Received February 24, 2026
Accepted March 10, 2026

Objective To evaluate whether vaginal–endometrial microbiome synchrony is associated with biochemical and clinical pregnancy outcomes in frozen embryo transfer (FET) cycles. Methods This prospective observational study included 100 women undergoing single Day 5 FET cycles. Vaginal and endometrial samples were collected during a mock FET cycle on progesterone day 5 and analyzed using a contamination-aware targeted 16S rRNA polymerase chain reaction (PCR) panel optimized for low-biomass reproductive tract samples. A Synchrony Index (SI), a similarity metric analogous to Jaccard-type indices, was calculated based on shared dominant microbial taxa between vaginal and endometrial compartments. An SI cutoff of ≥0.7 was pre-specified prior to outcome analysis based on biological plausibility and accepted similarity thresholds in microbiome research. Biochemical pregnancy (β-hCG positive) and clinical pregnancy rates were compared between high and low SI groups. A randomly selected subset underwent next-generation sequencing validation. Results Baseline demographic, ovarian reserve, embryo quality, and endometrial parameters were comparable between groups. Biochemical pregnancy rates were significantly higher in women with high SI compared with low SI (65.5% vs. 28.9%, p<0.001), as were clinical pregnancy rates (61.8% vs. 26.7%, p<0.001). On multivariable logistic regression adjusting for clinical confounders, SI remained independently associated with biochemical pregnancy (adjusted odds ratio 1.20 per 0.1 increase; 95% CI 1.06–1.36; p=0.004). PCR-derived SI demonstrated substantial agreement with sequencing-based validation. Conclusion Vaginal–endometrial microbiome synchrony is independently associated with biochemical pregnancy in Day 5 FET cycles. The PCR-based Synchrony Index warrants further evaluation as an investigational biomarker of reproductive tract receptivity in larger and independent cohorts.