NEONATAL OVARIAN RESERVE FOLLOWING CHEMOTHERAPY EXPOSURE, EX VIVO MURINE MODEL

Abstract

To determine the impact of exposure to maternal serum chemotherapy on formation of the ovarian reserve. Breast cancer complicates 1:3000 pregnancies, and current safety data is limited to congenital anomalies and early childhood outcomes. Chemotherapy is known to have significant germ cell toxicity, and yet little has been done to understand the transgenerational effect of in utero exposure. Murine ovarian reserve establishment mimics the human fetus but with a significant time shift to postnatal development. A randomized ex-vivo animal study with 100 postnatal day 0 C57BL/6 mouse ovaries was performed under IACUC approval. After sacrifice, sister ovaries were randomized to control (drug carrier alone) or drug exposure. Planned exposures were derived from widely used chemotherapy treatment regimens for maternal malignancy: doxorubicin, cyclophosphamide, paclitaxel, docetaxel, and cisplatin. Ovaries were cultured in hanging well organ culture media with addition of exposure on culture day 2. Drug dosing replicated known Cmax concentration. A mid dose of half the Cmax was also used to gain greater understanding of dynamics. A single drug exposure was utilized, and planned analysis occurred at 48 hours and 5 days. Immunofluorescence with TRA98 and VASA was used to quantify oocyte number and density. Data was then transferred to Graphpad Prism to generate descriptive statistics and apply a two-way ANOVA for each condition. Under control conditions, oocytes matured in culture with a moderate fall in density as oocyte size increased. Exposure to doxorubicin results in a loss of 95.2% of the oocyte density seen in the control at 7 days. At 7 days control ovaries have an oocyte density 570.3 oocytes/mm2, but Cmax exposure of doxorubicin results in a decrease to 27.2 oocytes/mm2. A two-way ANOVA confirms the significance of this relationship (p<0.0001). Exposure to cyclophosphamide resulted in less of a decrement. At 7 days the oocyte density decreased by 50.5% compared to the control to 282.2 oocytes/ mm2. Two-way ANOVA again confirmed significance (p=0.0004). Cisplatin, docetaxel and paclitaxel all demonstrated unique phenotypical changes on the ovaries and their oocytes, but none resulted in a significant decrease in oocyte density. The unique exposure of in utero chemotherapy may result in profound premature ovarian insufficiency. Our ex-vivo murine model suggests that maternal chemotherapy choices might be made to craft a less harmful chemotherapy regimen to ensure fetal health. By avoiding doxorubicin and cyclophosphamide, the delicate establishment of ovarian reserve in the fetus can continue even as the mother pursues life-saving chemotherapy treatment. Further work in vivo and in follow-up of human cohorts is needed to confirm these findings.