P–624 Doxorubicin exposure detrimentally effects establishment of early ovarian reserve, ex-vivo mouse model
Authors: Ozcan M , Woodman M , Chaqour J , Grive K 
Published: August 2021

Abstract

Study question
Does exposure to chemotherapy agents used to treat cancer during pregnancy cause significant decrease in early establishment of the ovarian reserve?

Summary answer
Significant oocyte loss is noted following exposure of early mouse ovaries to doxorubicin. This apoptotic decline is not seen with cisplatin, docetaxel or paclitaxel exposure.

What is known already
Chemotherapy has been shown to adversely affect the ovarian reserve of pre-pubertal and reproductive age females. However, little is known regarding the effects in the next generation following treatment in pregnancy beyond observational population studies that examined rates of birth defects and developmental delays. Reproductive function in offspring develops over time and would be difficult to quantify in these short-term studies. An ex-vivo mouse ovary culture offers the ability to closely mimic maternal treatment level serum concentrations and directly evaluate the impact on oocyte number and cell death markers.

Study design, size, duration
In mice, the ovarian reserve is matured postnatally, mimicking the biologic second trimester activity of the human ovary. Wild-type C57bl/6 pups were collected on postnatal day 0. Ovaries were cultured in hanging well organ culture media with addition of DMSO or a chemotherapy agent. Immunofluorescence was used to quantify oocyte number and density. Power calculation showed a N of 11 per drug would be needed to demonstrate a decrease of ⅔ or more. Participants/materials, setting, methods: 83 ovaries were cultured, sectioned and analyzed in duplicate. Planned analysis at serum max and mid concentrations was performed at 48 hours and 5 days. Given clinically variation in concentrations of cisplatin, additional concentration samples were added. After noting the degradation following exposure to doxorubicin, additional samples at earlier time points of 12 and 24 hours of exposure were added for dynamic evaluation. Means were calculated and then compared using a 2-way ANOVA.

Main results and the role of chance
Doxorubicin exposure during establishment of the ovarian reserve resulted in a significant loss of oocyte number and density. At 12 hours a 22% decrease was noted; this increased to a loss of 91% of oocytes by 48 hours of exposure. The oocyte density fell from 693 oocytes/ mm² in the control to only 63 oocytes/ mm² in the serum max concentration (p = 0.003). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed early impact within the stroma by 12 hours with widespread apoptotic changes by 24 hours.
Treatment with cisplatin resulted in a phenotypic change in the oocyte population with preservation of smaller, more peripheral cells. The average oocyte density remained similar to control even at the highest clinical concentrations, 536 oocytes/ mm² compared to 570 oocytes/ mm² (p = 0.772). Docetaxel and paclitaxel demonstrated an increase in oocyte number and density, though not enough to reach significance. The oocyte density 5 days following docetaxel exposure was 802 oocytes/ mm², a 41% increase (p = 0.12). The oocyte density 5 days following paclitaxel exposure was 780 oocytes/ mm², a 37% increase (p = 0.817). The drug exposure did impact stromal cells, as noted in TUNEL images.

Limitations, reasons for caution
This ex-vivo mouse model offers tight control of chemotherapy concentration, it does not account for filtration and modification by the placenta. Longer term cultures may also demonstrate that temporary arrest in small oocytes, such as those exposed to cisplatin, do not thrive and later progress to apoptosis.
Wider implications of the findings: Doxorubicin is employed as the most common chemotherapy during pregnancy. Utilization may be to the serious detriment of the younger generation. If other alternatives are clinically effective they should be considered. No other models have explored the effect of fetal ovary exposure to chemotherapy on the establishment of ovarian reserve.

Trial registration number
Not applicable