# Establishment of an optimized chemotherapy-induced mouse model for premature ovarian failure: protocol and findings

## TL;DR

This study establishes a reliable mouse model for premature ovarian failure using chemotherapy drugs Cyclophosphamide and Busulfan.

## Contribution

The study introduces an optimized single-dose regimen of Cyclophosphamide and Busulfan to induce premature ovarian failure in mice.

## Key findings

- The CTX 100 mg/kg + Bu 20 mg/kg regimen reliably induced POF within 3 weeks.
- No spontaneous ovarian recovery was observed during the 3-week post-induction period.
- The treatment protocol showed excellent safety with no mortality observed.

## 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.