Research Paper Advance Articles
Establishment of an optimized chemotherapy-induced mouse model for premature ovarian failure: protocol and findings
- 1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- 2 Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
- 3 Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran
- 4 Department of Anatomical Sciences, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
- 5 Department of Infertility, Yas Hospital, Tehran University of Medical Sciences, Tehran, Iran
Received: April 24, 2025 Accepted: September 30, 2025 Published: October 28, 2025
https://doi.org/10.18632/aging.206332How to Cite
Copyright: © 2025 Yavari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Objective: The aim of this study was to induce a practical premature ovarian failure (POF) mouse model using Cyclophosphamide (CTX) and Busulfan (Bu), considering both drug exposure duration and natural recovery time at the optimal dose.
Methods: Female NMRI mice (6-8 weeks) received single intraperitoneal injections of four CTX/Bu dose regimens. Controls were injected with a single dose of equal volume of saline (n=3/group). To evaluate natural ovarian recovery, treated mice were left without intervention for 3 and 4 weeks after the chemotherapeutic combination administration. In addition, follicle counting (in all groups) and hormonal analyses (in the optimal group) were performed to validate the recovery and model.
Results: Among all doses, the CTX 100 mg/kg + Bu 20 mg/kg regimen reliably induced POF within 3 weeks post-administration, as demonstrated by three key criteria: (1) persistent follicular decline in ovarian reserve (2) endocrine disruption (significantly elevated FSH and suppressed AMH/E2 levels and (3) sustained ovarian dysfunction throughout the 3-week post-induction observation period (until week 6 post-injection). No spontaneous ovarian recovery was observed during the 3-week post-induction period. Notably, the treatment protocol showed excellent safety profiles so that no mortality was observed compared with controls.
Conclusions: These results suggest that the single dose IP injection of the CTX 100 mg/kg + Bu 20 mg/kg can effectively induce POF within 3 weeks post-administration and POF model maintains for at least 3 weeks after induction.
Abbreviations
POF: Premature Ovarian Failure; Bu: Busulfan; CTX: Cyclophosphamide; bw: Body weight; POI: Premature Ovarian Insufficiency; AMH: Anti-Müllerian Hormone; E2: 17β-Estradiol; FSH: Follicle-Stimulating Hormone; ELISA: Enzyme-Linked Immunosorbent Assay; H\&E: Hematoxylin and Eosin; IACUC: Institutional Animal Care and Use Committee; IP: Intraperitoneal (injection); NMRI: Naval Medical Research Institute (mouse strain); PBS: Phosphate-Buffered Saline; PFA: Paraformaldehyde; SD: Standard Deviation; ANOVA: Analysis of Variance.