-derived Signature Protein(s) in Murine Vaginal Lavage Fluid: Investigating the Underlying factors of Infertility as a Consequence of Sperm Impairing Bacterium.

BACKGROUND

Bacterial infections are often an overlooked factor in female infertility. has been identified as the predominant vaginal pathogen in infertile women, with a prevalence of 57.33%. Previous studies showed that induces infertility in mice by sperm impairment, suggesting its asymptomatic vaginal colonization creates a hostile environment for sperm. While sperm immobilization factor (SIF) from culture supernatant has been identified, its production within host's environment remained unexplored.

OBJECTIVE

To unveil -derived signature protein(s) in vaginal lavage fluid (VLf).

DESIGN

Mass spectrometry combined with experimental studies.

METHODS

VLf was obtained from female mice administered either with sperm immobilizing (test group) or PBS alone (control group) and analyzed using nano-LC-MS/MS, gel filtration chromatography, SDS-PAGE, functional assays, and in silico studies.

RESULTS

Nano-LC-MS/MS yielded 5 distinct bacterial proteins in test group and no bacterial protein in control. Elution profile of test VLf revealed a single peak and indicated 1 protein band (~36 kDa) using SDS-PAGE that aligned with GMP reductase. VLf-protein showed impairment of sperm motility and viability in concentration-dependent manner and disrupted sperm morphology. Binding studies using FITC-labeled VLf-protein depicted presence of green fluorescence over entire surface of mouse spermatozoa. These results were akin to SIF, already isolated and characterized in our laboratory, from culture supernatant of , causing sperm impairment and hence, designated as vaginal lavage fluid-derived sperm immobilization factor (VLf-SIF). Through in silico analysis, superimposition of VLf-SIF and SIF, already known to show sequence homology to cysteine-tRNA ligase, revealed close structural alignment. Molecular docking analysis depicted energetically favorable binding between VLf-SIF and spermatozoa surface protein (Heat shock-related 70 kDa protein 2).

CONCLUSION

This study provides novel evidence of sperm-impairing signature proteins as key mediators of bacterial-induced infertility, paving way for diagnostic, and therapeutic advancements.