THU199 Using Primary Human Ovarian Stromal Cells To Elucidate Stromal Cell Influences On Egg Quality Across Pubertal Development

Disclosure: E. Tsui: None. M.M. Laronda: None. Follicles, the functional unit of the ovary with the potential to develop into mature eggs, are composed of a single germ cell and somatic support cells (i.e., granulosa and theca cells). While animal studies have demonstrated a positive effect of ovarian stromal cells to follicle development, the ovarian stromal compartment remains largely undescribed. Moreover, egg quality is maximized after the pubertal transition, but the impact of puberty on ovarian stromal cells and their influence on egg quality is not well understood. To elucidate the relationship between puberty, ovarian stromal cells, and follicle development, we utilized a follicle growth assay in which murine ovarian secondary follicles were cultured in three groups: alone (control), with pre-pubertal derived primary human ovarian stromal cells, or with post-pubertal derived primary human ovarian stromal cells. Follicles (n = 40 per group, N = 4 independent experiments) were monitored over 8 days by brightfield microscopy and evaluated for diameter and survival. No difference in follicle diameter or survival was detected between follicles grown with either pre-pubertal or post-pubertal derived stromal cells (diameter: p = 0.93 for days 2-8; survival: p > 0.9999 at day 8), however, each group of follicles co-cultured with stromal cells grew to larger diameters than follicles grown alone at days 2 (pre-pubertal and post-pubertal, p = 0.005), 4 (pre-pubertal, p = 0.0006; post-pubertal, p = 0.0002), 6 (pre-pubertal, p = 0.03; post-pubertal, p = 0.02), and 8 (pre-pubertal, p = 0.001; post-pubertal, p = 0.004). Survival between follicles grown alone or co-cultured with stromal cells trended towards significance (p = 0.0526 for both pre-pubertal and post-pubertal stroma groups), and all groups demonstrated average survival at day 8 above 70%. Follicle rupture was more commonly observed in follicles cultured with stromal cells (control, 10%; pre-pubertal, 40%; post-pubertal, 80%), with additional maturation studies in progress. Concurrently, the cellular landscape of ovarian stromal cells across puberty was evaluated with single cell RNA sequencing (n = 11, age range = 0.34 to 16.09 years, Tanner stages 1 and 3-5). Analyses revealed 12 sub-clusters representing five major cell types: endothelial cells, theca cells, vascular smooth muscle cells, granulosa cells, and immune cells. This data is screened through existing human secretome databases to identify candidate secreted factors that will be investigated in future follicle growth assays. These results demonstrate a benefit of human ovarian stromal cells in our follicle growth assay and, in conjunction with ongoing maturation and sequencing experiments, validate the utility of this assay to further understand how puberty affects the stromal microenvironment to impact egg quality, insight that can be used to develop and improve future reproductive technologies. Presentation: Thursday, June 15, 2023