Bacterial vaginosis toxins impair sperm capacitation and fertilization.

Abstract

Study question: What effect do toxins produced by bacterial vaginosis (BV) bacteria have on sperm function?

Summary answer: BV toxins dysregulate sperm capacitation and intracellular calcium homeostasis, and impair the ability of sperm to fertilize oocytes.

What is known already: In BV, 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, duration: This 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, methods: Motile 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 IVF 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 concentration was measured by using the calcium-sensitive dye Fluo-4 AM and fluorescence microscopy. The effects of LPS on sperm from CatSper knockout mice were assessed. Additionally, sperm were treated with a Toll-like receptor 4 (TLR4) antagonist and further exposed to LPS.

Main results and the role of chance: Exposure of mouse sperm to LPS or VLY significantly decreased IVF (P < 0.05). Under capacitating conditions, both toxins initially increased mouse (P < 0.001) and human (P < 0.05) 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 sperm intracellular calcium concentration. Effects of LPS, but not VLY, were prevented by polymyxin B, which binds LPS. The LPS-induced intracellular calcium increase required external calcium, but not the calcium channel CatSper, and was inhibited by a TLR4 antagonist.

Large scale data: N/A.

Limitations, reasons for caution: First, 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 findings: These studies suggest that BV-associated toxins contribute to infertility, in part, by impairing sperm capacitation and reducing their fertilizing ability.

Study funding/competing interest(s): This work was supported by the National Institutes of Health (grant number R01 HD069631 to C.M.S.). The authors declare that they have no conflict of interest.