Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing<em> In Vitro </em>Fertilization

Human ovary follicular contents, including follicular fluid (FF), oocytes, cumulus cells, and somatic cells, offer a powerful yet underutilized opportunity to study ovarian physiology. Assisted reproductive technologies (ART) routinely generate these biological byproducts, which are often discarded after mature oocytes are used for clinical care. However, they provide valuable insights into fertility, reproductive aging, and ovarian dysfunction. Here, we present a standardized protocol for the systematic collection and downstream processing of follicular components from women undergoing oocyte retrieval. Fully expanded cumulus-oocyte complexes (COCs) are microdissected to isolate cumulus cells without enzymatic denudation, preserving transcriptomic integrity. FF is centrifuged, with the supernatant analyzed for cytokines, hormones, metabolites, and environmental toxicants, while the pellet is cryopreserved and processed via density gradient centrifugation to isolate viable granulosa cells. A stepwise protocol enables the preparation of single-cell suspensions from fresh FF for single-cell RNA sequencing (scRNA-Seq), including enzymatic digestion, serial filtration, and red blood cell lysis, and yields >65% viable cells representing a diverse composition, including granulosa cells, macrophages, T cells, smooth muscle-like cells, and antigen-presenting cells. In addition, immature germinal vesicle (GV) oocytes can be matured in vitro, with 56% reaching the MII stage within 24 h. Together, these protocols yield high-quality RNA, enriched granulosa cell populations, and complex single-cell datasets that capture the heterogeneity of the ovarian microenvironment. They provide a framework to leverage discarded reproductive tissues for translational research in human fertility and reproductive biology.