Endocrine Abstracts

Introduction: Polycystic ovary syndrome (PCOS) is the leading cause of female infertility and is associated with high degree of comorbidities including type 2 diabetes and endometrial cancer. Hyperandrogenemia is a hallmark of PCOS and contributes to endometrial-related dysfunctions, including implantation failure and miscarriage. Whether cellular heterogeneity contributes to the functioning of the endometrium is not previously studied. Therefore, the aim is to reveal cell-type-specific disease signatures in the endometrium in women with PCOS at the single-cell level and to validate the role of molecular targets in endometrial organoids (EOs).

Method: Single nuclei were extracted from frozen endometrial biopsies collected from women with PCOS (n=12) and healthy controls (n=5) at cycle day 7-10. The nuclei RNA libraries were prepared following the 10x genomics protocol allowing us to sequence ~10,000 cells per sample and ~20,000 reads per cell. Sequencing data is processed using Cellranger count for further data integration, quality control and analysis with the Seurat package. In parallel, 3-dimensional (3D) EOs are established from fresh endometrial biopsies that are enzymatically dissociated and collected at cycle day 7-10. The cells are resuspended in Matrigel droplet and organoids are generated and maintained in a defined medium. Following several passages, EOs are cryopreserved to create a biobank to be used for future functional analyses of identified molecular targets by single-cell RNA-sequencing, immunofluorescence microscopy and Seahorse metabolic analysis.

Results: Single-nuclei were extracted from 17 endometrial samples, 12 from women with PCOS and 5 from healthy controls for 10x snRNA-sequencing. Our initial bioinformatic analyses show that the endometrial tissue of women with PCOS has a distinct single-cell transcriptomic profile, with cell-type specific differentially expressed genes that differ from the healthy controls. The EO protocol has been established and EOs from four women with PCOS have been cryopreserved with EOs successfully reestablished after thawing. Validation with immunofluorescent staining shows that the 3D EOs consist of an intact proliferative epithelial membrane with a consistent polarity across the whole EO.

Conclusion: This rigorous mapping of endometrial tissue samples will increase the understanding of the cellular complexity and dysfunction and will be linked to phenotypic features in women with PCOS. By successive formation of 3D PCOS-EOs, we can further study cellular and molecular mechanisms causing PCOS-specific endometrial dysfunction.