Eutopic echoes ectopic: organoid-based evidence of shared hormone dysregulation in endometriosis

Abstract Background Endometriosis is a highly prevalent, hormone-driven chronic disease characterized by the growth of endometrial tissue outside the uterine cavity. Despite its impact, the mechanisms underlying the disease’s pathogenesis remain poorly understood. We hypothesized that not only the ectopic endometrium of women with endometriosis differs from that of women without the disease, but also the eutopic endometrium. Accordingly, this study aimed to identify potential differences in basal expression of hormone receptors, their responses to hormonal treatment, as well as general morphology and growth factor dependencies among organoids derived from the endometrium of women without endometriosis and from the eutopic and ectopic endometrial tissues of patients with endometriosis. Methods Patient-derived organoids were established from the endometrium of women without endometriosis and from the eutopic and ectopic endometrial tissue of patients with endometriosis. To determine optimal culture conditions, primary isolates were seeded in parallel in medium containing the WNT signaling activators WNT3a and R-spondin-1 (RSPO1) or in medium supplemented with RSPO1 alone to evaluate WNT pathway requirements. Organoids were exposed to 17β-estradiol (E2), progesterone (P4) with cAMP, and the WNT inhibitor XAV939. The expression of estrogen receptor alpha and beta (ESR1, ESR2), progesterone receptor (PGR), and aromatase (CYP19A1) was measured via qPCR. Organoid morphology, growth, and aromatase expression were determined by brightfield and immunofluorescence imaging. Relative gene expression was calculated using the comparative CT method (ΔΔCT) to evaluate hormone responses across groups. Results Organoids derived from control donors and from eutopic endometrium of endometriosis patients exhibited robust long-term growth in medium supplemented with WNT3a and RSPO1, whereas ectopic endometriotic organoids proliferated best in medium containing only RSPO1, indicating reduced dependence on exogenous WNT pathway activation. Basal ESR1 and PGR expression was lower in lines from endometriosis patients than in control lines, indicating reduced E2 sensitivity and P4 responsiveness. ESR2 levels were uniformly low across groups. Upon E2 stimulation, ESR1 expression was consistently suppressed, whereas PGR expression was strongly induced in ectopic organoids (29.9-fold, ***p < 0.001), in eutopic organoids (13.5-fold, *p = 0.016), and to a lesser extent in organoids from women without endometriosis (5.2-fold, p = 0.069). P4-mediated downregulation of PGR required WNT inhibition. The effectiveness of amended hormonal stimulation with XAV939 in downregulating PGR in organoids was validated at the protein level via Western blotting (WB). Sporadic aromatase expression was detected exclusively in organoids derived from endometriosis patients. Conclusions These findings demonstrate that the eutopic endometrium of endometriosis patients exhibits altered responses to hormone stimulation. In this regard, it mirrors the characteristics of ectopic lesions more closely than those of endometrium from women without endometriosis. These results suggest that the endometrium plays an intrinsic role in the pathophysiology of endometriosis, providing a strong basis for future mechanistic studies.