Single-Cell Gene Expression Atlas of Human Follicular Aspirates: Impacts of Leukocytes on the Ovulatory Process. Single-Cell Gene Expression Atlas of Human Follicular Aspirates: Impacts of Leukocytes on the Ovulatory Process

Leukocytes are in situ regulators for ovarian functions. However, little is known about leukocyte subpopulations and functions in ovulatory follicles, especially in humans. This study performed single-cell RNA sequencing and bioinformatics analyses using follicular aspirates obtained from four IVF patients. Nineteen clusters were identified: six follicular cells, ten subsets of leukocytes, and one RBC/platelet. Using dominant follicle tissues collected throughout the periovulatory period from normally cycling women and cultured primary human granulosa/lutein cells, we determined the cell type-specific expression of PTPRC, CD68, IL1B, and IL1R1. RNA velocity analyses revealed developmental dynamics across five granulosa cell groups and two projections of granulosa cell differentiation. To understand how leukocytes promote periovulatory changes, we sought leukocyte-derived factors potentially involved in 1) granulosa cell function by activating their receptors, 2) tissue remodeling, and 3) angiogenesis in ovulatory follicles. Collectively, these findings identified diverse leukocyte-secreted factors that facilitate ovulation and luteinization in the human ovary. Overall design: Ovarian follicular aspirates obtained from four women undergoing the standardized IVF procedure