(27) Combinatory effects of unilateral oophorectomy plus cyclophosphamide treatment on ovarian reserve and fertility in a mouse model

Background: Ovarian tissue cryopreservation via unilateral oophorectomy (ooph) prior to receiving gonadotoxic treatment is the gold standard for fertility preservation in pre-pubertal girls. The deleterious effects of cyclophosphamide (Cy) on ovarian follicles have been well studied, but it is not known how unilateral ooph in combination with Cy may influence ovarian function and risk of premature ovarian insufficiency. The aim of this study was to investigate the combined effects of unilateral ooph + Cy on AMH and ovarian follicles using a mouse model.

Methods: 4–5 week-old B6 albino female mice underwent unilateral ooph or sham surgery and were treated with 75 mg/kg Cy or 0.9% saline via single intraperitoneal injection 10-11 days after surgery. Four groups were compared: 1) sham+saline, 2) sham+Cy, 3) ooph+saline, 4) ooph+Cy. Mice were sacrificed at 10 weeks old, approximately 30 days after injection, and ovary(ies) and blood were collected for histological analysis and AMH levels, respectively. Histologically ovarian follicles were counted, classified, and averaged in evenly spaced sections throughout the ovary.

Results: AMH levels and follicles were analyzed for 15 mice. AMH levels were lowest in ooph+Cy as compared to sham+saline, although there were no significant differences in AMH between groups at time of sacrifice (1a). While there were increased numbers of PMFs and PMF:growing follicle ratio in the sham+saline group compared to treatment groups, it was not statistically significant (1b-1d). At both timepoints, there were no significant differences in the number of pups per litter between groups (1e, 1f). TUNEL staining revealed higher intensity in the Cy groups as compared to the saline groups.

Conclusions: These experiments have demonstrated the feasibility of a mouse model to evaluate the effect of oophorectomy in addition to cyclophosphamide on ovarian histology and ovarian reserve markers. Repeated experiments at higher Cy doses (150 mg/kg, 250 mg/kg) are currently in progress.