SAT355 Single-Cell RNA Sequencing Pipeline for the Human Ovary Using IVF Tissue

Abstract

Abstract Disclosure: A.J. Hanson: None. S. Shah: None. C. Wai: None. R. Bauer: None. C. Boots: None. M. Urbanek: None. Objective: Polycystic Ovary Syndrome (PCOS) is the most common endocrinopathy of reproductive aged women, affecting 6-15% of women in this age group. Defined by hyperandrogenism and irregular menses, PCOS is a complex trait with a strong genetic component; its underlying cause remains poorly understood. To understand the molecular pathogenesis of PCOS, an appropriate experimental model of the human ovary is needed. The ovary is the primary source of androgen production and an essential regulator of ovulation and menstruation, the key processes disrupted in PCOS. Discard tissue from oocyte retrievals as part of assisted reproduction treatment (ART) is a plentiful source of human ovarian tissue, requires no additional risk to the patient, and is enriched for subjects with PCOS. Therefore, we developed a pipeline using discard ovarian aspirate tissue from reproductively healthy women and women with PCOS undergoing oocyte retrievals for ART to generate a single-cell (sc) transcriptomic atlas of human ovaries. We will use those maps to define the differences in gene expression between the two groups and determine the impact of genetic variation on human ovary function in women with PCOS. Materials and Methods: Our pipeline consists of i. harvesting oocyte retrieval discard tissue including follicular fluid, pieces of ovary, and blood from reproductively healthy women and women with PCOS ii. generating a sc suspension of ovarian cells, iii. constructing and sequencing a scRNA sequencing library, iv. building a transcriptomic atlas of the ovary, and v. identifying differences in cell abundance and expression between reproductively healthy women and women with PCOS. Results: We have processed samples from seven reproductively healthy women and eight women with PCOS using this scRNA sequencing pipeline. The sc isolation protocol resulted in a sc suspension with high viability (95%) and yield (19.6 x106 cells/mL), demonstrating that we can generate a large quantity of high-quality sc suspensions. The sequenced single cells were clustered into distinct groups based on their transcriptomes including cell types predicted to be affected by PCOS including theca, granulosa, and immune cells. Conclusions: We successfully created a scRNA sequencing pipeline from discard oocyte retrieval tissue and generated transcriptomic maps of the human ovary of reproductively healthy women and women with PCOS, demonstrating the feasibility of our approach. Impact Statement: By integrating our transcriptomic data with whole exome sequencing (WES) data to assess the impact of genomic variation on ovarian cellular composition and transcriptional profiles, we will, for the first time, evaluate the impact of an individual woman’s genetic variation on their ovarian functional profile, a critical first step towards understanding the underlying pathology of PCOS and personalized medicine. Presentation Date: Saturday, June 17, 2023