Fig. 1. Schematic of the ovarian fibrosis prevention and reversal experimental designs.

Fourteen- and 18-month-old mice were administrated metformin in drinking water (no metformin as control) for 6 months in prevention and reverse experimental settings, respectively. At the end point, 3- and 14-month-old control mice were also euthanized with the experimental mice to collect the ovaries for histology. The left ovary of the first three mice was used for single-cell analysis; single cells were flow sorted for viability and CD45 positivity and remixed at a ratio of 1:1 of immune (CD45⁺) and nonimmune (CD45⁻) cells for immune cell enrichment before multiplex barcoding and single-cell sequencing. Right ovaries were collected, formalin fixed, and paraffin embedded for second-harmonic generation (SHG) microscopy and with PSR staining assessed under polarized light microscopy. Together, histologic and transcriptomic data were used to analyze the effects of metformin on aged-associated ovarian fibrosis.