Gonadotoxicity of targeted therapies in young breast cancer patients and impact of carrying a germline BRCA mutation

Abstract Background: Breast cancer is the most common malignancy arising in women of reproductive age and its treatment often results in long-term sequelae and impaired quality of life (QoL). Among the possible negative consequences of anticancer therapies, treatment-induced gonadotoxicity leading to premature ovarian insufficiency (POI) represents one of the major sources of distress in this patient population. POI has a negative impact on global health and QoL of young breast cancer survivors being associated with several side effects including infertility. Carrying a germline deleterious BRCA pathogenic/likely pathogenic variant (PV) can add additional burden on this regard. Aim: The overall objective of this doctoral project was to evaluate the gonadotoxic potential of the targeted therapies Olaparib and Abemaciclib using a translational experimental approach based on human ovarian cortical tissue. Due to the limited availability of non- BRCA ovarian samples, the experimental phase was completed for Olaparib only, focusing on BRCA1/2- mutated ovarian tissues. Consequently, the lack of a non-BRCA control group prevented the evaluation of the second aim of the project namely, to determine whether the treatment exerts an impact on the ovarian reserve. Methods: Ovarian cortical fragments obtained from BRCA1/2 mutation carriers undergoing risk- reducing salpingo-oophorectomy were cultured for three days under four experimental conditions: control, Olaparib alone, standard chemotherapy (carboplatin and paclitaxel), and Olaparib combined with chemotherapy. Histological (H&E, Masson’s trichrome), immunohistochemical, and immunofluorescence analyses were performed to assess follicular morphology, collagen deposition, apoptosis, DNA damage, and activation markers, including γH2AX, TUNEL, cleaved Caspase-3, and Yap-1. In parallel, cytokine release in culture media was evaluated at 24, 48, and 72 hours using a multiplex immunoassay to explore treatment-related inflammatory and angiogenic responses. 5 Results: Exposure to Olaparib alone did not induce significant alterations in follicular health status, morphology, or stromal integrity compared with controls. Apoptotic and DNA damage markers remained at baseline levels, suggesting minimal direct ovarian toxicity. As expected, chemotherapy-treated tissues displayed clear signs of follicular atresia and increased apoptosis. The combination of Olaparib with chemotherapy resulted in a modest increase in apoptotic marker expression (γH2AX) compared with chemotherapy, indicating a potential additive effect on ovarian damage. Cytokine analyses revealed that control and Olaparib-treated tissues exhibited similar profiles with high levels of released cytokines, whereas chemotherapy and combined treatments consistently showed lower levels of the selected analytes. Conclusions: These findings suggest that Olaparib alone has no evident gonadotoxic effects in human ovarian tissue in vitro, whereas its combination with chemotherapy may enhance ovarian damage. Although preliminary, these results provide valuable insight into the reproductive safety of targeted therapies. Cytokine analyses revealed consistent differences between control/Olaparib and chemotherapy/combined treatment groups, reflecting respectively possible tissue stress and cytotoxic effects rather than physiological inflammation. While these data complement morphological and functional findings, further studies are necessary to validate these observations, clarify the biological significance of cytokine changes, and improve detection sensitivity. Additional research is also needed to elucidate the underlying molecular mechanisms and to extend the analysis to Abemaciclib to comprehensively assess the fertility impact of modern anticancer treatments in young women with breast cancer. Lastly, further investigations should clarify whether germline BRCA status influences reproductive toxicity.