Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

ABSTRACT Adult endometrial glands undergo cyclic regeneration and development during the menstrual cycle. Their secretions are vital for endometrial functions and early pregnancy, yet the mechanisms controlling gland development are not well understood. Although various 3D endometrial models exist, none fully replicate human gland development in vitro. This study establishes a robust 3D endometrial assembloid model by integrating human endometrial organoids (EOs) and human endometrial stromal cells (HESCs), successfully replicating tubular gland formation and illustrating essential stromal‐epithelial interactions. Transcriptomic analyses identify Wnt Family Member 7B (WNT7B) as an intrinsic inhibitor of gland formation and development, regulated extrinsically by transforming growth factor beta‐1 (TGFβ1) signaling through vitamin D receptor (VDR) interactions between EOs and HESCs. Endometrium‐specific WNT7B knockout mice exhibit enhanced gland development further supports WNT7B's inhibitory role in endometrial gland development. Estradiol facilitates tubular gland formation by suppressing WNT7B expression in vitro, which is confirmed in estradiol‐stimulated mouse models and clinical samples from women undergoing ovarian stimulation for in vitro fertilization. These findings elucidate the central roles of estradiol‐WNT7B signaling and stromal‐derived TGFβ1‐VDR crosstalk in endometrial gland development, providing a foundation for improved 3D endometrial models and identifying therapeutic targets for gland‐related disorders like endometriosis, infertility, and endometrial hyperplasia.