FRI393 Mapping Endometrial Cell-type-specific Disease Signatures A nd Endometrial Organoids In Polycystic Ovary Syndrome

Disclosure: G. Eriksson: None. C. Li: None. S. Risal: None. H. Pui: None. S. Torstensson: None. A. Linden Hirschberg: None. S. Petropoulos: None. Q. Deng: None. E. Stener-Victorin: None. Women with polycystic ovary syndrome (PCOS) suffer from reduced fertility linked to implantation failure and miscarriage, as well as endometrial cancer, all associated with endometrial dysfunction. We hypothesize that cell-type-specific endometrial dysfunction in insulin-resistant and hyperandrogenic women with PCOS contributes to the observed endometrial dysfunction and that treatment aimed at improving insulin sensitivity and decreasing androgen excess has the potential to reverse identified alterations.To uncover cell-type-specific disease signatures and molecular pathways for PCOS-specific endometrial dysfunction, we first extracted single-nuclei (sn) from frozen endometrial biopsies taken in the proliferative phase (day 7-10) from controls (n=5) and women with PCOS (n=10) at baseline and in PCOS after 16 weeks of metformin (n=7) or lifestyle management (n=3) for snRNA-sequencing. The 10x Genomics protocol allowed us to sequence ≈10,000 nuclei/sample and ≈20,000 reads/nuclei. A total of 248,694 nuclei were captured and 6 major cell types were identified. The 3 largest cell clusters were i) stromal cells (124,055 nuclei), ii) epithelial cells (105,095 nuclei) and iii) immune cells (13,596), whilst iv) uterine smooth muscle cells, v) endothelial and vi) lymphoid cell clusters consisted of <3000 nuclei. Subsetting the epithelial cells revealed the functional luminal, glandular and ciliated cell types as well as proliferative cells. In the immune cell cluster, both myeloid and lymphoid lineage cells were identified, of which uterine NK-cells (uNK) and macrophages (uM) were the largest populations. Several differentially expressed genes (DEGs) of the epithelial subtypes, uNKs, uMs and stromal cells were identified in women with PCOS compared with controls. DEGs in cell types between PCOS cases and controls were enriched in pathways related to cilium organization in the ciliated epithelium, extracellular matrix structure in stromal cells, and cysteine-type endopeptidase activity in uM type 1. Of note, both 16-week of metformin treatment or lifestyle management restored multiple DEGs in each subtype. Second, to validate the identified molecular target, we have established 3D endometrial organoids (EOs) from fresh endometrial biopsies of control and PCOS women. After several passages, EOs were cryopreserved to create a biobank for future functional analyses of the molecular targets. The EOs can be re-established after thawing and immunofluorescence staining shows that the EOs consist of an intact proliferative basolateral epithelial membrane. This rigorous mapping of the human PCOS endometrium improves our understanding of cellular complexity and specific cell types. It provides new mechanistic insights into disease-specific endometrial dysfunction(s), and several of the identified dysfunctions can be reversed by current first-line interventions. Presentation: Friday, June 16, 2023