O-084 Construction of human endometrial complex organoids and their application in endometrial injury repair and regeneration

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Abstract Study question Could human endometrial complex organoid be used to repair endometrial Injury and promote tissue regeneration? Summary answer The complex endometrial organoid we constructed can not only respond to hormonal stimulation but also promote the structural and functional repair and regeneration of endometrium What is known already The endometrium is a crucial site for embryo implantation and development. However, once the basal layer of the endometrium is damaged due to causes such as surgery or infection, it becomes difficult for the endometrium to repair and regenerate. This can lead to intrauterine fibrosis, scarring, and adhesions. Approximately 19% of women who experience miscarriages are diagnosed with intrauterine adhesions (IUA). Organoids can effectively mimic the complex microenvironment and functions of tissues in the body and hold promise as a new tissue regeneration technology for repairing and reconstructing large-area basal layer endometrial injuries. Study design, size, duration By utilizeing the key cell subpopulations and signaling pathways involved in the development and regeneration of endometrial tissue, we have induced and constructed complex human endometrial organoids with multiple cell components (epithelial, stromal, vascular endothelial, etc.) in vitro. We optimized the cell proportion of the complex organoid, and conducted in vitro identification and characterization (IHC staining, hormone induced gene expression), as well as validation in mice endometrial injury model in vivo. Participants/materials, setting, methods Materials and Methods: Primary cultured endometrial stromal cells, epithelial cells and HUVEC endothelial cells, embryonic stem cell differentiated endometrial stromal cells, epithelial cells. Matrigel was used as hydrogel to construct vascularized endometrial organoid. Animal model: After anesthesia and exposure of the uterus. In the lower segment of the uterus, a 2mm transverse incision was created and the endometrial layer was flipped out. The full endometrium was completely torn off using pointed forceps without damaging the muscle layer. Main results and the role of chance By analysis of the key cell subpopulations and signaling pathways involved in the development and regeneration of endometrial tissue, we have identified a pro-regenerative SFRP4+ endometrial stromal cell populations, which could promote the proliferation of endometrial epithelial cells in vitro and endometrial regeneration in vivo through IGF1 signaling. We also generated human pluripotent stem cells-derived Müllerian duct-like cells (MDLCs) using a defined and effective protocol. The MDLCs are bi-potent, can gradually differentiate into endometrial epithelial and stromal cells. We optimized the optimal cell proportion (endometrial stromal cells, epithelial cells and HUVEC endothelial cells) of the complex organoid. Through in vitro identification and characterization, as well as validation in animal models, we have demonstrated that the complex human endometrial organoid we constructed can not only respond to hormonal stimulation in vitro but also promote the structural and functional repair and regeneration of damaged endometrium in vivo. Limitations, reasons for caution Large animal model (e.g. pig) would be required to further confirm the efficacy of the complex human endometrial organoid on endometrial repair and regeneration. Wider implications of the findings These complex human endometrial organoids show potential for clinical application in repairing and reconstructing large-area basal layer endometrial injuries. Trial registration number No

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