{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,17]],"date-time":"2026-03-17T13:25:05Z","timestamp":1773753905818,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,7]]},"group-title":"Physiology","reference-count":56,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T00:00:00Z","timestamp":1741305600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Study question<\/jats:title>\n                  <jats:p>What effect do toxins produced by bacterial vaginosis (BV) bacteria have on sperm function?<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Summary answer<\/jats:title>\n                  <jats:p>Bacterial vaginosis toxins dysregulate sperm capacitation and intracellular calcium homeostasis and impair the ability of sperm to fertilize oocytes.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>What is known already<\/jats:title>\n                  <jats:p>\n                    In bacterial vaginosis, which is linked to infertility, overgrowth of\n                    <jats:italic>Prevotella<\/jats:italic>\n                    and\n                    <jats:italic>Gardnerella<\/jats:italic>\n                    in the vagina is accompanied by elevated concentrations of the toxins lipopolysaccharide (LPS) and vaginolysin (VLY).\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Study design, size, duration<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Participants\/materials, setting, methods<\/jats:title>\n                  <jats:p>\n                    Motile mouse and human sperm were isolated via swim-up and treated under non-capacitating or capacitating conditions. LPS from\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    was commercially available. VLY was produced by cloning the\n                    <jats:italic>Gardnerella<\/jats:italic>\n                    VLY protein in the ClearColi expression system. Mouse sperm were pre-incubated in\n                    <jats:italic>in vitro<\/jats:italic>\n                    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\n                    <jats:italic>Pisum sativum<\/jats:italic>\n                    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.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Main results and the role of chance<\/jats:title>\n                  <jats:p>\n                    Exposure of mouse sperm to LPS or VLY significantly decreased\n                    <jats:italic>in vitro<\/jats:italic>\n                    fertilization (\n                    <jats:italic>P<\/jats:italic>\n                    &lt; 0.05). Under capacitating conditions, both toxins initially increased mouse and human sperm hyperactivation, then significantly decreased sperm motility (\n                    <jats:italic>P<\/jats:italic>\n                    &lt; 0.05), hyperactivation (\n                    <jats:italic>P<\/jats:italic>\n                    &lt; 0.05), and acrosomal exocytosis (\n                    <jats:italic>P<\/jats:italic>\n                    &lt; 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.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Limitations, reasons for caution<\/jats:title>\n                  <jats:p>\n                    First, the commercially available LPS we used was isolated from\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    , rather than from the BV-associated bacteria\n                    <jats:italic>Prevotella bivia<\/jats:italic>\n                    . Second, we did not quantify the absolute sperm intracellular calcium concentration before or after LPS or VLY treatment. Third, all of our experiments were\n                    <jats:italic>in vitro<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Wider implications of the findings<\/jats:title>\n                  <jats:p>These studies suggest that BV-associated toxins contribute to infertility by, in part, impairing sperm capacitation and reducing their fertilizing ability.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Study funding\/competing interest(s)<\/jats:title>\n                  <jats:p>This work was supported by the National Institutes of Health (grant #R01 HD069631). The authors declare that they have no conflict of interest.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.01.640991","type":"posted-content","created":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T13:35:20Z","timestamp":1741354520000},"source":"Crossref","is-referenced-by-count":3,"title":["Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2278-3264","authenticated-orcid":false,"given":"Shweta","family":"Bhagwat","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leila","family":"Asadi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronald","family":"McCarthy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan","family":"Ferreira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ping","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ethan","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sariela","family":"Spivak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ariana","family":"Gaydon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vaka","family":"Reddy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christy","family":"Armstrong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sydney R.","family":"Morrill","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hillary","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amanda L.","family":"Lewis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Warren G.","family":"Lewis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Celia M.","family":"Santi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025031102051111000_2025.03.01.640991v1.1","doi-asserted-by":"publisher","DOI":"10.1097\/01.AOG.0000247627.84791.91"},{"key":"2025031102051111000_2025.03.01.640991v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2008.08.002"},{"key":"2025031102051111000_2025.03.01.640991v1.3","first-page":"1","article-title":"Membrane Potential Assessment by Fluorimetry as a Predictor Tool of Human Sperm Fertilizing Capacity","volume":"7","year":"2020","journal-title":"Front Cell Dev Biol"},{"key":"2025031102051111000_2025.03.01.640991v1.4","first-page":"1","article-title":"Lactobacillus iners and gasseri, prevotella bivia and hpv belong to the microbiological signature negatively affecting human reproduction","volume":"9","year":"2021","journal-title":"Microorganisms"},{"key":"2025031102051111000_2025.03.01.640991v1.5","doi-asserted-by":"crossref","unstructured":"Carlson AE , Westenbroek RE , Quill T , Ren D , Clapham DE , Hille B , Garbers DL , Babcock DF. 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