Longitudinal strain-level metagenomics reveals reciprocal Lactobacillus dynamics and outcome-associated protein variants during IVF treatment

The vaginal microbiome influences reproductive outcomes, yet strain-level dynamics during assisted reproductive treatment remain unexplored. Existing studies rely on species-level resolution that cannot distinguish within-species functional heterogeneity, leaving mechanistic questions unanswered. We conducted longitudinal shotgun metagenomic sequencing of vaginal samples from 41 women undergoing IVF/ICSI treatment across six timepoints (pre-stimulation baseline through pregnancy testing; 186 samples total). Strain-level analysis using inStrain revealed that Lactobacillus crispatus genomic stability and Lactobacillus iners adaptive flexibility both associate with pregnancy success, representing a reciprocal dynamic pattern. Functional protein analysis identified two outcome-associated targets: Inerolysin, a cholesterol-dependent cytolysin from L. iners , and L-lactate dehydrogenase (L-LDH) from L. crispatus . Structural modeling suggested that the Success-specific Inerolysin S429P mutation is predicted to disrupt cholesterol-binding, potentially attenuating cytolytic activity, while the L-LDH P169L mutation may enhance catalytic efficiency. Four orthogonal computational methods (FoldX thermodynamic calculation, ESM-1v evolutionary scoring, ConSurf conservation analysis, and 10 ns molecular dynamics simulation) provided convergent computational evidence supporting these structural interpretations. Critically, these protective variants were enriched at baseline (T1) rather than arising during treatment, indicating that pre-existing strain genetics predispose to differential responses under hormonal stress. A baseline 18-SNV prediction panel achieved an internal cross-validation AUC of 0.945 and an external validation AUC of 0.784 in an independent cohort ( n = 69) sequenced on a multiplex amplicon PCR platform. These findings reveal strain-level functional variants as candidate mechanistic determinants of reproductive success and potential biomarkers for personalized risk assessment in ART. The reciprocal dynamics of L. crispatus stability and L. iners adaptive flexibility, together with the dual-protein mutation pattern, suggest targets for strain-specific probiotic interventions. All computational predictions require experimental validation through biochemical and functional assays. Video Abstract