Decoding adenomyosis pathogenesis using an assembloid model

Adenomyosis remains a challenging gynecological disorder to investigate due to the absence of in vitro models that accurately replicate endometrial tissue dynamics across the menstrual cycle. To address this gap, we established an endometrial assembloid model that faithfully mimics cycle-dependent endometrial responses and captures key cellular and molecular hallmarks of adenomyosis, including ectopic lesion- specific epithelial and stromal heterogeneity. Single-cell transcriptomics revealed that ectopic epithelial cells shift toward a luminal- dominant, glandular-deficient transcriptional profile during the secretory-like phase. This transition correlated with ectopic stromal reorganization-specifically, loss of BMP4⁺ stromal cells and an accumulation of CRYAB⁺IL15⁺ stromal cells-which impaired BMP-mediated stromal-epithelial signaling while enhancing WNT activation. Additionally, ectopic epithelial and stromal cells demonstrated increased immunity and angiogenesis activities. Our assembloid platform not only provides a physiologically relevant model for investigating adenomyosis pathogenesis but also implicates aberrant WNT signaling as a potential therapeutic target, offering new opportunities for mechanism-driven treatment strategies.