Received December 01, 2025
Accepted December 05, 2025

Objective: This study investigated whether repeated cycles of controlled ovarian hyperstimulation (COH) influence the gut microbiota composition in female mice. Methods: Eight-week-old female Swiss mice (n = 20; 10 per group) underwent ten weekly COH treatments with exogenous gonadotropins or received saline injections as controls. One week after the final treatment, fecal samples were collected for gut microbiota profiling using 16S rRNA gene sequencing (V4 region amplification; primers 515F/806R) on the Illumina MiSeq platform. Sequencing data were processed in QIIME2 with DADA2 for denoising and Greengenes-based taxonomic assignment. Alpha diversity (Shannon, Simpson, observed OTUs, Faith’s PD) and beta diversity (Bray Curtis, Jaccard, unweighted/weighted UniFrac) metrics were calculated. Differential taxonomic abundance and biomarker analyses were performed using Random Forest and LEfSe. Statistical significance was set at p < 0.05. Results: No significant differences were observed between COH and control groups for alpha diversity indices (all p > 0.05) or beta diversity distances (PERMANOVA p > 0.05). Relative abundances across major taxonomic levels were similar, although the Firmicutes:Bacteroidetes ratio was lower in COH treated mice. Biomarker analyses identified Bacteroidales, Erysipelotrichaceae, and Parabacteroides as enriched in the COH group, while Rikenellaceae and Odoribacter were more abundant in controls. Conclusion: Repeated COH did not significantly alter overall gut microbial diversity in mice under the conditions tested. However, specific taxonomic shifts and biomarker signatures were detected, indicating associations—not causality—between COH exposure and changes in microbiota composition. Further studies are needed to determine the functional and physiological implications of these associations.