Preparation and Evaluation of Mouse Premature Ovarian Insufficiency Model.

Premature ovarian insufficiency (POI) is a critical condition leading to female infertility, necessitating reliable animal models for mechanistic and therapeutic research. Here, we present a standardized protocol for establishing and evaluating a cyclophosphamide (CTX)-induced POI mouse model. Six-to-eight-week-old female mice with regular estrous cycles were selected and subjected to intraperitoneal CTX injections: an initial dose of 100 mg/kg on day 1, followed by daily doses of 20 mg/kg for the subsequent 14 days. Dynamic changes in estrous cycles were monitored via vaginal smear cytology with Wright staining. Serum levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) were quantified using ELISA to assess endocrine alterations. Ovarian histopathology was evaluated through hematoxylin-eosin (H&E) staining of paraffin-embedded sections to quantify follicular atresia, while immunohistochemical analysis of cleaved caspase-3 was performed to detect granulosa cell apoptosis. Results demonstrated disrupted estrous cyclicity, significantly reduced E2 and AMH levels, elevated FSH concentrations, increased follicular atresia, and enhanced granulosa cell apoptosis in CTX-treated mice, confirming successful POI modeling. This model-building method can highly mimic the mechanism of chemotherapy-induced ovarian damage, presenting typical pathological features such as follicle reserve depletion and sex hormone disorders. It provides a reliable experimental platform for revealing the reproductive toxicity mechanism of chemotherapy, screening ovarian-protecting drugs, and optimizing fertility preservation strategies. Moreover, this model is relatively simple to operate, low-cost, and has a short production cycle, making it easy to carry out and popularize. The methodology aligns with the requirements of JoVE for visualizable, step-by-step experimental demonstrations.