FRI379 The Gonadotoxic Effects Of Cyclophosphamide And Busulfan On Ovaries In CD-1 Mouse Model

Abstract

Abstract Disclosure: M. Thonen: None. H. Jiang: None. Y. Luan: None. R. Dong: None. A. Wadhwa: None. S. Kim: None. Hematopoietic stem cell transplantation (HSCT) involves the transfer of healthy donor stem cells to replace a recipient's dysfunctional bone marrow. It can be used for both malignant diseases (eg lymphoma or leukemia) and non-malignant diseases (eg sickle cell anemia). Chemotherapy and/or radiation are usually required to deplete the recipient's bone marrow before transplant, also known as conditioning regimens. Alkylating agents including cyclophosphamide (CY) and busulfan (BU) are frequently involved in these regimens. Primary ovarian insufficiency in young female patients secondary to conditioning regimens presents a challenging fertility complication. Studies show that there may be unbalanced ovarian hormonal levels and decreased ovarian function in young female cancer survivors, however, limited studies have explained the mechanism of medication-induced ovarian toxicity in an animal model. This study aimed to investigate the gonadotoxic effects of CY and BU in CD-1 strain mice and to delineate the underlying mechanism of the gonadotoxic outcomes, eventually suggesting possible fertoprotectants for HSCT recipients’ fertility preservation.Sixteen one-month-old female CD-1 mice (n=4/group) were subcutaneously injected with solvent, BU, CY, or BU+CY, respectively, over the course of six days. The doses (144 mg/Kg BU and 300 mg/Kg CY) and injection plan were adjusted to clinical doses. One week after the last administration, the ovaries and female reproductive tracts were harvested. The samples were embedded in paraffin, then continuously sectioned into 5 μm thickness. The specimen was stained with hematoxylin and eosin (H&E) and TUNEL assays. Different stages of follicles were counted individually by two team members. The serum levels of anti-Mullerian hormone (AMH), 17-β-estradiol, and follicle-stimulating hormone (FSH) were measured. Additional immunofluorescence staining for AMH, inhibin α, and Ki-67 was also performed. All mice survived treatment and appeared grossly normal. The average ovary weight was significantly lower in BU-based treatment groups compared to control and CY alone groups. The primordial (p<0.0001) and primary follicles (p=0.01) in BU, CY, and BU+CY groups were lower than the control group. The average AMH level in the BU-based treatment groups was also significantly lower than in the control and CY-alone group. Our results indicate CY and BU regimens individually were gonadotoxic, reducing the ovarian reserve in CD-1 mice with a synergistic toxic effect when used in combination. Decreased AMH reveals the conditioning regimens are toxic to granulosa cells and the ovarian reserve. Future studies are needed to elaborate possible pathways of chemotherapy-induced ovarian follicle death and the toxicities of other regimens to improve fertility outcomes and better care for female patients receiving HSCT. Presentation: Friday, June 16, 2023