Abstract: Ethical reasons restrict our ability to study human embryo implantation in vitro. For this vital reason, our lab established an endometrial assembloid model which can be used in co-culture studies with human blastocysts. For our in vitro implantation assay, endometrial assembloids (n=12 biopsies) were hormonally stimulated with 8-bromo-cAMP, estradiol, and a progestin to induce decidualisation. Human 5 days post fertilisation (dpf), hatched blastocysts (n=78) were positioned on the surface epithelium of assembloids and cultured at 37°C, 6% CO2 and 5% O2. The average percentage of successful embryo attachment was 70%, ranging from 83.3 to 58.3%. We observed that early invasion of human blastocysts coincides with migration of surface epithelial cells away from the attachment site. We further showed that the described co-culture conditions promoted trophectoderm differentiation while the development of ICM lineages was suboptimal at 8 dpf. We set out to optimise culture conditions with emphasis on growth factor supplementation. Human blastocysts at 5 dpf (n=17) were co-cultured with decidualised assembloids (n=4 biopsies) at 5% O2 until 7 dpf and 20% O2 until 8 dpf. The basal co-culture medium was supplemented with IGF1 (50ng/μl) and/or ActivinA (100ng/μl). Attachment of embryos was assessed upon overnight co-culture by liquid disturbance. Embryo development and presence of embryonic lineages was confirmed by immunofluorescence antibody labelling for NANOG (epiblast), GATA4 (primitive endoderm) and GATA3 (trophectoderm), and 3D fluorescent visualization was performed using confocal microscopy. IGF1 supplementation resulted to a 3-fold increase in total ICM cell numbers. Supplementation of both IGF1 and ActivinA resulted to a 6-fold and 2-fold increase in epiblast and primitive endoderm average cell numbers, respectively. Moreover, post-implantation embryos were larger in diameter, from average 379μm to 578μm. Our findings signify that mTOR (IGF1) and TGFβ (ActivinA) signalling pathways ameliorate in vitro embryonic development at post-implantation stages. The ability to establish co-cultures using endometrial models and blastocysts is poised to lead to major advances in our understanding of mechanisms of reproductive success and failure, nonetheless, a plethora of technical hurdles remain.
Funding Source: - EUTOPIA funding between the University of Warwick and VUB for PhD project - Wetenschappelijk Fonds Willy Gepts (WFWG) funding by UZ Brussel for work at VUB
