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

Abstract

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