A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve

Abstract

Many chemotherapeutic agents, especially of the alkylating family, alter fertility in premenopausal females. However, it is not practically possible to quantify and characterize the impact of cancer drugs on ovarian reserve in a clinical setting. Thus, our specific aim was to develop a xenograft model to characterize the in vivo impact of chemotherapy agents on human ovary. Ovarian pieces from 24 weeks old abortuses were xenografted s.c. to severe combined immunodeficient mice (n = 52). Animals received either a single dose of 200 mg/kg of cyclophosphamide or the vehicle. Grafts were recovered from the control and treated mice 12 to 72 h after the cyclophosphamide injection and serially sectioned for primordial follicle counts. Apoptosis was assessed with terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Platelet/endothelial cell adhesion molecule 1 (PECAM-1) staining, as well as intra-vital fluorescein-conjugated lectin and Evans blue labeling were done to assess microvasculature by confocal microscopy. Although there was 12% reduction in primordial follicle density by 12 h following treatment (P > 0.05), the follicle loss increased significantly at 24 h (53%, P < 0.01) and peaked at 48 h (93%, P < 0.0001). TUNEL staining peaked at 12 h, earlier than the diminishment in follicle numbers, and decreased thereafter. Xenograft vascularization pattern was similar to non-xenografted tissue, indicating appropriate in vivo drug delivery. The impact of cyclophosphamide on primordial follicle reserve in our human ovarian xenograft model is consistent with the clinical gonadotoxicity of this drug. Human ovarian xenografting is a promising model to characterize the gonadotoxic effects of current and emerging cancer drugs without a need for lengthy clinical studies.