O-094 Novel stem cell-based twin embryo model reveals inner cell mass division and enhanced endometrial adhesion in monochorionic twin development

Abstract Study question How do monochorionic twin embryos arise and develop during the pre- and peri-implantation stage? Summary answer Twinning involves inner cell mass division during blastocyst expansion and heightened endometrial adhesion, with twin blastoids showing greater implantation potential. What is known already The occurrence of monozygotic twins is between 0.4 and 0.5% of livebirths globally from natural conception, of which the majority are monochorionic. Monochorionic twins are associated with increased risk of complications and higher mortality rates during pregnancy due to twin-to-twin transfusion syndrome, twin anemia-polycythemia sequence, developmental malformations and increased incidence of pregnancy-related diseases in the mother. The rarity of monochorionic twinning, combined with ethical constraints in human embryo research, renders the study of twinning practically infeasible, which presents in a significant knowledge gap regarding the origins and implications of early developmental monochorionic twin embryos development. Study design, size, duration In a stem cell-based model of the blastocyst-stage embryo (i.e., blastoid) conditions were identified that favor the generation of monochorionic twin blastoids. To obtain sufficient statistical power, screening experiments were performed using 3 culture wells per condition, each well containing at least 90 blastoids. Experiments were repeated 3 times and results were pooled. Blastoids were cultured for at least 3 days followed by 2 days on endometrium monolayers on-chip. Participants/materials, setting, methods Human naïve induced pluripotent stem cells and embryonic stem cells were used to form twin blastoids within custom microwell array platforms. Patient-derived endometrium epithelial cells were expanded as organoids and seeded as monolayers in microfluidic chips. Blastoids were infused into the chip, allowed to adhere for 2 days, followed by a controlled stepwise increase of flow rate after which the blastoid adhesion rate to the endometrium layer for both singleton and twin blastoids was quantified. Main results and the role of chance Twin blastoids contained a cystic GATA3+ single layered trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses revealed that twinning occurs during the blastocyst cavitation phase via division of the OCT4+ pluripotent cell mass. After three days of culture, twin blastoids measured 228±32 µm, which is in the upper range of the cyst diameter of a blastocyst. In addition, both sibling ICM-like clusters preserved similar epiblast and hypoblast cell proportions and were consistently positioned nearly 150° (146.6±26°) apart along the blastocoel lining (measured as 2D-projections and in 3D reconstructions), which suggests a minimal angular distance for establishing two separate condensed ICM clusters. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, indicative of implantation readiness. Under defined flow regimes, twin blastoids show increased adhesion capacity to endometrium monolayers compared to singleton blastoids (60% vs 38% at highest flow rate), suggestive of increased implantation potential possibly due to a larger adhesive surface area. Twin blastoids cultured on endometrium monolayers could develop into structures with two distinctive OCT4+ pluripotent cell clumps both delineated by SOX17+ hypoblast-like cells and as a whole encompassed by GATA3+ trophoblast-like cells, suggestive of post-implantation organization. Limitations, reasons for caution While twin blastoids adhered to the endometrium monolayer and maintained two separate OCT4 compartments after 48 hours in post-implantation culture, both singleton and twin blastoids did not establish organized 3D structures associated with later post-implantation development, including amniotic and yolk sac-like cavities. Wider implications of the findings Twin blastoids open new avenues for studying the various aspects of twinning, ranging from their formation processes to the effects of multiple ICMs on trophectoderm maturation and implantation potential. In the future, with more sophisticated endometrium platforms, this model may inform on how clinical complications arise in twin pregnancies. Trial registration number not applicable