The effect of “activated” cyclophosphamide on human and rat ovarian granulosa cells in vitro

Abstract

We investigated the mechanism of cyclophosphamide (CTX) induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on human and rat granulosa cell function in vitro. In previous experiments we demonstrated that in short-term incubations with rat granulosa cells, high (≥ 100 μg/mL) but not low (≤ 10 μg/mL) PCTX concentrations inhibited progesterone accumulation in vitro. In this study, human granulosa cells were obtained from patients undergoing follicle puncture for in vitro fertilization. PCTX at 10, 100, and 500 μg/mL, but not at 1 μg/mL, resulted in a dose-related reduction in progesterone accumulation in short-term human granulosa cell cultures. Rat granulosa cells were obtained from PMSG-primed immature rats and incubated for 24 h with PCTX concentrations of 1, 5, and 10 μg/mL. Ovine LH (10 ng/mL) was added in selected tubes. In comparison to control, progesterone accumulation was significantly reduced by PCTX concentration of 10 μg/mL. The above findings demonstrate that cyclophosphamide metabolites, at concentrations achievable in vivo during CTX therapy, decrease rat and human granulosa cell function in vitro. The effect of PCTX on granulosa cells is dependent on PCTX concentration and duration of exposure, as well as the species from which granulosa cells are obtained.