Metformin prevents age-associated ovarian fibrosis by modulating the immune landscape in female mice

Ovarian fibrosis is a pathological condition associated with aging and is responsible for a variety of ovarian dysfunctions. Given the known contributions of tissue fibrosis to tumorigenesis, it is anticipated that ovarian fibrosis may contribute to ovarian cancer risk. We recently reported that diabetic postmenopausal women using metformin had ovarian collagen abundance and organization that were similar to premenopausal ovaries from non-diabetic women. In this study, we investigated the effects of aging and metformin on mouse ovarian fibrosis at a single-cell level. We discovered that metformin treatment prevents age-associated ovarian fibrosis by modulating the proportion of fibroblasts, myofibroblasts and immune cells. We identified the emergence of a metformin responsive subpopulation of macrophages, unique to the aged mice treated with metformin. Our results demonstrate that metformin can modulate specific populations of immune cells and fibroblasts to prevent age-associated ovarian fibrosis and offers a new strategy to prevent ovarian fibrosis Overall design: In this study, we sought to determine if metformin can prevent and/or reverse the formation of ovarian fibrosis. Since ovarian fibrosis is substantially increased between 15 and 18 months of age in mice, we designed our prevention study by treating adult female mice C57BL/6 beginning at the age 14 months (pre-fibrosis development; 5/group) with 350mg/kg/day of metformin (Sigma) in their drinking water for 6 months (no drug as control). We also determined if existing fibrosis can be reversed by using naturally aged mice at 18 months of age, when collagen deposition is high. We treated the aged mice with the same metformin treatments in the drinking water for 6 months as a fibrosis reversal study. We selected this drug concentration in the drinking water as it achieves plasma concentrations similar to the doses taken by T2D patients (i.e. 5-10µM metformin) (76). At the end of the 6-month period, experimental mice were euthanized in static microisolators by CO2 asphyxiation delivered at a flow of 20-30% chamber volume per minute followed by cervical dislocation. We also euthanized two more experimental conditions based on age with no treatments, defined as young (n=5; 3 months old) and middle-aged (n=5; 14 months old). All mice were dissected to collect the ovaries. The left ovary of the first three mice of each group was used for single cell RNA sequencing while the right ovary of the same mice was collected for histologic analysis.