 and 29 genes that were down- and up-regulated in the poor group, respectively, in ICM. In TE, 64 and 20 genes were down- and up-regulated in the poor group, respectively. A comparison of these genes and DEGs identified by the pregnancy expectation found 14 common genes. By comparison with the elder group, we identified one gene (UCHL1) that was commonly up-regulated in TE. By comparison with the poor Gardner score group, one gene (CMTM7) was commonly down-regulated and three genes (LINC00458, DYNLRB1, and UBL4A) were commonly up-regulated in ICM, and 5 genes (LRIG3, SLC28A3, CXADR, LOC727896, and ATP1B1) were commonly down-regulated and 5 genes (UCHL1, BZW2, NABP1, HEXIM1, and PLEKHA6) were commonly up-regulated in TE. Limitations, reasons for caution Although we established an expected pregnancy rate concerning the degree of blastocyst development from retrospective clinical outcomes and used it as a surrogate marker for assigning biopsied blastocysts to different analysis groups, it remains unknown whether the gene expression profiles accurately reflect the pregnancy outcomes. Wider implications of the findings UCHL1 expression was commonly increased with lower blastocyst developmental potential, higher maternal age, and lower Gardner score. Since UCHL1 has been reported to be essential for blastocyst development in mice, our results suggest that UCHL1 may also be a marker of blastocyst quality in humans. Trial registration number not applicable

P-793 Human trophoblastic spheroid derived from human expanded potential stem cells (hEPSC) for the identification of adhesion molecules for embryo implantation.

Abstract Study question Are cell surface proteins on trophoblastic spheroid BAP-EB derived from hEPSC involved in the endometrial epithelial cell attachment? Summary answer E-cadherin (CDH1) and Neuropilin-1 (NRP1) are two potential embryonic surface molecules involved in the human implantation process. What is known already Study of the mechanisms of human implantation in vivo is challenging. The availability of high-quality human embryos for in vitro research is limited. We have recently derived morula-like hEPSC lines (hEPSC-em) from donated human embryos. Trophoblastic spheroids (em-BAP-EB) derived from hEPSC-em resemble human trophectoderm during the early implantation process. The em-BAP-EBs attach specifically onto receptive endometrial epithelial cells, suggesting its usefulness as human embryo surrogate for studying embryonic surface molecules involving in early implantation process. Study design, size, duration hEPSC-em was subjected to embryoid body formation and induced to differentiate into trophoblastic spheroids (em-BAP-EB) for 48h and 72h. Endometrial aspirates were collected from infertile women in their natural cycles 2 or 7 days after LH surge (LH + 2/+7). Endometrial epithelial cells (EEC) were isolated and cultured for the attachment assays. Biotin-labelled apical surface proteins on em-BAP-EB were identified by mass spectrometry. Antibody blocking assays were performed to confirm the biological functions. Participants/materials, setting, methods The attachment rates of em-BAP-EB onto EEC were compared between pre-receptive (LH + 2 day) and receptive (LH + 7 day) phases. The differentially expressed apical surface protein on em-BAP-EB-48h and -72h were subjected to plasma membrane proteins (PMP) identification and pathway analysis. The gene and protein expression patterns of the target proteins were analyzed by single cell RNA sequencing (scRNA-seq) and western blotting respectively. The functional roles of the target proteins were studied by antibody blocking during attachment Main results and the role of chance The attachment rates of em-BAP-EB-72h onto receptive stage (LH + 7, n = 17) EEC were significantly higher than those onto pre-receptive stage (LH + 2, n = 5) EEC. Gene Ontology analysis indicated that the PMP with induced expression levels on em-BAP-EB-72h when compared to -48h (72h>48h) were enriched for “protein binding” and “cadherin binding” in molecular functions, and “cell-cell adhesion” in biological processes. Among them, 28 PMP were trophectoderm-specific. CDH1 and NRP1 were selected for further analysis. Analysis based on a published dataset showed that significantly higher expressions of CDH1 and NRP1 were detected in polar trophectoderm as compared to mural trophectoderm of E7 human blastocyst. CDH1 and NRP1 were significantly up-regulated in trophectoderm-like em-BAP-EB by scRNA-seq analysis. Live-cell immunocytochemistry demonstrated more intense apical surface expression of CDH1 and NRP1 on em-BAP-EB-72h as compared to -48h. Antibody blocking experiments indicated that pre-treatment of the em-BAP-EB-72h with specific antibody individually against the ectodomains of CDH1 and NRP1 significantly reduced the attachment rates onto receptive endometrial epithelial cells. The data suggested that CDH1 and NRP1 are two potential apical surface molecules essential for blastocyst attachment. Limitations, reasons for caution The lack of embryonic compartment in the em-BAP-EB and the absence of other key players like decidualized stromal cells and immune cells in the coculture model might not fully represent the early attachment process. The limited sample size in this study may also limit the interpretation of the data. Wider implications of the findings Despite advancement in in vitro fertilization, the success rate remains unsatisfactory. It is not known if embryological factor(s) other than embryo aneuploidy are involved. The current data may suggest the potential roles of CDH1 and NRP1 as non-chromosomal embryological markers of its implantation potential. Trial registration number Not applicable

Identifying parental and cell-division origins of aneuploidy in the human blastocyst

Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%-100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n= 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.

Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts classified according to developmental potential, maternal age and morphology.

Selection of high-quality embryos is important to achieve successful pregnancy in assisted reproductive technology (ART). Recently, it has been debated whether RNA-sequencing (RNA-Seq) should be applied to ART to predict embryo quality. However, information on genes that can serve as markers for pregnant expectancy is limited. Furthermore, there is no information on which transcriptome of trophectoderm (TE) or inner cell mass (ICM) is more highly correlated with pregnant expectancy. Here, we performed RNA-Seq analysis of TE and ICM of human blastocysts, the pregnancy expectation of which was retrospectively determined using the clinical outcomes of 1,890 cases of frozen-thawed blastocyst transfer. We identified genes that were correlated with the expected pregnancy rate in ICM and TE, respectively, with a larger number of genes identified in TE than in ICM. Downregulated genes in the TE of blastocysts that were estimated to have lower expectation of pregnancy included tight junction-related genes such as CXADR and ATP1B1, which have been implicated in peri-implantation development. Moreover, we identified dozens of differentially expressed genes by regrouping the blastocysts based on the maternal age and the Gardner score. Additionally, we showed that aneuploidy estimation using RNA-Seq datasets does not correlate with pregnancy expectation. Thus, our study provides an expanded list of candidate genes for the prediction of pregnancy in human blastocyst embryos.

What 18028 biopsied blastocyst show us: biopsy technique, day of embryo biopsy and blastocyst stage are related to the risk of obtaining an inconclusive chromosomal assessment after trophectoderm biopsy.

Background and Aim: PGT-A is a reliable program in the IVF Unit but, occasionally, fails to yield a diagnostic result. The prevalence of inconclusive diagnoses reported in the literature ranges from 0.8 to 6% and, regardless of the etiology, are an inherent limitation of PGT-A that pose a challenge to both patients and clinicians. The objective of our work was to identify causative factors as blastocyst features and laboratory procedures possibly associated with the probability of obtaining an inconclusive result after blastocyst trophectoderm biopsy.

Correlation of the position and status of the polar body from the fertilized oocyte to the euploid status of blastocysts.

Polar bodies are tiny cells that are extruded during oocyte meiosis and are generally considered not essential for embryonic development. Therefore, polar bodies have been widely used as important materials for the preimplantation genetic diagnosis of human embryos. Recent studies have shown that polar bodies mediate embryonic development and that their morphology is related to embryo quality and developmental potential. However, the relationship between the emission of the polar body and embryonic euploidy remains unclear. In this study, a total of 1,360 blastocyst trophectoderm (TE) biopsies were performed, and blastocyst ploidy results were correlated with the state of polar bodies. The results showed that polar body angle size and polar body status are not directly related to whether the blastocysts are euploid, aneuploid, or mosaic (p > 0.05). Therefore, in the process of clinical embryo selection, embryologists should not predict the euploidy of blastocysts based on the state of polar bodies, thus affecting embryo selection.

Why Euploid Blastocysts Fail

Preimplantation genetic testing for aneuploidy improves implantation and reduces miscarriage rates, but still about 30 to 35% of euploid blastocysts fail to implant and 10 to 13% of pregnancies following euploid blastocyst transfer miscarry. Both endometrial and embryo factors may contribute to failed euploid embryo transfers. Endometrial factors include uterine structural abnormalities, altered timing of endometrial receptivity, glandular-stromal disynchrony, and inflammatory states. Recent studies question the clinical benefit of some recent assays for endometrial receptivity. Other recent studies question the value of surgical correction of uterine septae and fibroids to prevent further pregnancy loss. Embryo factors include aneuploidy, either undetected by PGT-A performed on the blastocyst trophectoderm (i.e. false negative test) or de novo non-disjunction. Sub chromosomal genetic variation, e.g. copy number variants and de novo retrotransposon insertions, may impair embryo viability. We employ a “genetics first” approach, which relies on genetic analysis of products of conception, obtained by uterine evacuation, to evaluate miscarriage after euploid blastocyst transfer. Non-invasive genetic analysis of first trimester pregnancies also may help characterize the cause of embryo loss after euploid embryo transfer. Early results suggest that analysis of cfDNA and trophoblast retrieval and isolation from the cervix (TRIC) may allow women experiencing early pregnancy loss to learn about the genetic state of their miscarriage, without an invasive surgical procedure to obtain products of conception.

Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development.

Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with invivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and invitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Morphology of inner cell mass: a better predictive biomarker of blastocyst viability.

BackgroundTransfer 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.MethodsThis 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.ResultsA 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).ConclusionThis 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.

Single-cell multiomics analyses of spindle-transferred human embryos suggest a mostly normal embryonic development

Mitochondrial DNA (mtDNA) mutations are often associated with incurable diseases and lead to detectable pathogenic variants in 1 out of 200 babies. Uncoupling of the inheritance of mtDNA and the nuclear genome by spindle transfer (ST) can potentially prevent the transmission of mtDNA mutations from mother to offspring. However, no well-established studies have critically assessed the safety of this technique. Here, using single-cell triple omics sequencing method, we systematically analyzed the genome (copy number variation), DNA methylome, and transcriptome of ST and control blastocysts. The results showed that, compared to that in control embryos, the percentage of aneuploid cells in ST embryos did not significantly change. The epiblast, primitive endoderm, and trophectoderm (TE) of ST blastocysts presented RNA expression profiles that were comparable to those of control blastocysts. However, the DNA demethylation process in TE cells of ST blastocysts was slightly slower than that in the control blastocysts. Collectively, our results suggest that ST seems generally safe for embryonic development, with a relatively minor delay in the DNA demethylation process at the blastocyst stage.

Primary specification of blastocyst trophectoderm by scRNA-seq: New insights into embryo implantation.

Mechanisms of implantation such as determination of the attachment pole, fetal-maternal communication, and underlying causes of implantation failure are largely unexplored. Here, we performed single-cell RNA sequencing on peri-implantation embryos from both humans and mice to explore trophectoderm (TE) development and embryo-endometrium cross-talk. We found that the transcriptomes of polar and mural TE diverged after embryos hatched from the zona pellucida in both species, with polar TE being more mature than mural TE. The implantation poles show similarities in cell cycle activities, as well as in expression of genes critical for implantation and placentation. Embryos that either fail to attach in vitro or fail to implant in vivo show abnormalities in pathways related to energy production, protein metabolism, and 18S ribosomal RNA m6A methylation. These findings uncover the gene expression characteristics of humans and mice TE differentiation during the peri-implantation period and provide new insights into embryo implantation.

The trophectoderm acts as a niche for the inner cell mass through C/EBPα-regulated IL-6 signaling.

SummaryIL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6.

Effect of trophectoderm biopsy for PGT-A on live birth rate per embryo in good prognosis patients

PurposeTo determine if blastocyst trophectoderm biopsy for PGT-A is associated with an increased rate of live birth per embryo in good prognosis IVF patients at a single center.MethodsWe performed a retrospective cohort study of good prognosis embryo transfer cycles at a single center from 1/1/2017 to 12/31/2019. We evaluated the rate of live birth per embryo with and without PGT-A for transfer of embryos in two groups of good prognosis patients: embryos from donor oocytes and embryos from autologous oocytes with maternal age less than 35 years at oocyte retrieval. Two-sided Fisher’s exact tests were used for comparisons between groups.ResultsAfter transfer of embryos created from donor oocytes the live birth rate per euploid embryo was 70.6% (24/34) compared to 34.3% (35/102) for untested embryos for a rate difference of 36.3% (95% CI 18.4–54.1%, p < 0.01). After transfer of embryos created from autologous oocytes with maternal age less than 35 years at oocyte retrieval the live birth rate per euploid embryo was 70.0% (49/70) compared to 52.5% (53/101) for untested embryos for a rate difference of 17.5% (95% CI 3.0–32.0%, p = 0.03).ConclusionsIn good prognosis patients at our center the live birth rate per euploid blastocyst was higher than for untested blastocysts.