Abstract
PCOS is a prevalent endocrine disorder, and the leading cause of female infertility, affecting 4-20% of reproductive-age women worldwide, characterised by cystic ovaries, irregular menstrual cycles, and hyperandrogenism, with diagnosis requiring at least two of these symptoms according to the Rotterdam criteria. This study aimed to optimise a letrozole-induced mouse model of polycystic ovary syndrome (PCOS) capable of sustaining pathophysiological features for extended durations (>80 days) to evaluate spironolactone's therapeutic efficacy. Through three sequential experiments, we first compared subcutaneous letrozole implants (4.5mg vs. 8mg), which successfully induced persistent diestrus (***p < 0.001) and increased androgen receptor-positive cells in the arcuate nucleus (***p < 0.001) but failed to maintain PCOS-like symptoms beyond 40-50 days. Subsequently, we developed a novel oral administration method delivering letrozole via drinking water at low (0.04mg/kg/day) and high (1mg/kg/day) concentrations, which significantly improved phenotype sustainability throughout the experimental period. The high-dose regimen produced pronounced effects on reproductive parameters (persistent diestrus, *** p < 0.001) and metabolic outcomes (increased bodyweight **p<0.01, impaired insulin tolerance *p<0.05, elevated adiposity *p<0.05, and elevated testosterone concentrations **p<0.01). This model fulfilled all major diagnostic criteria for clinical PCOS. In our final experiment, we evaluated subcutaneous spironolactone implants (35mg/pellet, 23.33mg/kg/day) in this optimised model using the 1 mg/kg/day dose. Spironolactone initially reduced testosterone levels by 42.6% (**p<0.01) and 61.0% (**p<0.01) at first and second measurements, respectively, but this effect diminished by the final measurement. No significant improvements were observed in other reproductive or metabolic parameters. These findings represent a significant methodological advancement in PCOS research, establishing a robust model that recapitulates comprehensive PCOS pathophysiology for extended durations, while suggesting limitations in spironolactone's efficacy against severe hyperandrogenism induced by high-dose letrozole administration. Future research should explore modified dosing strategies to identify optimal therapeutic windows for anti-androgenic interventions in PCOS.