The human ovary : a characterization of cell types and adverse ovarian side effects of chemotherapy

Sammanfattning: The human ovary has a major role in the body’s endocrine and reproductive system. Understanding its cell type composition is crucial in order to study underlying mechanisms of hormonal balance and ovarian follicle maturation in health and disease. The outer lining of the ovary, the cortex, harbors the ovarian reserve. This reserve is made up of follicles that have formed before birth. The prevailing dogma of female fertility sees this reserve as being limited in humans, with follicle numbers decreasing over time. Once the ovarian reserve is depleted, women enter menopause which marks the end of their reproductive period. Premature ovarian insufficiency is characterized by an early depletion of the ovarian reserve and reveals itself, among others, with an altered hormone profile and early menopause onset. Anti-cancer treatment is one of the causes of premature ovarian insufficiency. However, mechanisms underlying chemotherapy-induced altered ovarian tissue morphology and function are not yet fully understood. Therefore, effective treatments to prevent or cure the damage are still missing. One possibility for novel treatments could be offered by oogonial stem cells (OSCs) that have been recently reported to exist in the adult human ovaries. These cells can be isolated with DDX4 antibody (Ab), show a gene expression profile similar to germline stem cells in the developing embryo and have the potential to mature into oocytes. The overall aim of this thesis is to assess the effect of first-line chemotherapy on ovarian follicles and stroma by using a tissue collection from fertility preservation programs and to dissect the structure of human ovarian cortex at single-cell resolution by using advanced single- cell profiling technology. In study I, the effects of first-line chemotherapy that is not considered a risk for future fertility on ovarian follicles and stroma cells from pediatric and young adult ovaries were investigated. Exposure to chemotherapy prior to ovarian tissue cryopreservation revealed an impact on follicle and stroma health. Early ovarian follicles (primordial and intermediary) appeared more atretic and were smaller, while healthy growing follicles (primary) decreased in numbers. Steroid production by treated ovarian tissue in culture decreased and DNA damage as well as fibrotic lesions in the ovarian stroma increased. In study II, we assessed the cellular composition of the adult human ovarian cortex at a single- cell level. Transcriptome and surface proteome analysis revealed six major cell types consistently detected in the ovarian cortex from Cesarean section and gender reassignment patients, namely oocytes, immune cells, granulosa cells, endothelial cells, perivascular cells and stroma cells. In order to investigate the existence of the previously reported OSCs, ovarian cortex cells positive for DDX4 Ab were isolated and analyzed. DDX4 Ab-positive cells did not show a germline-like profile, neither at a transcriptional nor protein level, but instead could be identified as perivascular cells. Furthermore, when integrating published single-cell sequencing data from fetal germ cells, we could not identify any cortex cell with a transcriptome profile of germline stem cells. In conclusion, we demonstrate that first-line chemotherapy treatment has negative effects on the health of ovarian cortex and exposure should be limited before cryopreservation of ovarian tissue, if possible. Adult ovarian cortex is made up of various cell types, but no evidence for the existence of germline-like stem cells was found.