Abstract

The human endometrium has a remarkable capacity for cyclical regeneration throughout the menstrual cycle. However, surgical trauma or other factors that damage the basal layer would impair endometrial repair, which significantly threaten reproductive health in women. While organoid-based therapy has emerged as a promising regenerative approach, the efficiency of current models in reconstructing severe deep endometrial damage is limited by their restricted cellular diversity. In this study, we engineered a endometrial regenerative organoid (ERO) model by optimizing the cell composition and culture method to recapitulate the in vivo endometrial histological structure and function. Histology, immunofluorescence, and single-cell RNA-seq analyses revealed that the constructed organoids possess structural and hormonal response functions similar to those of in vivo endometrial tissues. In vivo cell tracking experiments confirmed the successful integration of transplanted EROs into the luminal epithelium, glandular epithelium, and stromal compartments of the damaged endometria of immunodeficient mice. To further assess their regenerative potential under normal immunological circumstances, EROs were transplanted into a mouse model of full-thickness endometrial injury. The results demonstrated efficient structural regeneration, improved endometrial receptivity and considerable restoration of pregnancy rates, highlighting the feasibility of functionally reconstructing severe deep endometrial injuries. Therefore, ERO, a multicellular endometrial organoid, not only provides a biomimetic platform for studying endometrial physiology in vitro but also proposes a novel regenerative strategy for endometrial repair. Significance statement The human endometrium has a remarkable capacity for cyclical regeneration throughout the menstrual cycle. However, surgical trauma or other factors that damage the basal layer would impair endometrial repair, which significantly threaten endometrial