Protective effect of metformin on the ovarian reserve of prepubertal mice under cyclophosphamide chemotherapy

Abstract Study question Can metformin reduce the side effects of cyclophosphamide on the ovaries of prepubertal mice treated with cyclophosphamide? Summary answer Yes, metformin, as an inhibitor of mTOR and, by affecting the HIPPO pathway, can exert its protective effect in the chemotherapy mice model. What is known already PI3K/AKT and HIPPO pathways are influential pathways in the process of activation of primordial follicles and proliferation of cell growth, which are affected by various agents and drugs such as cyclophosphamide. One of the side effects of cyclophosphamide is the reduction of the ovarian reserve through abnormal changes in the PI3K/AKT and HIPPO pathways. Metformin is a drug that is used to treat type 2 diabetes and at the same time regulates PI3K/AKT and HIPPO pathways and prohibits the proliferation of cancer cells. Study design, size, duration Controlled experimental study. After metformin and cyclophosphamide dose adjustment, 12 female NMRI 14-day-old mice were randomly divided into four groups: the control group (CONT), metformin group (MET), cyclophosphamide group (CYC) and metformin with cyclophosphamide group (MET-CYC). Participants/materials, setting, methods Mice were treated by intraperitoneal injection of a dose of 150mg/kg of metformin for 11 consecutive days, and/or a dose of 65mg/kg of cyclophosphamide every three days (The third, sixth and ninth days). The mice were sacrificed 24 hours after the last metformin injection or 3 days after the last cyclophosphamide injection. The ovaries were collected for stereological evaluation, and expression of Pten, Mtor, Yap-1, p53, Bcl-2, and Bax genes by qRT-PCR method. Main results and the role of chance The stereological evaluation showed that the ovarian volume and primordial follicle number in the CYC group were decreased compared to the other groups (P < 0.05), and also in the MET-CYC group reduced compared to the CONT and MET groups, but increased significantly compared to the CYC group (P < 0.05). A significantly increased number of growing follicles was observed in the CYC group compared to the other groups (P < 0.05). The molecular analysis showed that the Pten expression in the CYC group was lower than CONT and MET groups (P < 0.05). Mtor expression in the CYC group was significantly increased compared to other groups (P < 0.05). The expression of Yap-1 was significantly decreased in the MET group compared to other groups (P < 0.05), and was lower in the MET-CYC than in the CYC group (P < 0.05). The of P53 expression in the MET-CYC group was higher than CONT and MET groups (P < 0.05), and was increased in the CYC compared to the CONT group (P < 0.05). Bcl-2 expression was higher in CONT and MET groups than in MET-CYC and CYC groups (P < 0.05). The expression of Bax in the CYC group was significantly increased compared to CONT and MET groups (P < 0.05). Limitations, reasons for caution Due to ethical issues in less use of mice, dose of metformin was obtained according to the articles used in adult mice and also by using the human to animal dose conversion formula. Wider implications of the findings It can be concluded that metformin during cyclophosphamide treatment can maintains ovarian volume, reserve, and activity in prepubertal mice by regulating PI3K/AKT and HIPPO pathways. Considering the relatively acceptable cost and long-term safety of metformin, it is a promising factor to be used to preserve fertility during chemotherapy. Trial registration number not applicable