ABSTRACT: Cyclophosphamide (CP) could cause severe gonadotoxicity via imbalanced activation of primordial follicles through PI3K/ AKT/mTOR activation. Whether metformin, a widely prescribed anti-diabetes agent with mTOR inhibitory effect, could preserve ovarian
function against CP toxicity is unknown. Female C57BL/6 mice were randomized into seven groups (n¼11), including control, CP-alone, CPþ metformin, CPþsirolimus or everolimus, metformin-alone and sirolimus-alone groups. The duration of pharmaceutical treatment
was 4 weeks. CP treatment significantly impaired ovarian function and fertility in mice. CPþ metformin treatment significantly attenuated the gonadotoxicity comparing to CP-alone treatment (primordial follicle count: 17.664.2 versus 10.362.7 follicles/high-power field;
P¼0.027). CPþ metformin treatment also tended to increase antral follicular count (5.461.1 versus 2.561.6 follicles/section), serumAMH levels (4.661.2 versus 2.060.8 ng/ml) and the litter size (4.261.3 versus 1.561.0 mice per pregnancy), compared withCP-alone group. Expression of phospho-mTOR and the number of TUNEL-positive granulosa cells increased after CP treatment anddecreased in the CPþ metformin groups, suggesting the mTOR inhibitory and anti-apoptotic effects of metformin. In in-vitro granulosa cell experiments, the anti-apoptotic effect of metformin was blocked after inhibiting p53 or p21 function, and the expression of p53 mRNA
was blocked with AMPK inhibitor, suggesting that the anti-apoptotic effect was AMPK/p53/p21-mediated. In conclusion, concurrent metformin treatment during CP therapy could significantly preserve ovarian function and fertility and could be a promising novel fertility preserving agent during chemotherapy. The relatively acceptable cost and well-established long-term safety profiles of this old drug mightprompt its further clinical application at a faster pace.