Establishment of a Novel Human Endometrial Organoid.

Purpose: Human implantation is difficult to study in vivo because of ethical constraints, making the development of in vitro models essential for mechanistic investigations. Here, we applied the technique previously established in mice to develop a novel human endometrial organoid. Methods: Human endometrial tissues were digested and cultured as adherent monolayers without extracellular matrix (ECM). After 10 days, self-organized aggregates appeared and were transferred to low-attachment plates for three-dimensional culture. To assess hormonal responsiveness, organoids were treated with medroxyprogesterone acetate and cyclic adenosine monophosphate for 4 days. Results: The resulting organoids consisted of EpCAM-positive epithelial cells forming an outer layer and PDGFRα-positive stromal cells occupying the inner region. MUC1 and acetylated α-tubulin, proteins localized on the apical surface of endometrial epithelial cells in vivo, were expressed on the outer surface of the epithelial layer in our organoids. Hormonal stimulation altered the expression of receptivity markers, including increased PAEP and decreased PGR in epithelial cells and increased FOXO1 in both epithelial and stromal cells, reflecting in vivo implantation-phase responses. Conclusion: We established a novel ECM-free human endometrial organoid that recapitulates key structural and hormonal characteristics of the human endometrium, providing a promising platform for studying human implantation mechanisms.