PCOS endometrium-derived epithelial organoids as a novel model to study endometrial dysfunction

Are we able to establish endometrium epithelial organoids (EEOs) from endometrial samples obtained from women with PCOS, and do they differ from non-PCOS EEOs? We were able to establish, for the first time, PCOS EEOs which capture endometrial abnormalities present in women with PCOS, including increased inflammation and decreased receptivity-related gene expression. Patient-derived EEOs could serve as a tool to study endometrial dysfunction, as diseased tissue-derived organoid models typically retain the disease-related traits. In PCOS, endometrial dysfunction likely contributes to subfertility and pregnancy complications, yet previous research on the endometrial epithelial compartment has been scarce and, so far, no PCOS-derived EEOs have been established. EEOs were established from endometrial biopsies from two cohorts of women with PCOS-including overweight/obese (O-PCOS, n = 4) and lean (L-PCOS, n = 4)-along with BMI-matched controls (overweight/obese control (O-Ctrl), n = 4; lean control (L-Ctrl), n = 4). EEOs were exposed to combinations of steroid hormones (β-estradiol (E2), progesterone, cAMP, and the Wnt/β-catenin signaling (WNT) inhibitor XAV-939) for 6 days to simulate the proliferative or secretory phases of the menstrual cycle, with or without simultaneous androgen exposure with dihydrotestosterone (DHT). Bulk RNA-sequencing was conducted to identify variations in gene expression between PCOS and Ctrl EEOs, while reverse-transcription quantitative PCR RT-qPCR was employed to validate these results. Morphological assessment of EEOs was performed using hematoxylin and eosin staining and immunostaining. The size of EEOs was evaluated after 6 days of hormonal exposure. PCOS EEOs from both BMI groups demonstrated increased inflammation-related gene expression (including increased expression of Oncostatin M Receptor (OSMR) and Intercellular Adhesion Molecule 1 (ICAM1)) and showed a reduced diameter compared to their respective control EEOs. The O-PCOS EEOs displayed an aberrant response to steroid exposure with E2 and progesterone (including reduced expression of receptivity-related genes progestagen-associated endometrial protein and leukemia inhibitory factor) as compared to control EEOs. Addition of DHT to the culture media did not affect EEO transcriptome, aligning with the minimal androgen receptor (AR) expression in the EEOs. Sequencing data are available from the corresponding author upon request. The study should be replicated with a larger number of samples and with other PCOS phenotypes apart from different weight categories. Furthermore, as this work is the first one to establish PCOS EEOs, future studies should focus on incorporating other endometrial cell types, including immune cells, in a co-culture system. This novel in vitro organoid model for PCOS captures the endometrial abnormalities present in the two weight categories of women with PCOS, thereby providing a valuable tool to gain insights into PCOS-related endometrial dysfunction. Our findings propose potential links to the increased risk of pregnancy complications in women with PCOS, such as the role of altered receptivity and implantation environment including increased inflammation, which may contribute to aberrant placentation and subsequent placental dysfunction. Jusélius Foundation, Novo Nordisk Foundation, Research Council of Finland, Horizon 2020 Marie-Curie MATER Innovative Training Network (all to T.T.P.), Fund for Scientific Research Flanders-Belgium (FWO, G0A6719N to J.V. and GO99023N to H.V.); KU Leuven Research Fund (C14/21/116 to H.V. and C14/24/152 to J.V.), University of Oulu Scholarship Foundation Grant (to L.L.), and PhD grant of China Scholarship Council (CSC, to M.W.). The authors have no conflicts of interest to declare.