Abstract P4-10-16: Increased chemotherapy-induced ovarian reserve loss and impaired oocyte quality in a mouse ATM knock-down model: An augury for women with ATM-pathway-pathogenic-variants-associated breast cancer?

Abstract

Abstract Rationale: Breast cancer chemotherapy commonly causes the depletion of ovarian primordial follicle reserve, which results in premature ovarian insufficiency and infertility. It is critical to understand the mechanism of this ovarian damage to be able to develop pharmacological fertility preservation methods. We have previously shown that the main mechanism of ovarian follicle loss induced by cyclophosphamide (C) and doxorubicin (A) is via the induction of double-strand DNA breaks (DSBs) and the resulting apoptotic death of primordial follicles. We have also shown that some oocytes may repair this damage by activating the ATM-mediated DNA DSB repair pathway (ATM-Pathway). We have also demonstrated that the oocytes of women with BRCA1 pathogenic variants are more prone to DNA damage and such women are more likely to lose ovarian reserve after AC-based chemotherapy. BRCA1 is a key member of the ATM-Pathway. An increasing number of women are diagnosed with breast cancer associated with mutations of the other members of the ATM-Pathway. This includes ATM, which is the orchestrator of the ATM-Pathway. Therefore, we hypothesized that ATM-deficient oocytes may be more prone to chemotherapy-induced damage. In addition, given our recent findings of the role of declining BRCA1 function in oocyte aging, we hypothesized that ATM function is important in oocyte and embryo quality. Having both increased liability to chemotherapy-induced loss and accelerated ovarian aging can be a double-whammy for women with breast cancer that are associated with the pathogenic variants of the ATM-Pathway genes. Methods: Germinal Vesicle (GV) stage oocytes were collected from 6-wk old mice 48h after ovarian stimulation. Oocytes were then microinjected with 50uM ATM small interfering RNA (siRNA). The scrambled-siRNA-injected and un-injected oocytes served as negative controls. In separate experiments, effective ATM-knockdown (~70%) was confirmed by single-cell real-time-qPCR. Then, oocytes were subjected to subsequent in vitro maturation followed by parthenogenesis or 4h exposure to the active metabolite of cyclophosphamide, 4-hydroperoxycyclophosphamide (4-HC, 20µg/mL). Experiments were repeated ≥ thrice. One-way ANOVA and non-parametric Kruskal-Wallis posthoc tests were used for statistical analysis.Results: ATM-knockdown resulted in a significant reduction of oocyte maturation compared with scrambled or uninjected (29%, n=157 vs. 78%, n=172 vs. 84%, n=151) (p<0.001 siATM vs. the controls), suggesting altered oocyte quality. The siATM parthenotes had lower cleavage and blastocyst rates (15% and 0%, n=20,) compared with scrambled (63% and 18%, n=22), and uninjected controls (66% and 24%, n=21) (p<0.05 compared to either control), and demonstrated impaired morphology. Moreover, ATM-knock-down resulted in increased oocyte sensitivity to chemotherapy; with significantly lower survival rates (15%, n=37) compared to scrambled (55%, n=48) or un-injected controls (57% n=48; p<0.001) after the 4-HC exposure. Conclusions: This mouse model indicates that intact ATM function is critical in both oocyte resistance against chemotherapy and the maintenance of egg and embryo quality. Clinical studies are justified to determine if women with ATM pathogenic variants have impaired egg and embryo quality and are more prone to chemotherapy-induced loss of ovarian reserve and fertility. Citation Format: Kutluk H Oktay, Xiu Tan, Katarzyna Szymanska-Vandendriessche. Increased chemotherapy-induced ovarian reserve loss and impaired oocyte quality in a mouse ATM knock-down model: An augury for women with ATM-pathway-pathogenic-variants-associated breast cancer? [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-10-16.