75 A two-step protocol for the generation of a three-dimensional implantation model in vitro

Women’s empowerment has become a focal point for development efforts worldwide. However, this advancing gender equality process is causing important social challenges that include female age-related infertility. Currently, an increasing number of women tend to postpone pregnancy, having their first child after age 35. Although the improvements in ART allow for the generation of embryos, implantation and pregnancy rates are significantly reduced for women over the age of 40. The molecular mechanisms undelaying implantation failure is still not fully understood and need to be clarified. To this end, we developed a three-dimensional (3D) in vitro model that mimics the interactions between the blastocyst and the endometrium, using the pig as a model: (1) Preparation of the endometrial compartment. The 3D endometrial model was obtained plating 1 × 10⁶ stromal cells onto polystyrene scaffold inserts at Days 0, 7, and 9. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 2 mM ascorbic acid at 37°C and 5% CO₂ for 14 days. On Day 14, 2 × 10⁶ endometrial epithelial cells were layered on top of the stromal compartment and maintained in DMEM/F12 supplemented with 5% fetal calf serum, at 37°C and 5% CO2 for 21 days. (2) Development of trophoblast (TR) spheroids. The TR organoids were generated encapsulating dermal fibroblasts into microbioreactors and exposing them to 5-azacytidine to increase cell plasticity. A cocktail containing BMP4 and inhibitors of the Activin/Nodal/ERK signaling pathways was then used to drive trophoblastic differentiation. Histological analysis demonstrated that by Day 7 fibroblasts within the scaffold started to grow and showed a large, flat, elongated (spindle-shaped) morphology, creating robust foundation for epithelial cells to grow. On Day 21 epithelial cells displayed a cuboidal morphology and create a closed monolayer overlaying the stromal cell mass. In parallel, development of TR organoids was confirmed by the expression of the distinctive mature markers KRT7, GCM1, PPAG3, PAG6, HSD17B1, CYP11A1, and IFNG, undetectable in untreated fibroblasts. The results obtained demonstrate the possibility to generate the two cellular components involved in implantation and pave the way to the creation of a useful model to recreate in vitro the main dynamics involved in implantation. The system created will be also advantageous to investigate the impact of aging on embryo/endometrium recognition or communication.

This research was supported by the Carraresi Foundation, HORIZON-WIDERA-2021 project n#101079349 (OH-Boost), PSR2021.