Spatially Organized Human Ovarian Spheroids Instruct Endometrial Morphogenesis

ABSTRACT The human ovarian‐endometrial axis operates through coordinated, dynamic endocrine signaling that cannot be faithfully reproduced by static hormone supplementation. While endometrial organoids (EMOs) respond to exogenous estradiol and progesterone, whether a spatially organized human ovarian endocrine unit can instruct epithelial morphogenesis through physiologically integrated signaling remains unknown. Here, we engineered a multilayered human follicle‐like spheroid composed of primary granulosa cells and stromal‐derived theca‐like cells (SPHEGaT), recreating architectural and steroidogenic features of the ovarian follicle. These constructs generated sustained biologically active concentrations of estradiol and progesterone while maintaining high viability and low hypoxic burden. When co‐cultured with EMOs, SPHEGaTs induced progressive epithelial remodeling characterized by folding morphogenesis, increased progesterone receptor expression, and upregulation of secretory/progesterone‐responsive genes including PAEP, SPP1 and HSD17B2. Strikingly, partial steroid depletion did not abolish organoid folding, whereas static supplementation with exogenous estradiol and progesterone failed to restore the differentiation phenotype. These findings suggest that endometrial remodeling is influenced not merely by hormone concentration, but also by additional signaling cues present within the SPHEGaT‐derived microenvironment. Together, this work establishes a human 3D platform in which integrated ovarian‐derived signaling supports epithelial morphogenesis, providing new framework for studying ovarian‐endometrial communication and highlighting the limitations of hormone‐only models in reproductive bioengineering.