Blastocyst Trophectoderm Research Articles

P-138 Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts with expected pregnancy rates

Abstract Study question Is it possible to identify the molecular factors that contribute to the implantation potential of blastocysts? Summary answer Genes correlated with expected pregnancy rate in trophectoderm (TE) and inner cell mass (ICM) respectively were identified, and aneuploidy alone couldn’t predict the pregnancy expectation. What is known already The selection of suitable embryos for transfer is critical for achieving successful pregnancy outcomes in assisted reproductive technology (ART). Although pre-implantation genetic testing for aneuploidy (PGT-A) as well as morphological and chronological evaluation of embryos, have been conducted in clinical practice, they do not fully guarantee 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. Study design, size, duration To determine the correlation between blastocyst evaluation and pregnancy rate, we retrospectively analyzed 1,890 cases underwent frozen-thawed blastocyst transfer from March 2018 to December 2020. A total of 13 blastocysts that were cryopreserved for clinical use between February 2011 and September 2018, then scheduled for disposal and with consented for research, were subjected to RNA-seq without distinguishing between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). 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 and ICM cells were collected from blastocysts by using a micromanipulator and then subjected to RNA-seq. Gene expression analysis and digital karyotyping using RNA-seq were performed simultaneously for TE and ICM cells, respectively. One-way analysis of variance, chi-square test and Tukey's multiple comparison test were used for this study. Main results and the role of chance Blastocysts were classified into three groups to correlate with pregnancy rates based on the diameter of the blastocyst and the time to reach this size: those taking less than 130 h to reach a diameter of > 170 μm (Group 1, n = 676), those taking more than 140 h to reach a diameter of < 180 μm (Group 2, n = 158), and the rest (Group 3, n = 1,056). The pregnancy rates of Groups 1, 2 and 3 were 59.0%, 16.5%, and 34.2%, respectively (p < 0.01). Assessing the differences in overall transcripts correlated between Group 1 (n = 5), Group 2 (n = 4), and Group 3 (n = 4), 26 and 67 differentially expressed genes (DEGs) were identified in ICM and TE cells, respectively. Importantly, downregulated genes in TE of blastocysts with lower expectation of pregnancy included tight junction-related genes, such as CXADR, CLDN10, and ATP1B1, which were implicated in peri-implantation development. Digital karyotyping revealed karyotypic abnormalities and mosaicism in all groups with no common abnormalities observed, suggesting that aneuploidy alone cannot predict the pregnancy expectation. Limitations, reasons for caution Although 93 genes potentially related to implantation have been identified, it is still unclear how these genes are involved in implantation. In vitro implantation models using human embryos and artificial embryos currently under development are expected to contribute to the elucidation of the functions of these genes. Wider implications of the findings Our results provide reliable candidates for genes that could allow for non-invasive selection of high-quality blastocysts for ART and add to the knowledge base of transcriptional events in human peri-implantation development. Trial registration number not applicable

P-420 Dysregulated microRNAs in uterine fluid from women with recurrent implantation failure are linked to endometrial receptivity and implantation

Abstract Study question Is the microRNA content different in uterine fluid (UF) from women with recurrent implantation failure (RIF) compared to healthy fertile women? Summary answer The miRNA content is altered in the UF of women with RIF compared to healthy fertile women. What is known already Previous studies indicate that microRNAs secreted from endometrial cells into the UF are involved in endometrial receptivity and embryo implantation. Moreover, endometrial miRNAs are dysregulated in women with RIF and poor endometrial receptivity has been suggested as a putative cause of the condition. Study design, size, duration This is a descriptive experimental case-control study where microRNA abundancy in UF was compared between women with RIF (n = 34) and healthy fertile women (n = 17). Study participants were recruited at two university clinics in Stockholm, Sweden, and Tartu, Estonia. UF samples were collected vaginally in the receptive phase on day LH + 7-9 by flushing the uterine cavity with sterile saline. RIF was defined as three failed in vitro fertilization (IVF) cycles with good-quality embryos. Participants/materials, setting, methods To identify miRNAs in UF we performed small RNA sequencing. Differential expression analysis (DESeq2) was used to compare the abundancy of miRNAs in UF between the two groups. Dysregulated miRNAs were externally validated using relevant published datasets and further analyzed using tools such as target gene prediction (miRTarBase) and biological KEGG pathway analysis (g:Profiler). Technical validation was performed on two miRNAs with quantitative real-time PCR (RT-PCR). Main results and the role of chance In total, we identified 61 differentially abundant UF microRNAs with a false discovery rate of < 0.05 and fold change (FC) of -2 or 2. Out of these, 34 were up-regulated and 27 were down-regulated. External validation based on published literature showed that several of the dysregulated miRNAs are expressed in endometrial epithelial cells and have been described in extracellular vesicles from the endometrium and in the context of endometrial receptivity and RIF. Using publicly available single-cell transcriptome data, we found that the predicted target genes of our microRNAs are expressed both in the trophectoderm of human blastocysts and in epithelial cells of mid-secretory endometrium. Furthermore, the predicted miRNA target genes were significantly enriched in 25 pathways critical for endometrial receptivity and embryo implantation. Technical validation of hsa-miR-486-5p (FC -20.32; p-value=0.004) and hsa-miR-92b-3p (FC -9.72; p-value=0.004) were successfully in line with the sequencing data. Limitations, reasons for caution The sample size of this descriptive study was limited. A larger study cohort should be used to validate the differentially abundant microRNAs. Moreover, further in-vitro and in-vivo studies are needed to establish the role of identified miRNAs and their predicted target genes and enriched pathways in the pathogenesis of RIF. Wider implications of the findings RIF represents a true challenge in the IVF clinic. We show that total miRNAs can be comprehensively mapped in UF and constitute a promising source of non-invasive biomarkers for RIF that could be further evaluated for its clinical utility. Our findings also give insight into the molecular mechanisms of RIF. Trial registration number Not applicable

P-518 Do mosaic embryos still stand a chance for pregnancy?

Abstract Study question To investigate the effect of embryo mosaicism on clinical IVF outcomes in single and double frozen embryo transfer (FET). Summary answer There is no statistical difference between the low/high mosaic and euploid embryos in regards to the pregnancy and miscarriage rate, in single and double FETs. What is known already During mitosis and meiosis, mis-segregation of chromosomes causes aneuploidy. Recent studies have shown that next-generation sequencing (NGS) based pre-implantation genetic test for aneuploidy (PGT-A) improve clinical IVF outcomes by detecting chromosomal abnormalities in human embryos. Euploid embryos have been confirmed to achieve a higher pregnancy, implantation and live birth rate than aneuploid embryos. However, mosaic embryos, which contain two or more cell lines with different chromosomal set, are yet controversial in the assisted reproductive medical field. Hence, comprehensive studies on the clinical IVF outcomes of mosaic embryo transfers (METs) are necessary to resolve the concerns of embryo mosaicism. Study design, size, duration This single centre retrospective study included 93 low mosaic embryos, which were transferred in 64 FET cycles (35 single and 29 double FET), and 27 high mosaic embryos from 18 FET cycles (9 single and 9 double FET), from January 2018 to October 2021. The pregnancy and miscarriage rate of the METs were compared to an euploid control group, which comprised of 184 euploid embryos from 124 FET cycles (64 single and 60 double FET). Participants/materials, setting, methods All embryos were cultured to blastocyst stage and trophectoderm (TE) biopsy was performed on day 5 or 6. NGS based PGT-A was conducted to detect chromosomal abnormalities. According to the Preimplantation Genetic Diagnosis International Society guideline, mosaic embryos were categorized into 2 groups: low (20-39% mosaicism) and high mosaic embryos (40-80% mosaicism). Pearson chi-square test was performed to compare the pregnancy and miscarriage rate between low/high METs and euploid group, in single and double FETs. Main results and the role of chance In single FET cycles, patients with low mosaic embryos transferred achieved a higher pregnancy rate than the patients with euploid (60% versus 42%) and high mosaic (60% versus 33%) embryos transferred. In addition, the miscarriage rate for low mosaic embryos (9%) was the lowest, compared to the the euploid (11%) and high mosaic (22%) embryos. On the other hand, the highest pregnancy and lowest miscarriage rate in double FET cycles were achieved by patients with euploid embryos transferred. Results showed that patients with euploid embryos transferred possessed a pregnancy rate of 68%, whereas patients with low and high mosaic embryos transferred showed a pregnancy rate of 48% and 56% respectively. As mentioned above, the euploid embryos resulted in a lower miscarriage rate than the low (3% versus 10%) and high mosaic (3% versus 11%) embryos in double FET cycles. However, it is important to highlight that the above mentioned differences between the low/high METs and euploid control group in respect of the pregnancy and miscarriage rate were not statistically significant, in both single and double FET cycles (p < 0.05). Limitations, reasons for caution The factors of patient’s age and blastocyst grading were not taken into account in this study. Hence, a larger-scale randomized trial is a need to further identify and resolve the concerns of embryo mosaicism in the assisted reproductive medical field . Wider implications of the findings In the PGT-A analysis, only a fraction of the TE within an embryo was tested. Thus, misclassification of embryos might occur. It is suggested that clinical management of mosaic embryos should be carefully reviewed in the case of patients with no euploid embryos to be transferred. Trial registration number NA

P-520 Factors associated with embryonic mosaicism: a systematic review and meta-analysis

Abstract Study question What factors are involved in chromosomal mosaicism in human blastocysts? Summary answer Progenitors’ age and embryo biopsy day are associated with mosaicism rate. What is known already Chromosomal abnormalities are common in embryos analyzed in preimplantation genetic testing for aneuploidy (PGT-A) cycles. Mosaicism is a usual event in embryos derived from IVF cycles. With the development of Next-Generation Sequencing (NGS) techniques, the ability to detect mosaicism in embryos has been improved. Several studies show that mosaic embryos have reduced potential to reach term compared to euploid. Furthermore, the existing scientific evidence regarding the cause of embryonic mosaicism is scarce with conclusions generating considerable controversy. Study design, size, duration A systematic review and meta-analysis were conducted following the PRISMA guidelines on Medline and Google Scholar (January 2016 to December 2021). Studies addressing factors associated with embryo mosaicism were comprehensively analyzed. As inclusion criteria: mosaicism should have been detected from a blastocyst trophectoderm biopsy on day 5,6 o 7 and analyzed by NGS. Embryo quality, maternal and paternal age, day of biopsy and seminal quality were the outcomes variables included for analysis. Participants/materials, setting, methods A comprehensive database search resulted in 195 articles, 18 of which were included for abstract reading. Search, screen, and data extraction were performed by two reviewers independently based on inclusion criteria. The study by meta-analysis of the effect of the different factors associated with embryonic mosaicism was carried out using the inverse variance method. Depending on the existence of heterogeneity between studies, the fixed effects method or the random effects method were used. Main results and the role of chance After critically and thoroughly reading the selected papers, 10 of them were included in the meta-analysis. Data from our reproductive clinic were also enclosed in the analysis (2513 cycles, 7242 embryos). The results of the meta-analysis show that neither embryo quality nor seminal quality (male factor) were related to mosaic embryo rate (OR: 1.15; 95%CI: 0.98-1.35 and OR: 1.01; 95%CI: 0.80-1.27, respectively). In contrast, a positive association with embryo mosaicism was observed for the variable “biopsy day” (OR: 1.06; 95% CI: 1.01-1.11): in embryos biopsied on day 6 or 7 of embryonic development, a small increase in the mosaicism rate was observed, in comparison with day 5. A significative positive association was also observed when we studied “paternal age” factor (OR: 1.13; 95% CI: 1.02-1.26): embryo mosaicism increases with paternal age. On the other hand, maternal age showed a negative association with mosaicism (OR: 0.84; 95% CI: 0.74-0.95). Interestingly, in opposite to what happens with the aneuploidy rate, embryo mosaicism is higher in young women. Limitations, reasons for caution The main limitation is the low number of papers analyzed in the meta-analysis. Limitations also include the retrospective design and heterogeneity of studies, limiting comparison and pooling of data. Nonetheless, our conclusions were based on studies with low risk of bias. Wider implications of the findings Our result, combined with the body of medical evidence available suggest that embryo mosaicism rate is influenced by trophectoderm biopsy day and maternal and paternal age. This information will add in the knowledge for elucidating the uncertainties surrounding the factors by which mosaicism is generated in embryos. Trial registration number Not applicable

Comparison of chromosomal status in reserved multiple displacement amplification products of embryos that resulted in miscarriages or live births: a blinded, nonselection case\u2013control study

ObjectiveTo analyze chromosomal status in reserved multiple displacement amplification (MDA) products of embryos that result in miscarriages or live births.MethodsPatients who underwent preimplantation genetic testing for monogenic disorders (PGT-Ms) without aneuploidy screening were included. The case group included 28 cycles that resulted in miscarriages. Controls included 56 cycles with live births. Comprehensive chromosomal screening (CCS) using next-generation sequencing (NGS) was performed on reserved MDA products from previous blastocyst trophectoderm biopsies. The incidence and type of chromosomal abnormalities in embryos resulting in miscarriages or live births were analyzed.ResultsOf 28 embryos resulting in miscarriages in the case group, the rate of chromosomal abnormalities was 53.6%, which was significantly greater than 14.3% for those resulting in live births in control group (P < 0.001). Whole-chromosome aneuploidy was not found in the control group but was noted in 25.0% of embryos in the case group. Although the rates of segmental abnormality and mosaicism were also greater in the case group, no significant differences were detected. One chaotic embryo in the control group progressed to live birth.ConclusionChromosomal abnormalities were the main reason leading to early pregnancy loss. However, abnormalities, such as segmental aneuploidy and mosaicism, should be managed cautiously, considering their undermined reproductive potential.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTIONHow does the human embryo breach the endometrial epithelium at implantation?SUMMARY ANSWEREmbryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer.WHAT IS KNOWN ALREADYA significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown.STUDY DESIGN, SIZE, DURATIONFresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm–epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation.MAIN RESULTS AND THE ROLE OF CHANCEThe majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGβ secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion.LARGE SCALE DATANo omics datasets were generated in this study, and those used from previously published studies are cited.LIMITATIONS, REASONS FOR CAUTION In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models.WIDER IMPLICATIONS OF THE FINDINGSUnderstanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Blastocyst trophectoderm endocytic activation, a marker of adverse developmental programming.

The mouse preimplantation embryo is sensitive to its environment, including maternal dietary protein restriction, which can alter the developmental programme and affect lifetime health. Previously, we have shown maternal low-protein diet (LPD) causes a reduction in blastocyst mTORC1 signalling coinciding with reduced availability of branched-chain amino acids (BCAAs) in surrounding uterine fluid. BCAA deficiency leads to increased endocytosis and lysosome biogenesis in blastocyst trophectoderm (TE), a response to promote compensatory histotrophic nutrition. Here, we first investigated the induction mechanism by individual variation in BCAA deficiency in an in vitro quantitative model of TE responsiveness. We found isoleucine (ILE) deficiency as the most effective activator of TE endocytosis and lysosome biogenesis, with less potent roles for other BCAAs and insulin; cell volume was also influential. TE response to low ILE included upregulation of vesicles comprising megalin receptor and cathepsin-B, and the response was activated from blastocyst formation. Secondly, we identified the transcription factor TFEB as mediating the histotrophic response by translocation from cytoplasm to nucleus during ILE deficiency and in response to mTORC1 inhibition. Lastly, we investigated whether a similar mechanism responsive to maternal nutritional status was found in human blastocysts. Blastocysts from women with high body-mass index, but not the method of fertilisation, revealed stimulated lysosome biogenesis and TFEB nuclear migration. We propose TE lysosomal phenotype as an early biomarker of environmental nutrient stress that may associate with long-term health outcomes.

Mitochondrial maturation in the trophectoderm and inner cell mass regions of bovine blastocysts

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.

MOSAICISM AND ANEUPLOIDY LEAD TO GRADUAL LOSS OF BIOCHEMICAL IDENTITY AND INDUCE APOPTOSIS IN HUMAN BLASTOCYSTS

To investigate the transcriptomic consequences of mosaicism and aneuploidy in the inner cell mass (ICM) and the trophectoderm (TE) of human blastocysts. Prospective study comparing the transcriptomic identities of the ICM and the TE of blastocysts classified as euploid (n=5), mosaic (n=8) and aneuploid (n=6) by preimplantation genetic testing for aneuploidy. Mosaicism was defined in the range 30%-<70%. For RNA-seq, differentially expressed genes were calculated with DESeq2 package [Benjamini-Hochberg (BH)-adjusted p<0.01 & abs(log2FoldChange)>2 significant]. Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology (GO) terms (BH-adjusted p<0.01 significant). ICM and TE of euploid blastocysts displayed expected lineage-specific transcriptomic differences. Cell cycle and 9 GO processes related to embryogenesis, including organogenesis and apoptosis involved in anatomical structure development, were significantly upregulated in the ICM. In contrast, telomere lengthening and 67 GO processes involved in energy production and biosynthesis, metabolism and transport of nucleic acids, carbohydrates, proteins and steroids were significantly upregulated in the TE. In mosaic and aneuploid blastocysts, several developmental and metabolic processes were also upregulated in the ICM and the TE, respectively. However, aneuploidy led to a stepwise impairment of cellular functions in both lineages. In the ICM, mosaic and aneuploid blastocysts significantly downregulated cell growth and upregulated inflammatory and extrinsic apoptotic pathways. This anti-survival effect was exacerbated in aneuploid blastocysts, which upregulated other stress response pathways in the ICM, including cell death due to oxidative stress and intrinsic apoptosis. In the TE, the number of significantly-upregulated metabolic processes decreased in mosaic and aneuploid blastocysts (48 and 5 GO processes, respectively), suggesting a gradual loss of biochemical features in response to aneuploidy. Transcriptomic identities of ICM and TE reflect their different roles in normal embryo development. While the ICM proliferates under a relatively quiescent metabolism, the TE serves as an active provider of energy and metabolites. Aneuploidy gradually disrupts TE biochemical functions and activates specific cell death programs in the ICM. This is consistent with the known dual role of apoptosis as an effector of both physiological and stress-driven pathological programs during embryogenesis.

Excessive endoplasmic reticulum stress drives aberrant mouse trophoblast differentiation and placental development leading to pregnancy loss.

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1tm1RjK ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number and developmental delay and also is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1+/tm1RjK heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions. Women with pre-gestational health conditions (e.g. obesity, diabetes) or gynaecological problems (e.g. endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, pre-eclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPRER ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1tm1RjK mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPRER pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPRER pathway inhibitors rescued this effect. Although the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1tm1RjK blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1tm1RjK homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1tm1RjK homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1+/tm1RjK heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions.

P–523 Whole-chromosome aneuploidies revealed by transcriptome of trophectoderm biopsied from human pre-implantation blastocyst

Abstract Study question Whether mRNA transcriptome of biopsied trophectoderm (TE) in human pre-implantation blastocyst can predict embryo karyotype? Summary answer mRNA transcriptome of biopsied TE can precisely predict whole-chromosome aneuploidies but not mosaicism or segmental aneuploidies. What is known already Karyotype of human pre-implantation blastocyst is well recognized by PGT-A. However, genome can’t demonstrate gene expression level which might infer the development potential of euploidy. Transcriptome of blastocyst by singe-cell RNA-seq has revealed the lineage segregation of human pre-implantation blastocyst. It is not known whether transcriptome of biopsied TE used in PGT-A can infer the karyotype of human pre-implantation blastocyst. Study design, size, duration A total of 74 TE samples were biopsied from 26 blastocysts which were donated from patients who underwent PGT at our Reproductive Medicine Center. All of these embryos have been previously diagnosed as aneuploidies (n = 19) or euploidies (n = 7) with monogenic disorder. Participants/materials, setting, methods The DNA and mRNA of all biopsied TEs were separated independently using a modified oligo-dT bead capture, followed by PGT-A of DNA and smart2-sequencing of mRNA (G&amp;T-seq). Karyotype of biopsied TEs were confirmed with PGT-A performed in MiSeq system (Illumina) in our PGT laboratory with the use of next-generation sequencing. Data of transcriptome was analyzed using Rstudio and R package InferCNV to predict aneuploidies by referring to euploidies which were inferred with corresponding PGT-A results. Main results and the role of chance In human pre-implantation blastocyst, all whole-chromosome aneuploidies could be inferred by transcriptome of biopsied TE, which were consistent with PGT-A result. But chromosomal mosaicism or segmental aneuploidies were hard to be predicted precisely by transcriptome of TE. Limitations, reasons for caution The main limitation of this study lies in the inability to retrieve the exact copy number variations from mRNA transcription. Gene expression is in a great imbalance in such an early development of human pre-implantation blastocyst. Wider implications of the findings: Our data suggest that mRNA transcriptome is enough for prediction of whole-chromosome aneuploidies. The method and value for predicting mosaicism and segmental aneuploidies by transcriptome should be further investigated. Trial registration number Not applicable

P-523 Whole-chromosome aneuploidies revealed by transcriptome of trophectoderm biopsied from human pre-implantation blastocyst

Abstract Study question Whether mRNA transcriptome of biopsied trophectoderm (TE) in human pre-implantation blastocyst can predict embryo karyotype? Summary answer mRNA transcriptome of biopsied TE can precisely predict whole-chromosome aneuploidies but not mosaicism or segmental aneuploidies. What is known already Karyotype of human pre-implantation blastocyst is well recognized by PGT-A. However, genome can’t demonstrate gene expression level which might infer the development potential of euploidy. Transcriptome of blastocyst by singe-cell RNA-seq has revealed the lineage segregation of human pre-implantation blastocyst. It is not known whether transcriptome of biopsied TE used in PGT-A can infer the karyotype of human pre-implantation blastocyst. Study design, size, duration A total of 74 TE samples were biopsied from 26 blastocysts which were donated from patients who underwent PGT at our Reproductive Medicine Center. All of these embryos have been previously diagnosed as aneuploidies (n = 19) or euploidies (n = 7) with monogenic disorder. Participants/materials, setting, methods The DNA and mRNA of all biopsied TEs were separated independently using a modified oligo-dT bead capture, followed by PGT-A of DNA and smart2-sequencing of mRNA (G&amp;T-seq). Karyotype of biopsied TEs were confirmed with PGT-A performed in MiSeq system (Illumina) in our PGT laboratory with the use of next-generation sequencing. Data of transcriptome was analyzed using Rstudio and R package InferCNV to predict aneuploidies by referring to euploidies which were inferred with corresponding PGT-A results. Main results and the role of chance In human pre-implantation blastocyst, all whole-chromosome aneuploidies could be inferred by transcriptome of biopsied TE, which were consistent with PGT-A result. But chromosomal mosaicism or segmental aneuploidies were hard to be predicted precisely by transcriptome of TE. Limitations, reasons for caution The main limitation of this study lies in the inability to retrieve the exact copy number variations from mRNA transcription. Gene expression is in a great imbalance in such an early development of human pre-implantation blastocyst. Wider implications of the findings Our data suggest that mRNA transcriptome is enough for prediction of whole-chromosome aneuploidies. The method and value for predicting mosaicism and segmental aneuploidies by transcriptome should be further investigated. Trial registration number not applicable

P–758 The efficacy, safety and proven security of microSecure vitrification offers “peace of mind” and reliability during a global pandemic

Study questionUnder deadly pandemic conditions involving the novel SARS-CoV–2 corona virus, could biopsied blastocysts be safely cryopreserved, stored and utilized for subsequent warming cycles?Summary answerBlastocysts were securely stored, effectively warmed and safely transferred to yield normal pregnancy outcomes under uncertain laboratory conditions subject to unprecedented policy changes.What is known alreadyBy April 2020, every IVF lab worldwide was implementing deep cleaning/disinfecting procedures in their laboratory and patient-contact areas, thorough hand-washing policies and mandatory PPE to reduce the chance of contact transmission and spread of the potentially deadly SARS-CoV–2 coronavirus. Furthermore, we know that safeguards like oil overlay culture dishes and pipetting dilution factors provide insurance against possible contamination. However, knowing that the trophectoderm of blastocysts possessed the ACE–2 binding receptor, potential concern existed regarding the continuation of laser zona opening and biopsy procedures that could possibly expose cryopreserved embryos to the coronavirus in liquid nitrogen storage (vapor or liquid).Study design, size, durationBetween March 8 and December 22, 2020, 508 patients performed FET cycles involving the use of single (n = 490) or dual (n = 18) euploid microSecure vitrified blastocysts. In this retrospective analysis, we compared clinical pregnancy outcomes to a 5 year dataset (2015–2019) encompassing 2768 single and 272 dual embryo transfer FET cycles. All blastocysts were vitrified using a closed microSecure system and Innovative Cryoenterprise (ICE; NJ, USA) non-DMSO, glycerol-EG solutions. Differences were assessed by Chi-square analysis (p < 0.05).Participants/materials, setting, methodsDeep cleaning was performed with Simple Green Pro3+ Virucide in non-lab areas (e.g., ET rooms, waiting room) and 6% H2O2 & OoSafe solutions to disinfect lab surfaces and equipment. Group embryo cultures were performed in MCO–5M humidified incubators under low oxygen tri-gas conditions with varying CO2 levels (5.3–6.0%; pH = 7.3–7.35) using 25µl droplets of LifeGlobal medium+7.5%LGPS+1%sodium hyaluronate, before changing to 10µl droplet/GPS dishes post-biopsy. FET cycles involved 4-step sucrose dilutions and transvaginal ultrasound-guided embryo transfers.Main results and the role of chanceWhile ICSI fertilization rates were unchanged in 2020 (79.4% 2PN vs 77.3%), blastocyst utilization rates tended to be slightly lower than past years (56.4% vs 59.9%) but within an acceptable range. Of 529 blastocysts warmed, 527 (99.7%) survived completely for transfer, being comparable to the 99.4% experienced over 5 years. Furthermore, there was no differences detected in single embryo transfer pregnancy outcomes. The implantation and ongoing clinical pregnancy/live birth rates were 69% and 66.53% compared to 70.4% and 65.1%, respectively. Under pandemic conditions we did not observe an increase in biochemical pregnancies (10.3%) nor spontaneous miscarriage rates (7.8%). Although it is possible that our rigorous disinfection practices could have attributed to lower blastocyst production, the viability of those embryos was not compromised. Importantly, we were able to feel comfortable performing micromanipulation and cryopreservation procedures throughout the year knowing that we were effectively eliminating possible vertical transmission of coronavirus to an exposed trophectoderm layer in cryostorage by applying mircoSecure vitrification. Post-FET clinical check-ups revealed no patient reporting any fever or other Covid–19 symptoms in the weeks following their transfers. We are fortunate to say that our Lab staff, physicians and patients have remained healthy throughout 2020.Limitations, reasons for cautionBlastocyst survival and viability are independent of possible viral exposure. Previously, the risk of disease transmission via liquid nitrogen or vapor exposure was considered highly unlikely (Pomeroy et al., 2010), but that was at a time when embryos were primarily zona-enclosed. Today’s ART standards have us re-evaluating safer approaches.Wider implications of the findings: We have effectively mitigated avoiding performing zona opening procedures by employing our standard practice of aseptic, closed vitrification. In combination with standard preventative measures (PPE, hand hygiene, distance awareness) and routine deep cleaning practices, we sustained a contamination-free environment and healthy patients, capable of sustaining high levels of pregnancy success.Trial registration numberNot applicable

P–248 Statistical estimation for incidence of blastocyst trophectoderm vesicles (TVs) and efficacy of assisted hatching (AH)

Abstract Study question The aim of this study is to analyse the association between blastocyst diameter and TVs development, and to examine the efficacy of AH. Summary answer Blastocysts with a diameter of more than 170 μm leads to high incidence of TVs and AH applied from the incidence should be effective. What is known already TVs are protrusion of trophectoderm cells often observed in expanding blastocyst stages. TVs can be observed in expanding blastocysts regardless of Intracytoplasmic sperm injection (ICSI) and Conventional-IVF (C-IVF), when the internal pressure of blastocysts increase. The rate of TVs incidence in blastocysts inseminated by ICSI is higher than that by C-IVF, due to penetration of the needle into the zona pellucida. Moreover, it has been reported that TVs may inhibit blastocyst hatching. However, the developmental timing of TVs is still unclear, and there is no study that has analysed the association between blastocyst diameter and the incidence of TVs. Study design, size, duration 1) Diameters and TVs incidence of blastocysts by ICSI and C-IVF were measured, and the cut-off value and the area under the curve (AUC) of the receiver operating characteristic (ROC) curve were calculated to estimate the timing of TV incidence. 2) We analysed the clinical pregnancy rates of blastocysts with TVs treated by AH compared to those of blastocysts by C-IVF not subjected to AH. Participants/materials, setting, methods This study included 821 transferred frozen blastocysts ranging from March 2018 to November 2019. The embryos were cultured in a dry incubator after insemination by ICSI or C-IVF. Blastocyst freezing conditions were set at day5 to day7 with a diameter of more than 150 μm in inner diameter of zona pellucida, and this was measured before freezing. The ROC curve was performed using EZR statistical analysis software. Main results and the role of chance 1) The incidence of TVs in blastocysts by ICSI and C-IVF was 27.5% (117/424) and 14.6% (58/397) respectively. The rate of the incidence of TVs in blastocysts inseminated by ICSI and C-IVF; 8.6% (12/140) and 0.95% (1/105) in 150–159 μm, 12.7% (14/110) and 8.2% (6/73) in 160–169 μm, 40.6% (28/69) and 10.5% (6/57) in 170–179 μm, 55.6% (30/54) and 25.5% (13/51) in 180–189 μm, 66.7% (20/30) and 35.7% (10/28) in 190–199 μm, and 68.4% (13/19) and 26.8% (22/82) in the diameter of more than 200 μm. The cut-off value of the ROC curve was respectively 170 μm (sensitivity 78.6% and specificity 73.0%) and 176 μm (sensitivity 84.5% and specificity 59.6%) in the diameter; the AUC was 0.8 [95%CI:0.752–0.848] and 0.74 [95%CI:0.687–0.793] respectively. 2) The clinical pregnancy rate of TVs blastocyst vs C-IVF blastocyst was 52.7% (88/167) vs 57.8% (37/64) respectively. There is no significant difference between the two clinical pregnancy rates (P = 0.556). Limitations, reasons for caution The findings of this study have to be seen in light of some limitations. Since this study aimed to analyse the incidence of TVs based on blastocyst size, we did not take into account the grade according to the Gardner classification and the number of trophectoderm cells. Wider implications of the findings: Blastocysts inseminated by ICSI and C-IVF were highly likely to have TVs above 170 μm and 176 μm respectively. The clinical pregnancy rates of the blastocyst with TV treated by AH was similar to those of the C-IVF blastocyst. Trial registration number Not applicable

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells.

PurposeThis study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells.MethodsTE 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.ResultsDiscordance 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.ConclusionChromosomal 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.

Cell-Cell Communication at the Embryo Implantation Site of Mouse Uterus Revealed by Single-Cell Analysis.

As a crucial step for human reproduction, embryo implantation is a low-efficiency process. Despite rapid advances in recent years, the molecular mechanism underlying embryo implantation remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of embryo implantation sites. By analyzing inter-implantation sites of the uterus as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Additionally, we predicted signaling interactions between uterine luminal epithelial cells and mural trophectoderm of blastocysts, which represent the key mechanism of embryo implantation. We also predicted signaling interactions between uterine epithelial-stromal crosstalk at implantation sites, which are crucial for post-implantation development. Our data provide a valuable resource for deciphering the molecular mechanism underlying embryo implantation.

Next‐generation sequence‐based preimplantation genetic testing for monogenic disease resulting from maternal mosaicism

ABSTRACTBackgroundMosaicism poses challenges for genetic counseling and preimplantation genetic testing for monogenic disorders (PGT‐M). NGS‐based PGT‐M has been extensively used to prevent the transmission of monogenic defects, but it has not been evaluated in the application of PGT‐M resulting from mosaicism.MethodsFour women suspected of mosaicism were confirmed by ultra‐deep sequencing. Blastocyst trophectoderm cells and polar bodies were collected for whole genome amplification, followed by pathogenic variants detection and haplotype analysis based on NGS. The embryos free of the monogenic disorders were transplantable.ResultsUltra‐deep sequencing confirmed that the four women harbored somatic mosaic variants, with the proportion of variant cells at 1.12%, 9.0%, 27.60%, and 91.03%, respectively. A total of 25 blastocysts were biopsied and detected during four PGT cycles and 5 polar bodies were involved in one cycle additionally. For each couple, a wild‐type embryo was successfully transplanted and confirmed by prenatal diagnosis, resulting in the birth of four healthy infants.ConclusionsMosaic variants could be effectively evaluated via ultra‐deep sequencing, and could be prevented the transmission by PGT. Our work suggested that an NGS‐based PGT approach, involving pathogenic variants detection combined with haplotype analysis, is crucial for accurate PGT‐M with mosaicism.

Human naive epiblast cells possess unrestricted lineage potential.

SummaryClassic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.

First live birth in Greece after blastocyst trophectoderm biopsy and preimplantation genetic testing for hereditary angioedema

Preimplantation genetic testing for monogenic/single-gene defects (PGT-M) is a well established tool in assisted reproduction. A couple, where the male was carrier of the c.550G&gt;A (p.Gly184Arg) mutation of the SERPING 1 gene causing hereditary angioedema (HAE) attended our clinic for PGT-M. Employing a strategy of preserving embryos after blastocyst trophectoderm biopsy by vitrification cryopreservation, we managed to screen 10 embryos collected from a single IVF cycle. Three embryos were found unaffected after preimplantation genetic analysis for the paternal mutation. The transfer of one normal blastocyst post-thaw resulted in a healthy and uneventful pregnancy and in the live birth of a male neonate on the 38th week of pregnancy. This is the first report of a live birth in Greece after blastocyst trophectoderm biopsy and preimplantation genetic analysis for hereditary angioedema.

Chromosomal analysis for embryos from balanced chromosomal rearrangement carriers using next generation sequencing.

We aimed to use next generation sequencing (NGS) to investigate chromosomal abnormalities in blastocyst trophectoderm (TE) samples, and reproductive outcomes with the different types of chromosomal rearrangements (CR) and for each sex of CR carrier. A total of 1189 blastocyst TE samples were evaluated using NGS to detect chromosomal unbalanced translocations as well as aneuploidy, including blastocytes from 637 blastocysts from carriers of balanced CRand 552 blastocysts from carriers of normal chromosomes. The optimal embryos had lower chromosomal abnormality rates compared to the poor-quality embryos. The experimental group had significantly reduced rates of normal embryos and euploidy, and higher rates of total abnormalities, aneuploidy and unbalanced chromosomal aberrations. Carriers of reciprocal translocationshad a reduced rate of normal embryos and an increased percentage of embryos with total abnormalities and unbalanced chromosomal aberrations compared with carriers of Robertsonian translocations. Couples with female carriers of chromosomal abnormalities had significantly reduced rates of normal embryos and euploidy, and a higher percentage of embryos with total abnormalities, aneuploidy, and unbalanced chromosomal aberrations compared with couples of male carriers. Our preimplantation genetic testing (PGT) study identified higher rates of chromosomal abnormalities, including chromosomal unbalanced translocations and aneuploidy, in blastocysts from CR carriers, especially from the female carriers, in a Chinese population. The PGT cycles successfully improved clinical outcomes by increasing the fertilization rate and reducing the early spontaneous abortion rate compared with the in vitro fertilization and intracytoplasmic sperm injection cycles, especially for CR carriers.