Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Single-cell RNA-sequencing of retrieved human oocytes and eggs in clinical practice and for human ovarian cell atlasing.

With the advancement of single-cell separation techniques and high-throughput sequencing platforms, single-cell RNA-sequencing (scRNA-seq) has emerged as a vital technology for understanding tissue and organ systems at cellular resolution. Through transcriptional analysis, it is possible to characterize unique or rare cell types, interpret their interactions, and reveal novel functional states or shifts in developmental stages. As such, this technology is uniquely suited for studying the cells within the human ovary. The ovary is a cellularly heterogeneous organ that houses follicles, the reproductive and endocrine unit that consists of an oocyte surrounded by hormone-producing support cells, as well as many other cell populations constituting stroma, vasculature, lymphatic, and immune components. Here we review studies that have utilized scRNA-seq technology to analyze cells from healthy human ovaries and discuss the single-cell isolation techniques used. We identified two overarching applications for scRNA-seq in the human ovary. The first applies this technology to investigate transcriptional differences in oocytes/eggs from patients undergoing in vitro fertilization treatments to ultimately improve clinical outcomes. The second utilizes scRNA-seq for the pursuit of creating a comprehensive single-cell atlas of the human ovary. The knowledge gained from these studies underscores the importance of scRNA-seq technologies in unlocking a new biological understanding of the human ovary.

Fibroinflammatory Signatures Increase with Age in the Human Ovary and Follicular Fluid.

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. "Fibroinflammation" is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7-44.8 years). This signature (IL-3, IL-7, IL-15, TGFß1, TGFß3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFß3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFß3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.