L26/P-917 Investigating progesterone resistance in endometriosis and adenomyosis using endometrial assembloids

Abstract Study question Do endometrial assembloids derived from eutopic endometrium from endometriosis and adenomyosis patients exhibit altered molecular and functional responses to progesterone compared with healthy controls? Summary answer Endometrial assembloids derived from endometriosis and adenomyosis patients show impaired and heterogeneous progesterone responsiveness compared with healthy controls. What is known already Endometriosis and adenomyosis are estrogen-dependent disorders characterized by progesterone resistance. This phenomenon is not fully understood, but contributes to lesion persistence, impaired endometrial receptivity and reduced hormone therapy responses. In physiological conditions, progesterone-responsive stromal cells regulate epithelial differentiation and limit paracrine estrogenic activity. In progesterone-resistant endometrium, this mechanism is faulty, causing defective epithelial function. In diseased endometrium, epithelial cells secrete pro-inflammatory cytokines that affect stromal cells, curtailing progesterone receptor expression and inducing epigenetic changes harming decidualization. By combining epithelial organoids with stromal cells, endometrial assembloids model this reciprocal crosstalk and provide a relevant system to study hormone responsiveness. Study design, size, duration This experimental study involved endometrial assembloids generated from eutopic endometrial biopsies from healthy controls (n = 5), endometriosis patients (n = 5) and adenomyosis patients (n = 5). Assembloids were cultured in hormone-free, estrogenic or progesterone-stimulated conditions to mimic proliferative and secretory cycle phases. Morphological and molecular analyses were performed after five days of hormone exposure. Participants/materials, setting, methods Premenopausal women undergoing gynecological surgery were recruited after providing written informed consent. Endometrial epithelial organoids and primary stromal cells were isolated and recombined to create three-dimensional assembloids following 2-6 passages in culture. Progesterone responsiveness was assessed by histology, immunohistochemistry, ELISA and RT-qPCR, evaluating secretory transformation, glycodelin, leukemia inhibitory factor, prolactin, HSD17β2, Ki67 and steroid receptor expression. Main results and the role of chance Assembloids were successfully generated from endometrial biopsies from healthy, endometriosis and adenomyosis patients and reproduced epithelial and stromal organization of native endometrium. In healthy controls, progesterone stimulation initiated secretory morphological changes and significant upregulation of progesterone-responsive markers, including glycodelin secretion and HSD17β2 and PRB mRNA expression, confirming maintained progesterone responsiveness. By contrast, assembloids derived from endometriosis and adenomyosis patients showed attenuated and heterogeneous responses to progesterone. Endometriosis-derived assembloids exhibited weakened secretory transformation and glycodelin induction, and markedly reduced PRB expression. Adenomyosis-derived assembloids displayed defective stromal regulation of LIF after progesterone stimulation, suggesting altered endometrial receptivity. Progesterone-induced HSD17β2 expression was absent or highly variable in both disease groups. Ki67 analyses indicated greater stromal proliferation in disease-derived assembloids, consistent with deficient progesterone-mediated growth inhibition. Statistically significant differences were observed for secretory transformation, glycodelin expression, HSD17β2 induction and PRB mRNA expression in healthy assembloids. Responses in disease-derived assembloids were more variable, suggesting reduced progesterone responsiveness rather than random variation. Overall, these results indicate possible disease-specific impairments in progesterone-driven secretory differentiation and receptivity, with interindividual variability emphasizing the potential of assembloids as a model to study human endometrial dysfunction. Limitations, reasons for caution This study is limited by its small sample size, interindividual variability and heterogeneous menstrual cycle phases at biopsy. Absence of immune, vascular and innervation components, variable cell passage numbers and use of Matrigel further hinder direct extrapolation to in vivo endometrial physiology. Wider implications of the findings Endometrial assembloids may provide a physiologically relevant human model to investigate disease-specific progesterone resistance mechanisms. Indeed, this will help inform development of personalized therapeutic strategies for infertility and treatment resistance in endometriosis and adenomyosis. Trial registration number No