Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro

Abstract How chemotherapy affects dormant ovarian primordial follicles is unclear. The ‘burnout’ theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. This study assed how the cyclophosphamide metabolites, (4-hydroxycyclophosphamide [4-OHC] and phosphoramide mustard [PM]), affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24−48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17β-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured ( P = 0.04 to P < 0.0005) and uncultured controls ( P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twice the levels of AMH and 17β-oestradiol in treated compared with untreated samples (AMH with 4-OHC 3 µmol/l; P = 0.04). All follicles stained positively for AMH included treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro . Study findings support the ‘burnout’ theory as the mechanism of chemotherapy-induced ovarian toxicity.