Co-Culture of Mouse Blastocysts on A Human Recellularized Endometrial Scaffold: An In Vitro Model for Future Implantation Studies.

Objective: This study evaluates the interaction of mouse blastocysts as a surrogate embryo on a recellularized endometrial scaffold by seeding human endometrial mesenchymal cells (hEMCs).

Materials and methods: In this experimental study, prepared decellularized human endometrial tissues were characterized by morphological staining, DNA content analysis, and scanning electron microscopic (SEM) analysis. The scaffolds were subsequently recellularized by hEMCs. After seven days of cultivation, the mouse blastocysts were co-cultured on the recellularized scaffolds for 48 hours. Embryo attachment and implantation within these scaffolds were evaluated at the morphological, ultrastructural, molecular, and hormonal levels.

Results: There was no morphological evidence of cells and nuclei in the decellularized scaffold. DNA content significantly decreased by 89.92% compared to the control group (P<0.05). Both decellularized and native tissues had similar patterns of collagen bundles and elastin fibers, and glycosaminoglycan (GAGs) distribution in the stroma. After recellularization, the hEMCs attached to the scaffold surface and penetrated different parts of these scaffolds. In the co-cultured group, the embryo attached to the surface of the scaffold after 24 hours and penetrated the recellularized endometrial tissue after 48 hours. We observed multi-layered organoid-like structures formed by hEMC proliferation. The relative expressions of epithelial-related genes, ZO-1 and COL4A1, and SSP1, MMP2, and PRL, as decidualizationrelated genes, were significantly higher in the recellularized group on day 9 in the presence of the embryo compared to the other groups (P<0.05). Beta human chorionic gonadotropin (β-hCG) and prolactin were statistically increased in the recellularized group on day 9 group (P<0.05).

Conclusion: hEMCs and mouse embryo co-cultured on a decellularized endometrial scaffold provides an alternative model to study embryo implantation and the earlier stage of embryo development.