MODELLING EMBRYO IMPLANTATION IN VITRO USING 3D ENDOMETRIAL ASSEMBLOIDS AND HUMAN BLASTOCYSTS

Human pregnancy involves implantation of chromosomally diverse embryos in a uterine mucosa – the endometrium – subjected to cyclical breakdown and regeneration. Upon implantation of high-fitness embryos, the endometrium transforms into the decidua of pregnancy, which anchors and supports the placenta throughout gestation. Perturbations in these processes are believed to underpin prevalent reproductive disorders while, the mechanisms that account for persistent reproductive failure remain poorly understood. There is a vital need for a model
involving the endometrium and human embryos since ethical reasons limit our ability to study human embryo implantation in vitro. For this, endometrial assembloids were established which are physiologically similar to endometrial tissue in morphology and function. Assembloids consist of epithelial compartments including gland-like organoids and a luminal epithelium which is the embryo attachment side, and a stromal compartment rich in fibroblasts, immune and endothelial cells. Hormonal stimulation results in the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes. Aiming to mimic implantation, assembloids underwent a 3-day long differentiation in a chemically defined medium, 8-br-cAMP, estradiol, and a progestin. Subsequently, assembloids were overlaid with single epithelial cells from the matching patient. Following a 24-hour long incubation period, human hatched day 6 blastocysts were positioned on the surface epithelium. Co-cultures were fixed on day 8 and immunofluorescent antibody labelling using markers for the epiblast (OCT4), primitive endoderm (GATA4) and trophectoderm (GATA3) were used to visualise the co-culture. Secretion of βhCG was detected on day 9 while by day 10, βhCG detection was enhanced and Cytokeratin7 was also detected around the embryo marking the syncytiotrophoblast and cytotrophoblast populations,
respectively. The proposed system is an innovative method to recapitulate apposition, adhesion, and invasion during implantation. While many technical hurdles remain, the ability to establish patient-specific endometrial models is poised to lead to advances in our understanding of mechanisms of reproductive failure and the development of personalised therapeutic interventions.