Metformin-mediated amelioration of the ovarian microenvironment in autoimmune premature ovarian insufficiency

Premature ovarian insufficiency (POI), characterized by diminished ovarian function in women under 40, has immune factors as one of the significant etiological factors. Metformin, a frontline medication for type 2 diabetes and it may also modulate immune responses, suggesting potential immunomodulatory and anti-inflammatory properties. This study investigates the therapeutic potential of metformin in autoimmune POI. We established an autoimmune POI mouse model by immunizing with the peptide zona pellucida 3 (pZP3) and administered metformin via drinking water to assess its impact on ovarian and immune functions. Flow cytometry revealed an increase in the number of Treg cells in the POI group (pZP3), while the metformin-treated group showed a decrease close to the control group (CON) levels. Under transmission electron micros-copy, the pZP3 exhibited collapse and thinning of the zona pellucida, vacuolization of granulosa cells, and mitochondrial damage, with improvements observed in the metformin-treated group. In paraffin sections of mouse ovaries stained with Sirius Red, the pZP3 displayed a greater number of strong orange signals indicative of fibrosis, and in immunofluorescence, the macrophages in the pZP3 showed a clear polarization ab-normality in M1/M2, which was significantly reduced in the metformin-treated group. Our findings indicate that metformin significantly enhances ovarian function in pZP3-induced autoimmune POI mice and mitigates inflammation and fibrosis within the ovarian microenvironment. Transcriptome sequencing of mouse ovaries revealed 15,250 co-expressed transcription factors (TFs) across all groups. Comparative analysis identified 1098 differentially expressed genes (DEGs) between the CON and pZP3, with 403 upregulated and 695 downregulated genes. Notably, upregulated genes like S100a9 were associated with the IL-17 signaling pathway, while downregulated genes such as Cyp17a1 were linked to steroid metabolism pathways. These results indicate that metformin's therapeutic effects in autoimmune POI are mediated through pathways in-volving hormone biosynthesis, steroid hormone synthesis, and cytokine metabolism. Consequently, metformin's action on the ovarian microenvironment and hormone metabolism could improve both ovarian and immune state in pZP3-induced autoimmune POI mice, presenting a promising therapeutic strategy for POI sufferers.