The ovarian stroma includes all cells and tissue outside the follicles e.g. immune cells, fibroblasts, blood vessels, nerves, extracellular matrix. Macrophages are the primary innate immune cells of the ovary and are involved in the tissue remodeling that occurs with every ovulation cycle. In our mouse models of aging and obesity, ovarian fibrosis increased, preventing follicle growth and oocyte release. This fibrosis was associated with ovarian stromal oxidative stress and an altered macrophage phenotype, towards pro-fibrotic CD68+ cells. Based on these findings, we investigated macrophage phenotypes within human ovaries and their localisation relative to collagen deposition.
Ovarian tissue was obtained following informed consent from 44 women (median age 42, range 37-79) undergoing gynaecological surgery for benign conditions. Cortical fibrosis was measured using Picrosirius Red staining, and oxidative damage was assessed by immunohistochemistry (IHC) for 4-HNE, a marker of oxidised lipids. For each sample, the percentage area positive for medium-thick collagen (orange-red), and 4-HNE (threshold based on IgG negative control) was measured in 4 areas of cortex using ImageJ, then averaged. Cortical fibrosis and 4-HNE was present but variable across samples. We used IHC to count macrophage populations in these non-pathological human ovaries, focusing on the cortex close to ovarian surface epithelial cells (OSE), and the medial stroma. IBA1, a pan-macrophage/monocyte marker, CD68, a marker of macrophages associated with fibrosis, and CD206, a marker of pro-repair macrophages, were used. Fewer macrophages were present in the dense, fibrotic cortex than the looser medial tissue. Macrophages were in close contact with OSE, foamy macrophages were present in follicular cysts, and CD206+ macrophages were more abundant than pro-fibrotic CD68+ macrophages. These results highlight that diverse immune cell populations reside in the ovarian stroma and contribute to our understanding of the localisation and functions of immune cells in the human ovary.