Human stem cell derived embryo surrogates for human implantation study

Abstract

Implantation failure is one of the major causes leading to failed pregnancy and low success rates of assisted reproductive technology treatments. However, there is no reliable tool for assessing endometrial receptivity. Interactions between human embryo and endometrium during implantation are poorly understood. Trophoblastic spheroids (BAP-EB) derived from human stem cells resembling human blastocyst polar trophectoderm (pTE) was established in our laboratory. It was hypothesized that BAP-EB can be used for predicting clinical pregnancy outcomes and studying molecular interactions at the embryo-maternal interface. The predictive value of BAP-EB attachment rate onto endometrial epithelial cells (EEC) on the cumulative live birth rate of an in vitro fertilization (IVF) cycle was evaluated by a prospective observation study. The results showed that BAP-EB attachment rates on EEC isolated from women aged 35 years who had a cumulative live birth within 6 months of ovarian stimulation were significantly higher than those without a live birth. No such difference was observed for women with younger ages. The data indicated that the attachment of BAP-EB onto EEC modestly predicted cumulative livebirth of women aged 35 or over. The surface molecules involved in the BAP-EB-EEC attachment process were identified. The conditioned medium of attachment competent BAP-EB induced the expressions of catenin delta 1 (CTNND1) and E-cadherin (CDH1) on EEC. The current study was the first to report CTNND1 which maintained the cell surface integrity and stabilized CDH1 expression on EEC played a role in the early implantation processes. CDH, but not CTNND1, was located at the apical surface of EEC. Knockdown of CTNND1 and CDH1 in EEC reduced cell adhesiveness and BAP-EB attachment. Human blastocyst secretory factors like human chorionic gonadotropin (HCG) induced the translocation of CDH1 from the adherent junction to the apical surface of EEC. Most critically, the expressions of CTNND1 and CDH1 in EEC were correlated with the live birth and repeated implantation failure. Mass spectrometry analysis identified CDH1 as the embryonic binding partner of endometrial CDH1. Intense apical CDH1 signal was located on receptive EEC and pTE-like BAP-EB. Homophilic binding of trophectodermal CDH1 with endometrial CDH1 was confirmed by atomic force microscope and antibody blocking assays. It was found that CDH1 homophilic binding was a major mediator of the attachment of BAP-EB onto EEC. The use of expanded potential stem cell derived from human embryos further confirmed the role of CDH1 during BAP-EB attachment. In addition, trophectodermal Neuropilin 1 was identified as another molecule potentially involved in the attachment process during embryo implantation. Three-dimensional implantation models were reconstructed to better mimic the in vivo conditions of embryo implantation. While ex vivo explant culture failed to maintain the structure and viability of endometrium, an UP-CELL 3D implantation model with columnar epithelium-like Ishikawa cells stacking on multi-layered stromal cells was established. Attachment and early invasion of BAP-EB onto the Ishikawa cells were observed. The setup might potentially be used for studying the mechanistic pathways leading to implantation failure. In summary, BAP-EB represented an embryo surrogate for predicting endometrial receptivity and studying embryo-endometrium communications during embryo implantation.