Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization

Abstract

Study questionWhat effect do toxins produced by bacterial vaginosis (BV) bacteria have on sperm function? Summary answerBacterial vaginosis toxins dysregulate sperm capacitation and intracellular calcium homeostasis and impair the ability of sperm to fertilize oocytes. What is known alreadyIn bacterial vaginosis, which is linked to infertility, overgrowth of Prevotella and Gardnerella in the vagina is accompanied by elevated concentrations of the toxins lipopolysaccharide (LPS) and vaginolysin (VLY). Study design, size, durationThis was a laboratory study in which human semen samples were collected from consenting healthy donors with normal semen parameters. Mouse sperm samples were obtained from the caudal epididymis. Participants/materials, setting, methodsMotile mouse and human sperm were isolated via swim-up and treated under non-capacitating or capacitating conditions. LPS from Escherichia coli was commercially available. VLY was produced by cloning the Gardnerella VLY protein in the ClearColi expression system. Mouse sperm were pre-incubated in in vitro fertilization medium with LPS or VLY and then co-cultured with ovulated cumulus-oocyte complexes. The effects of LPS and VLY on sperm motility and hyperactivation were assessed with computer-assisted sperm analysis. Effects on viability were assessed by Hoechst staining. Acrosomal exocytosis was assessed in sperm from transgenic Acr-eGFP mice and in human sperm stained with Pisum sativum agglutinin FITC. Intracellular calcium dynamics were assessed by staining sperm with the calcium-sensitive dye Fluo-4 AM and fluorescent imaging several sperm at the single-cell level. The effects of LPS on sperm from CatSper knock-out mice were assessed. Additionally, sperm were treated with a toll-like receptor 4 antagonist and further exposed to LPS. Main results and the role of chanceExposure of mouse sperm to LPS or VLY significantly decreased in vitro fertilization (P < 0.05). Under capacitating conditions, both toxins initially increased mouse and human sperm hyperactivation, then significantly decreased sperm motility (P < 0.05), hyperactivation (P < 0.05), and acrosomal exocytosis (P < 0.01). These changes were accompanied by a rapid and irreversible increase in intracellular calcium concentration. Effects of LPS, but not VLY, were prevented by polymyxin-B, which aggregates LPS. The LPS-induced intracellular calcium increase required external calcium but not the calcium channel CatSper and was inhibited by the Toll-like receptor 4 antagonist. Limitations, reasons for cautionFirst, the commercially available LPS we used was isolated from Escherichia coli, rather than from the BV-associated bacteria Prevotella bivia. Second, we did not quantify the absolute sperm intracellular calcium concentration before or after LPS or VLY treatment. Third, all of our experiments were in vitro. Wider implications of the findingsThese studies suggest that BV-associated toxins contribute to infertility by, in part, impairing sperm capacitation and reducing their fertilizing ability. Study funding/competing interest(s)This work was supported by the National Institutes of Health (grant #R01 HD069631). The authors declare that they have no conflict of interest.