Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization

Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization View ORCID Profile Shweta Bhagwat , Leila Asadi , Ronald McCarthy , Juan Ferreira , Ping Li , Ethan Li , Sariela Spivak , Ariana Gaydon , Vaka Reddy , Christy Armstrong , Sydney R. Morrill , Hillary Zhou , Amanda L. Lewis , Warren G. Lewis , Celia M. Santi doi: https://doi.org/10.1101/2025.03.01.640991 Shweta Bhagwat 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shweta Bhagwat Leila Asadi 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ronald McCarthy 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Juan Ferreira 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ping Li 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ethan Li 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sariela Spivak 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ariana Gaydon 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vaka Reddy 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christy Armstrong 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sydney R. Morrill 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hillary Zhou 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amanda L. Lewis 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Warren G. Lewis 2 Department of Obstetrics , Gynecology, and Reproductive Sciences, Glycobiology Research and Training Center, University of California San Diego , La Jolla, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Celia M. Santi 1 Department of Obstetrics and Gynecology, Washington University School of Medicine , Saint Louis, MO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: santic{at}wustl.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Study question What effect do toxins produced by bacterial vaginosis (BV) bacteria have on sperm function? Summary answer Bacterial vaginosis toxins dysregulate sperm capacitation and intracellular calcium homeostasis and impair the ability of sperm to fertilize oocytes. What is known already In 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, 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 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 chance Exposure 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 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 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. Introduction Bacterial vaginosis (BV) is characterized by a disruption of the vaginal microbiota, with a decrease in protective Lactobacillus species and overgrowth of anaerobic bacteria, including Gardnerella and Prevotella ( Ravel et al ., 2011 ). BV affects approximately 29% of reproductive-aged women in the U.S. ( Allsworth and Peipert, 2007 ), and is associated with various adverse health outcomes, such as sexually transmitted infections, pelvic inflammatory disease ( Ravel et al ., 2021 ; Turpin et al ., 2021 ), and preterm birth ( Salminen et al ., 2008 ). Moreover, BV has been linked to female infertility through several mechanisms ( Ravel et al ., 2021 ; Cocomazzi et al ., 2023 ). First, elevated concentrations of pro-inflammatory cytokines and chronic inflammation in the reproductive tract lead to tissue damage and impaired implantation ( Murphy and Mitchell, 2016 ; Ravel et al ., 2021 ). Second, BV-associated bacteria and their metabolites induce endometrial dysfunction ( Łaniewski and Herbst-Kralovetz, 2021 ), interfere with embryo implantation, and cause early pregnancy loss ( Eckert et al ., 2003 ; Oostrum et al ., 2013 ; Haahr et al ., 2018 ; Hong et al ., 2020 ). Third, imbalances in vaginal pH and microbiota ( Criteria and Associations, 1983 ; Mania-pramanik et al ., 2008 ), along with the toxins produced by Gardnerella and Prevotella , adversely affect sperm quality and motility ( Li et al ., 2016 ; O’Doherty et al ., 2016 ). Prevotella is an opportunistic pathogen in the female genital tract ( George et al ., 2024 ). Like other Proteobacteria, it produces lipopolysaccharide (LPS) ( Aroutcheva et al ., 2008 ), a heat-stable toxin (also known as endotoxin). LPS reaches 2-3 orders of magnitude higher concentrations in the vagina of those with BV than in those without BV. These higher LPS concentrations strongly correlate with the abundance of Prevotella in BV-positive individuals ( Aroutcheva et al ., 2008 ). LPS-mediated inflammation has been linked with female infertility, affecting ovarian and pituitary function ( Magata et al ., 2023 ). Additionally, in several animal models, LPS challenge can trigger preterm birth and injure the fetal brain ( Wang et al ., 2006 ; Firmal et al ., 2020 ). Unlike Prevotella , Gardnerella does not make LPS but instead produces the toxin vaginolysin (VLY). VLY is a cholesterol-dependent cytolysin that forms pores in human cell membranes and is active against erythrocytes, as well as vaginal and cervical cells in vitro ( Yoo and Lee, 2016 ; Ragaliauskas et al ., 2019 ; Campisciano et al ., 2021 ). We hypothesize that LPS and VLY contribute to female infertility by disrupting sperm capacitation, which occurs in the female genital tract and is necessary for a sperm to be able to fertilize an egg. One component of capacitation is hyperactivation, which causes flagellar beating to increase in amplitude, decrease in frequency, and become asymmetrical ( Suarez, 2008 ). Capacitation also involves acrosomal exocytosis ( Molina et al ., 2018 ) in which the outer acrosomal membrane at the sperm head fuses with the sperm plasma membrane to release the acrosomal content in response to a stimulus ( Sosa et al ., 2014 ). Hyperactivation and acrosomal exocytosis are necessary for sperm to penetrate the cumulus and zona pellucida layers surrounding the oocyte and fuse with the oocyte. Here, we show that both LPS and VLY caused mouse and human sperm to hyperactivate early during capacitation. Prolonged exposure to LPS and VLY decreased sperm motility, hyperactivation, and fertilization capability. Furthermore, sperm acrosomal exocytosis was inhibited by the two toxins. In defining the underlying mechanisms, we found that LPS and VLY caused rapid and irreversible increases in intracellular calcium (Ca 2+ ) concentration in sperm. Although hyperactivation and acrosomal exocytosis require an increase in intracellular Ca 2+ , excessive intracellular Ca 2+ concentration impairs mitochondrial function, leading to a loss of motility, activation of apoptotic cascades, and premature loss of the acrosome, in mammalian sperm ( Liu and Baker, 1990 ). Finally, we show that the LPS-induced Ca 2+ entry involved Toll- like receptor 4 (TLR4). VLY-induced Ca 2+ entry is likely due to the membrane pores formed by this toxin. Together, our data show that toxins present in BV dysregulate capacitation and intracellular Ca 2+ homeostasis, impairing the ability of sperm to fertilize oocytes. This mechanism may contribute to infertility in patients with BV. Materials and methods Reagents All reagents used to prepare Human Tubal Fluid (HTF) and Toyoda–Yokoyama–Hosi (TYH) media, as well as the Ca 2+ ionophore A23187, polymyxin B (PMB), TAK-242 (TLR4 receptor inhibitor), progesterone, Methyl-ß-cyclodextrin, and immunoglobulin-free bovine serum albumin (BSA) were from Millipore Sigma (St. Louis, MO, USA). Dulbecco’s Phosphate Buffered Saline (DPBS) tissue culture grade was from GIBCO Life Technologies (Gaithersburg, MD, USA); Hoechst 33342 was from Cayman Chemicals (Ann Arbor, MI, USA); Fluo-4 AM was from Invitrogen TM Thermo Fisher Scientific (Waltham, MA, USA); paraformaldehyde was from Electron Microscopy Sciences (Hatfield, PA, USA); and LPS from E. coli strain O112:H10 was from Associates of Cape Cod Inc. (E. Falmouth, MA, USA). Animals and ethics statement All mouse procedures were performed according to the National Institutes of Health Guiding Principles for the care and use of laboratory animals. These procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis (St. Louis, MO, USA) (protocol # 20-0126). C57BL/6 and CatSper1 knock-out male mice (CatSper KO) (60–90 days old) were purchased from Jackson Labs and kept at a constant temperature of 22 ± 2 °C under a 12/12 h dark/light cycle with free access to food and water. Oocytes for in vitro fertilization experiments were obtained from 4–8-week-old C57BL/6 female mice. Mouse sperm collection and capacitation Sexually mature male mice (60–90 days old) were euthanized via cervical dislocation, and sperm were isolated from the cauda epididymis. Sperm were allowed to swim-up in non-capacitating (NC) TYH media buffered with HEPES (in mM: 119.3 NaCl, 4.7 KCl, 1.71 CaCl 2 .2H 2 O, 1.2 KH 2 PO 4 , 1.2 MgSO 4 .7H 2 O, 25.1 NaHCO 3 , 5.56 glucose, 0.51 sodium pyruvate, 10 HEPES) at pH 7.4 for 15–20 min at 37 °C, unless specified. The motile fraction of the sample was removed from the tube. To induce capacitation, sperm were incubated in capacitating (CAP) TYH medium supplemented with 5 mg/mL BSA and 15 mM NaHCO 3 at 37 °C for 60-90 min. NC sperm were incubated in TYH without BSA and NaHCO 3 unless specified otherwise. Human participants and sperm preparation Human semen samples were obtained by masturbation after 3 to 5 days of abstinence. Samples were left over from the Washington University Fertility and Reproductive Medicine Center and were deidentified. The clinic confirmed that all samples met normal semen parameters according to the World Health Organization (≥15 million sperm per mL, ≥40% total motility, ≥32% progressive motility). Samples were allowed to liquefy for 1 h at room temperature, then sperm were purified by swim-up as previously described ( Molina et al ., 2020 ). Briefly, within 2 h of production, sperm were allowed to swim up in non-commercial HTF (in mM: 98 NaCl, 4.7 KCl, 0.4 KH 2 PO 4 , 2 CaCl 2 , 0.2 MgSO 4 , 20 HEPES, 3 glucose, 21 lactic acid, 0.3 sodium pyruvate; pH adjusted to 7.4 with NaOH) for 1 h at 37 °C without CO 2 . Production of Gardnerella vaginolysin (VLY) protein A truncated mutant vly gene from Gardnerella ATCC14019 ( Randis et al ., 2009 ) was cloned into a pET28a vector, which was transformed into ClearColi BL21(DE3) cells (Biosearch Technologies, Teddington, UK) with detoxified LPS. Protein was expressed and purified as previously described ( Morrill et al ., 2023 ). Cytolytic activity on human red blood cells was verified by serial dilution and assay of hemoglobin release measured at 545 nm. In parallel, an empty vector was used to prepare a mock expression and purification that lacked VLY but contained all other reagents. VLY and mock preparations were stored in 20% glycerol at -20 °C. LPS precautions and testing LPS from Associates of Cape Cod, Inc. (E Falmouth, MA) was dissolved according to the manufacturer’s instructions. Dilutions were performed in a manner that avoided loss of LPS, avoiding multiple dilutions in plastic that would allow adsorption. Because LPS can contaminate reagents, especially recombinant proteins, pyrogen-free reagents and labware were used when available. Clean glassware was dry baked at 200 °C for 2 h to destroy LPS. The LPS content of reagents was quantified by using a chromogenic Limulus Amoebocyte Lysate assay from Associates of Cape Cod. Protein preparations and buffers (TYH and HTF) contained <1 EU/mL (approximately <0.1 ng/mL) of LPS at the concentrations used in sperm preparation and assays. In vitro fertilization (IVF) protocol Sperm from C57BL/6 male mice (4–8 weeks old) were prepared as described above and capacitated for 3 h in TYH-CO 2 buffered media supplemented either with 0.75 mM Methyl-ß-cyclodextrin ( Nakagata, 2010 ) or 5 mg/mL BSA at 37 °C and 5 % CO 2 to induce capacitation, with or without LPS or VLY. C57BL/6 female mice (4–8 weeks old) were superovulated by intraperitoneally injecting 5-7.5 IU pregnant mare serum gonadotropin (ProSpec Cat# HOR-272). 48 h later, these mice were injected with 5-7.5 IU human chorionic gonadotropin (Millipore/Sigma CG5-1VL). 12-15 h later (at the 3-h sperm capacitation timepoint), female mice were euthanized, and oocyte-cumulus complexes were isolated in 200 µL of high Ca 2+ HTF ( Nakagata, 2010 ). Cumulus-oocyte complexes were washed four times with 100 µl HTF, then overlayed with Ovoil. The media in the dish was allowed to equilibrate for at least 1 h in an incubator at 37 °C and 5% CO 2 , before the addition of oocytes. Fertilization wells containing 20–30 eggs were mixed with CAP sperm (final concentration of 2 × 10 6 sperm/mL) and incubated for 4-6 h. Eggs were then washed three times and placed in a drop of HTF medium with a disposable soda-lime glass 50 µL microcapillary pipette (Kimble Cat# 71900-50) attached to an aspirator tube assembly (Millipore/Sigma A5177-5EA). Oocytes were counted, and the dish was incubated overnight at 37 °C and 5% CO 2 . The percentage of fertilized eggs was calculated by dividing the number of two-cell embryos by the total number of oocytes. Computer-Assisted Sperm Analysis (CASA) Mouse and human sperm were suspended in their respective CAP media and exposed to 1 µg/mL LPS or VLY (mouse sperm) and 0.1 µg/mL LPS or VLY (human sperm) from the start of capacitation. Sperm motility and hyperactivity were measured at time 0 and at 0.5-1 h intervals. Sperm samples (3 μl) from each condition were placed into 20-micron Leja standard count four-chamber slides, pre-warmed at 37 °C, and a minimum of 200 cells were counted. CASA was performed with a Hamilton–Thorne digital image analyzer (HTR-CEROS II v.1.7; Hamilton–Thorne Research, Beverly, MA, USA). CASA settings were as previously described ( Molina et al ., 2020 ). For mouse sperm, hyperactivated motility was defined as curvilinear velocity (VCL) > 150 μm/s, lateral head displacement (ALH) > 7.0 μm, and linearity coefficient (LIN) of 32% at 60 Hz.( Mortimer et al ., 1998 ) For human sperm, hyperactivated motility was defined as VCL ≥ 150 μm/s, ALH ≥ 7 μm, LIN ≤ 50% at 60 Hz. Percent hyperactivation was calculated by dividing the number of hyperactivated sperm by the number of motile sperm. Total and progressive motility values were obtained as percentages from the CASA software. Sperm viability assessment Hoechst 33342 (900 nM) was added to sperm. After 2 to 5 min, fluorescence intensity was measured with an Aurora 4L 16V-14B-10YG-8R Flow Cytometer and a V3 or 458 nm filter ( Lyon et al ., 2023 ). The excitation wavelength of Hoechst 33342 is 350 nm, and its emission wavelength is 461 nm. At least 10,000 sperm were recorded for each sample. The fluorescence cutoff was selected by measuring the fluorescence of sperm killed by treatment with 3 to 6% sodium hypochlorite for 2 to 5 min. Sperm intracellular calcium concentration measurement After sperm swim-up in NC or CAP conditions for 15–20 min, motile sperm were collected and incubated with 10 μM Fluo-4 AM and 0.05% Pluronic F-127 at 37 °C for 60 min. Sperm were centrifuged at 300-400 g for 5–10 min, resuspended in the corresponding media, and allowed to attach to Poly-l-lysine (0.1%) coated coverslips for 5 min. A local perfusion device with an estimated exchange time of 10 s was used to apply test solutions. Sperm [Ca 2+ ] I was recorded at room temperature with a Leica AF 6000LX system connected to a Leica DMi8000 inverted microscope, equipped with a 63X objective (HC PL FluoTar L 63X/0.70 Dry) and an Andor-Zyla-VCS04494 camera. A halogen lamp was used with a 488 ± 20 nm excitation filter and a 530 ± 20 nm emission filter. Data were collected with Leica LasX 2.0.014332 software. Acquisition parameters used were: 20 ms exposure time, 4x4 binning, 1024 x 1024 pixels resolution ( Ferreira et al ., 2021a ). For each condition, an initial baseline reading was followed by the addition of LPS or VLY and a final perfusion with 5 μM Ionomycin (Iono) at the end of every experiment. Whole images were collected every 10 s. LAS X, ImageJ, Clampfit 10 (Molecular Devices), and GraphPad were used for data analysis. A region of interest was selected in the sperm head ( Chávez et al ., 2014 ). To compare fluorescence across different samples and experimental conditions, Fluo-4 fluorescence (F) in response to LPS or VLY for each sperm was normalized to its respective F Iono (F/F iono ), after background subtraction. Cells with [Ca 2+ ] I increases of >10% of that obtained with Iono were counted as responsive. Percentages of cells responding to LPS or VLY were calculated by dividing the number of cells responding to the respective toxin by the total number of cells that responded to Iono. Clampfit 10 software was used to measure amplitude and Tau (time taken to exhibit the response) in responding sperm ( Ferreira et al ., 2021a ). Acrosomal exocytosis For mouse sperm, acrosomal exocytosis was evaluated in both NC and CAP sperm from transgenic mice that express green fluorescent protein in the acrosome ( Hasuwa et al ., 2010 ). Acrosomal exocytosis was induced by incubating sperm with 15 µM Ca 2+ ionophore A23187 or 50 mM KCl (Millipore Sigma, St. Louis) for 30 min before the end of capacitation. LPS or VLY (1 µg/mL) were added to CAP and NC samples either at the start of capacitation or with the inducers A23187 and KCl. Sperm from all conditions were fixed in 4% paraformaldehyde and centrifuged at 300-400 g for 5–10 min. Supernatants were removed, the sperm were washed with phosphate buffered saline (PBS), and 15 μL of each sample was smeared onto a glass slide. Slides were air-dried and coverslips were mounted with 1:1 glycerol: pyrogen-free DPBS at room temperature. Sperm were examined at 40X on an EVOS FL Cell Imaging System epifluorescence microscope (Thermo Fischer Scientific). Sperm were counted as acrosome-reacted if the acrosomal region lacked GFP fluorescence and non-reacted if they retained GFP fluorescence. A minimum of 200 sperm were evaluated in each experiment. Percent acrosomal exocytosis was calculated by dividing the number of reacted sperm by the number of total sperm per sample. Human sperm acrosomal exocytosis was measured as previously described ( Lyon et al ., 2023 ). Briefly, swim-up sperm were incubated in both NC and CAP media (HTF supplemented with 25 mM HCO 3 and 5 mg/mL BSA) for 3 h. Acrosomal exocytosis was induced by adding 10 µM Ca 2+ ionophore A23187 or 10 µM progesterone for 30 min. LPS or VLY (0.1 µg/mL) were added immediately after swim-up or during acrosomal exocytosis induction. After induction, sperm were fixed with 3% paraformaldehyde in DPBS at 4 °C, smeared on slides, and stained with FITC-labelled Pisum sativum agglutinin (Millipore Sigma). Fluorescence was imaged as described for mouse sperm acrosomal exocytosis. Sperm with bright, uniformly stained acrosomes were counted as intact, and sperm with equatorial or no staining in the acrosomal region were counted as reacted. The minimum number of sperm and method for calculating percent acrosomal exocytosis were as described for mouse sperm. Statistical analyses GraphPad Prism version 10.1.0 for Windows (GraphPad Software, La Jolla, CA) was used for all statistical analyses. An unpaired two-tailed Student’s t-test (alpha < 0.05) was used to compare sperm samples from different individuals, whereas a paired two-tailed t-test was used to compare control and treatment in the same sample, unless otherwise indicated. For time series data analysis, where time and the treatment groups were the two variables, two-way ANOVA with Bonferroni’s multiple comparison test was performed. Data are expressed as the mean ± SD. P < 0.05 was considered statistically significant. Results LPS and VLY interfere with mouse sperm fertility and capacitation To determine whether LPS or VLY impair the ability of sperm to fertilize oocytes, we capacitated mouse epididymal sperm in the presence and absence of 1 µg/mL LPS or 1 µg/mL VLY for 3 h. We then performed mouse in vitro fertilization. When sperm were capacitated in the presence of LPS, the fertilization rate was 34.93 ± 8.57%, significantly lower than the rate of 60.57 ± 4.28% in control conditions. Similarly, fertilization rates decreased from 76 ± 16% to 44 ± 16% in the absence and presence of VLY, respectively ( Fig. 1A, B ) . The addition of LPS or VLY to the fertilization drop after capacitation had no effect on fertilization rates ( Fig. S1 and Fig. S2 ). Download figure Open in new tab Figure 1: LPS and VLY impair mouse sperm fertility, hyperactivation, total motility, and acrosomal exocytosis in capacitating conditions. Percentage of oocytes that reached the two-cell stage within 24 h after in vitro fertilization (IVF) using sperm (n=5 biological replicates) previously treated for 3 h with 1 µg/mL (A) LPS or (B) VLY were calculated. CASA measurements were obtained for (C, D) hyperactivated motility, ( E, F ) total motility, and ( G, H ) progressive motility of mouse sperm incubated under capacitating (CAP) conditions in the presence and absence of 1 µg/mL ( C, E, G ) LPS and (D, F, H) VLY (n=9 biological replicates). Here 0 min is the time-point of BSA + sodium bicarbonate addition to initiate in vitro capacitation. Acrosomal exocytosis (AE) was quantified in non-CAP (NC), and CAP sperm (200 each, from 5 biological replicates) incubated with (I) LPS and (J) VLY, for 0.5 and 1.5 h. Similarly, AE induced by 15 µM A23187 or 50 mM KCl, was also quantified in CAP sperm. Sperm viability for NC and CAP sperm (n≥3 biological replicates) was analyzed in the presence of 1 µg/ml (K) LPS or (L) VLY (10,000 sperm each). Data are presented as mean and standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.001. For A-B , a paired t-test was used. For C-L , two-way ANOVA with Bonferroni’s multiple comparison test was used. Next, we wanted to determine which outcomes of capacitation – hyperactivation, acrosomal exocytosis, or both – were affected by these toxins. Mouse sperm were incubated in capacitating (CAP) media with or without 1 µg/mL LPS or VLY, and motility was measured every 30 min for 120 min. At 30 min, the percentages of hyperactivated sperm were significantly higher in the presence of LPS or VLY than in the controls ( Fig. 1C, D ) . However, at 90 and 120 min, sperm hyperactivation and total motility were significantly lower in the presence of LPS or VLY than in controls ( Fig. 1C–F ). No effect was observed on progressive motility in CAP ( Fig. 1G, H ) or in non-CAP conditions ( Fig. S3 ). These results suggest that LPS and VLY impair sperm hyperactivation, which is required for fertilization. To measure the impact of LPS and VLY on acrosomal exocytosis, mouse sperm were exposed to 1 µg/mL LPS or VLY in CAP conditions, and acrosomal exocytosis was induced by adding 15 µM Ca 2+ ionophore A23187 (A23) or 50 mM potassium chloride (KCl).(De La Vega-Beltran et al ., 2012 ) As reported earlier (De La Vega-Beltran et al ., 2012 ), about 40–60% of mouse sperm underwent induced acrosomal exocytosis in control CAP conditions. The presence of LPS or VLY significantly inhibited A23- and KCl-induced sperm acrosomal exocytosis but had no significant effect on acrosomal exocytosis in non-CAP conditions ( Fig. 1I, J ) , which is also defined as spontaneous acrosome reaction ( Liu and Baker, 1990 ). Next, we assessed whether these effects were specific to hyperactivation and acrosomal exocytosis or caused by a decrease in sperm viability. We found that mouse sperm viability did not significantly differ in the absence or presence of either toxin over 180 min ( Fig. 1K, L ). To test whether the effects were specifically due to LPS, we capacitated sperm in the presence of LPS plus polymyxin B (PMB), which forms aggregates with LPS and hampers its activity. In this condition, LPS had no effect on mouse sperm hyperactivation ( Fig. 2A ). PMB alone also had no effect on hyperactivation ( Fig. 2B ). As a control for VLY specificity, we treated sperm with protein generated in parallel using an empty vector control. This treatment did not impair hyperactivation ( Fig. 2C ). Additionally, the changes in hyperactivated motility in the presence of VLY plus PMB ( Fig. 2D ) were similar to those observed with VLY alone. Download figure Open in new tab Figure 2: LPS and VLY effects on mouse sperm are specific. CASA measurements were obtained for hyperactivated motility of mouse sperm incubated under capacitating (CAP) conditions in the presence of (A) 1 µg/ml LPS or 100 µg/ml PMB + 1 µg/ml LPS, (B) vehicle or 100 µg/ml PMB alone, (C) vehicle, 1 µg/ml VLY, or VLY empty vector control, and (D) 1 µg/ml VLY or 100 µg/ml PMB + 1 µg/ml VLY. Here, 0 min is the time-point of BSA + sodium bicarbonate addition to initiate in vitro capacitation. Data are presented as mean and standard deviation (n≥4 biological replicates). *P<0.05, ****P<0.0001. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. Because capacitation media contains 15 mM HCO 3 − , we wanted to determine whether the effects could be explained by changes in osmolarity. Therefore, we increased the external medium osmolarity with 15 mM NaCl and found that neither LPS nor VLY induced hyperactivated motility in this condition ( Fig. S4 ). We conclude that the effects on hyperactivation were due to changes in the capacitation signaling pathway induced by HCO3 − and not by changes in external osmolarity ( Ferreira et al ., 2021b ). LPS and VLY interfere with human sperm capacitation To determine whether LPS and VLY also impair human sperm function, we obtained leftover sperm samples from patients with normal semen parameters being treated at the Reproductive Endocrinology and Infertility Clinic at Washington University in Saint Louis. As the median concentration of LPS in patients with BV is approximately 3000 EU/mL ( Aroutcheva et al ., 2008 ), we used 1000 EU/mL LPS, corresponding to 0.1 µg/mL LPS, for human sperm. The physiologically relevant concentration of VLY is unknown, so we used the same concentration as LPS. Immediately after adding 0.1 µg/mL LPS or 0.1 µg/mL VLY to sperm in CAP conditions, the percentage of hyperactivated sperm was significantly greater than in untreated controls ( Fig. 3A, B ). However, by the end of the 180-min treatment period, human sperm treated with LPS or VLY showed significantly lower hyperactivated, total, and progressive motility than untreated sperm ( Fig. 3A–F ). LPS and VLY had no effect on human sperm hyperactivation or total or progressive motility in non-CAP conditions ( Fig. S5 ). PMB prevented the dose-dependent increase in human sperm hyperactivation induced by LPS (Fig. S6) . Download figure Open in new tab Figure 3: LPS and VLY impair human sperm hyperactivation, total motility, progressive motility, and acrosomal exocytosis in capacitating conditions. CASA measurements were obtained for ( A, B ) hyperactivated motility, ( C, D ) total motility, and ( E, F ) progressive motility of human sperm incubated under capacitating (CAP) conditions in the presence and absence of 0.1 µg/mL (A, C, E) LPS (n=5 biological replicates) and (B, D, F) VLY or VLY vector control (n≥5 biological replicates). Here 0 min is the time-point of BSA + sodium bicarbonate addition to initiate in vitro capacitation. Acrosomal exocytosis (AE) was quantified in non-CAP (NC), and CAP sperm (200 each, n=3 biological replicates) incubated with (G) LPS and (H) VLY for 0.5 and 3.5 h. Similarly, AE induced by 10 µM A23187 or progesterone (P4), was also quantified in CAP sperm. Sperm viability for NC and CAP sperm was analyzed in the presence of 0.1 µg/ml (I) LPS or (J) VLY (10,000 sperm each, n≥3 biological replicates). Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test and are presented as mean and standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Next, we measured the effect of a ten-fold lower dose of LPS and VLY (0.1 µg/mL) on human sperm acrosomal exocytosis induced by addition of the Ca 2+ ionophore A23187 or progesterone. As reported earlier ( Baro Graf et al ., 2020 ), about 15–30% of human sperm underwent induced acrosomal exocytosis in control conditions ( Fig. 3G, H ) . Both LPS ( Fig. 3G ) and VLY ( Fig. 3H ) significantly reduced the percentage of acrosomal exocytosis induced by A23187 or progesterone in CAP conditions but had no effect in non-CAP conditions. At 180 min of treatment, LPS ( Fig. 3I ) and VLY ( Fig. 3J ) significantly reduced human sperm viability by 13% and 21.2%, respectively, in non-CAP conditions. Under CAP conditions, LPS and VLY reduced the viability by 7.7% and 18.3%, respectively; however, this was not statistically significant. We conclude that LPS and VLY impair human sperm function in a similar manner as they impair mouse sperm function. LPS and VLY rapidly and irreversibly induce increases in intracellular Ca 2+ concentration in CAP mouse and human sperm Because both hyperactivated motility and acrosomal exocytosis are dependent on Ca 2+ influx and increased intracellular Ca 2+ concentration ([Ca 2+ ] I ), we wondered whether LPS and VLY affected sperm [Ca 2+ ] I . To test this idea, we loaded mouse sperm with the cell-permeant Ca 2+ -sensitive fluorescent dye Fluo-4 AM. In control conditions, mouse sperm [Ca 2+ ] I reached maximum after addition of CAP media, with a time of initiation of the response (tau) of 164.2 ± 61.91. Addition of LPS and VLY increased [Ca 2+ ] I more rapidly, with a tau of 53.68 ± 36.36 and 20.01 ± 14.13, respectively (Fig. S7-8) . Also, the amplitude of the increase in [Ca 2+ ] I was significantly larger in the presence of LPS ( Fig. S8 ). We next compared the LPS and VLY effects between non-CAP and CAP conditions. Between 10% and 40% of mouse sperm in non-CAP conditions and 50-100% of sperm in CAP conditions reached maximum [Ca 2+ ] I after addition of LPS or VLY ( Fig. 4A–F ). The amplitude of the Ca 2+ response was greater and the tau was shorter in CAP than in non-CAP conditions ( Fig. 4G–J ) (see traces of LPS- and VLY- induced [Ca 2+ ] I response in Fig. S9 ). Experiments using human sperm produced similar results ( Fig. 5 , Fig. S10). Washout experiments indicated that the LPS- and VLY-induced Ca 2+ responses were irreversible for both mouse and human sperm ( Fig. 6 ). Therefore, we showed that both LPS and VLY induced rapid and irreversible increases in [Ca 2+ ] I , and these increases were significantly larger in capacitated conditions than in non-capacitated conditions. Download figure Open in new tab Figure 4: LPS and VLY cause rapid increases in [Ca 2+ ] I in capacitating mouse sperm. Representative traces of normalized Fluo4-AM fluorescence in responding (red) versus non-responding (black) mouse sperm with 1 µg/mL (A, C) LPS and (B, D) VLY, incubated under ( A, B ) Non-CAP (NC) and ( C, D ) CAP conditions. Each trace was normalized to its respective ionomycin (Iono, 2-5 µM) response. Percentages of mouse sperm responding to (E) LPS and (F) VLY, in NC and CAP conditions were calculated. ( G, H ) Amplitude and ( I, J ) tau of the [Ca 2+ ] I response in NC and CAP sperm, with 1 µg/mL (G, I) LPS and (H, J) VLY. Data are presented as mean and standard deviation (n=4 biological replicates). *P<0.05, ***P<0.001, ****P<0.0001, by unpaired t-test for E-J . Download figure Open in new tab Figure 5: LPS and VLY cause rapid increases in [Ca 2+ ] I in capacitating human sperm. Representative traces of normalized Fluo4-AM fluorescence in responding (red) versus non-responding (black) human sperm with 0.1 µg/mL (A, C) LPS and (B, D) VLY, incubated under ( A, B ) Non-CAP (NC) and ( C, D ) CAP conditions. Each trace was normalized to its respective ionomycin (Iono, 2-5 µM) response. Percentages of human sperm responding to (E) LPS and (F) VLY, in NC and CAP conditions were calculated. ( G, H ) Amplitude and ( I, J ) tau of the [Ca 2+ ] I response in NC and CAP sperm, with 0.1 µg/mL (G, I) LPS, and (H, J) VLY was quantified. Data are presented as mean and standard deviation (n≥4 biological replicates). **P<0.01, ***P<0.001, ****P<0.0001, by unpaired t-test for E-J . Download figure Open in new tab Figure 6: LPS- and VLY-induced [Ca 2+ ] I increases in mouse and human sperm are irreversible. Representative traces of normalized Fluo4-AM fluorescence in CAP ( A, B ) mouse and ( C, D ) human sperm, perfused with ( A, C ) LPS and ( B, D ) VLY, followed by media (Wash, denoted in blue), and subsequent perfusion with 2-5 µM ionomycin (Iono, denoted in black). Each trace was normalized to its respective ionomycin response. Data are presented as mean and standard deviation (n=3 biological replicates). LPS-induced Ca 2+ response requires TLR4 and external Ca 2+ but not CatSper The main mechanism of [Ca 2+ ] I increase during normal capacitation is entry of external Ca 2+ through the sperm specific Ca 2+ channel, CatSper. To determine whether the effects of LPS required external Ca 2+ , we exposed mouse sperm to CAP conditions in media that lacked Ca 2+ . In this condition, LPS did not cause a [Ca 2+ ] I increase ( Fig. 7A ). Similarly, LPS did not increase [Ca 2+ ] I in human sperm ( Fig. 7B ). To determine whether CatSper was required for the LPS- induced [Ca 2+ ] I increase, we measured [Ca 2+ ] I in sperm from wild-type ( Fig. 7C ) and CatSper knock-out ( Fig. 7D ) mice. Neither the percentages of sperm responding to LPS ( Fig. 7E ) nor the amplitude (Fig. S11A) or tau (Fig. S11B) of their responses differed significantly between sperm from wild-type and CatSper knock-out mice. Moreover, the [Ca 2+ ] I changes in LPS- responding cells were similar for wild-type ( Fig. 7F ) and CatSper knock-out ( Fig. 7G ) mouse sperm. These data indicate that LPS triggered an increase in [Ca 2+ ] I through a mechanism independent of CatSper. Download figure Open in new tab Figure 7: LPS-induced increase in sperm [Ca 2+ ] I is dependent on external Ca 2+ , independent of the CatSper ion channel, and mediated by the TLR4 receptor. Representative traces of normalized Fluo4-AM fluorescence from capacitating (CAP) (A) wild- type (WT) mouse, and (B) human sperm perfused with 0 mM Ca 2+ , 0 mM Ca 2+ with LPS, and ionomycin (Iono) with 2 mM Ca 2+ (n=3 biological replicates). Representative traces for LPS responding (red) and non-responding (black) (C) WT and (D) CatSper knock-out (KO) mouse sperm in the presence of 2 mM extracellular Ca 2+ . (E) Percentages of LPS-responding sperm with increased [Ca 2+ ] I from WT and CatSper KO mice (n=5 biological replicates). The LPS-induced [Ca 2+ ] I responses are shown on the traces obtained from (F) WT and (G) CatSper KO mouse sperm. Red curves calculated as a standard exponential fit. Representative traces of Fluo4-AM fluorescence from CAP (H) WT mouse, and (I) human sperm perfused with TAK-242 (TLR4 inhibitor), TAK-242 with LPS, and ionomycin (Iono), in the presence of 2 mM extracellular Ca 2+ throughout the experiment (n=3 biological replicates). Each trace was normalized to its respective ionomycin (Iono, 2-5 µM) response. Data are presented as mean and standard deviation. ns= non-significant, by unpaired t-test in (E). Given that LPS binding to the TLR4 receptor induces [Ca 2+ ] I increases in other cell types ( Tauseef et al ., 2012 ), we wondered whether TLR4 was required for LPS effects on [Ca 2+ ] I in sperm. To test this idea, we exposed sperm to LPS in the presence of the specific TLR4 inhibitor TAK-242 ( Takashima et al ., 2009 ; Yuko et al ., 2020 ). The LPS-induced Ca 2+ response did not occur in mouse ( Fig. 7H ) or human ( Fig. 7I ) sperm in the presence of TAK-242. These data show that the [Ca 2+ ] I increase induced by LPS in human and mouse sperm is mediated by the TLR4 receptor. Discussion Bacterial vaginosis (BV) is more prevalent in infertile women than in fertile women, and 37.4% of females with unexplained infertility are diagnosed with BV ( Salah et al ., 2013 ). Although the cause of infertility among patients with BV is unclear, several mechanisms have been proposed. One possibility is that BV-related bacteria can induce immune activation and increase concentrations of proinflammatory cytokines, resulting in mucosal inflammation in the genital tract (van Teijlingen et al ., 2020 ). Higher cervical concentrations of the cytokines interleukin (IL)-1β, IL-6, and IL-8 have been observed in women with infertility and BV ( Spandorfer et al ., 2001 ). Another possibility is that sialidases and other mucinases produced by microorganisms impair cervical mucus integrity ( Wiggins et al ., 2001 ) by degrading mucins. This could weaken the natural barrier against microbial invasion and facilitate bacterial adhesion and colonization, possibly leading to upper reproductive tract disease and infertility. Finally, women with BV have a 3.4 and 4.1-fold, respectively, elevated risk of acquiring the sexually transmitted infections with Chlamydia trachomatis and Neisseria gonorrhoeae ( Wiesenfeld et al ., 2003 ), which can impair fertility. Additionally, BV is associated with increased rates of upper genital tract infections and pelvic inflammatory disease, both of which are linked to infertility ( Ravel et al ., 2021 ). Here, we present three lines of evidence supporting a new mechanism by which BV contributes to female infertility: the BV-associated toxins LPS and VLY interfere with sperm capacitation, which is required to fertilize an egg. First, both LPS and VLY treatment reduced the fertilizing ability of mouse sperm. Second, LPS and VLY impaired hyperactivation, motility, and acrosomal exocytosis in both mouse and human sperm. Third, LPS and VLY caused premature and irreversible increases in intracellular Ca 2+ concentration ([Ca 2+ ] I ) in both mouse and human sperm. Our results regarding the impact of LPS on mouse sperm are consistent with findings reported by Fujita et. al. ( Fujita et al ., 2011 ). They reported decreased mouse sperm motility, increased sperm apoptosis, and decreased fertilization ability after 6 h of incubation with LPS. We observed that incubation with LPS for over 90 min led to decreased sperm motility and a nearly 50% reduction in the formation of two pronuclei-stage embryos. We also noted a slight decrease in viability after 3 h in capacitated media. However, the largest effects we observed were on hyperactivated motility and acrosomal exocytosis, which were not explored by Fujita and co- workers. Li et. al . also found that LPS decreased human sperm motility and intracellular cAMP concentrations ( Li et al ., 2016 ). Inconsistent with our findings, Li et. al. reported that LPS did not affect sperm [Ca 2+ ] I , acrosomal exocytosis, or capacitation ( Li et al ., 2016 ). These negative results may have been due to contamination of controls or reagents with LPS. Alternatively, LPS can adhere to tubes upon dilution and be lost. We used methods to prevent these problems and found that the effects of LPS were irreversible. Given this irreversibility, we speculate that LPS in the vagina in patients with BV could affect sperm fertility. Consistent with our findings, Sahnoun et. al. reported that LPS led to reduced percentages of sperm acrosomal exocytosis in infertile males ( Sahnoun et al ., 2017 ). Additionally, LPS was reported to decrease human sperm motility and curvilinear movement, inhibit phosphorylation of NF-κB inhibitor alpha, and cause increased β-oxidation of fatty acids ( Li et al ., 2023 ). Previous results regarding the effects of LPS on human sperm viability have been contradictory. In one report, 0.1 to 100 µg/mL LPS had no effect on viability ( Li et al ., 2016 ), whereas in another report, 50 µg/mL LPS caused 80% reduction in human sperm viability ( Galdiero et al ., 1988 ). With our validated concentrations of high-quality LPS and precautions to avoid loss of LPS through adsorption, we observed only a small decrease in sperm viability after 180 min of treatment. This small effect is unlikely to account for the effects of LPS on motility or acrosomal exocytosis. Both sperm hyperactivation and acrosomal exocytosis are finely regulated by intracellular Ca 2+ . Calcium entry is required for both the initiation and maintenance of hyperactivation through direct regulation of axonemal machinery components ( Carlson et al ., 2003 ). However, excessive increases in [Ca 2+ ] I in mammalian sperm are associated with impaired fertilization potential. This includes mitochondrial failure, motility loss, activation of apoptotic cascades, and premature loss of the acrosome, known as spontaneous acrosome reaction ( Liu and Baker, 1990 ). Prematurely inducing the human acrosome reaction with the Ca 2+ ionophore A23187 decreases sperm-zona pellucida binding, causing fertilization failure in vitro ( Liu and Baker, 1990 ). Additionally, spermatozoa deficient in the Ca 2+ pump PMCA4 cannot extrude Ca 2+ and thus experience Ca 2+ overload, resulting in male infertility ( Okunade et al ., 2004 ). Furthermore, high concentrations (10–20 μM) of the Ca 2+ ionophore A23187 initially increase the sperm flagellar beat, then immobilize sperm after 10 min. Concentrations of 5–10 μM A23187 rapidly immobilized sperm by elevating [Ca²⁺] I , whereas lower concentrations (0.5 and 1 μM) caused smaller [Ca²⁺] I increases and resulted in hyperactivation. Notably, A23187 in Ca²⁺-free medium did not immobilize sperm. These findings suggest that excessive Ca²⁺ influx affects motility and, at high concentrations, can lead to sperm immobilization. Observing faster and higher-amplitude [Ca²⁺] I increases in sperm exposed to LPS and VLY compared to controls, we speculate that both toxins cause sperm Ca 2+ overload. However, the mechanisms by which this overload occurs in response to LPS and VLY might be distinct. We found that LPS caused Ca 2+ influx in both human and mouse sperm. In mammalian sperm, the Ca 2+ influx into the flagellum is primarily regulated by CatSper ( Kirichok et al ., 2006 ), but we found that LPS-induced Ca 2+ influx in mouse sperm was independent of CatSper. Fujita et. al. reported that the negative effects of LPS on sperm motility and fertility were blocked in TLR4 knockout mice ( Fujita et al ., 2011 ), suggesting that TLR4 participates in LPS-mediated Ca 2+ signalling. Our experiments with the TLR4 inhibitor support the idea that the LPS-induced [Ca 2+ ] I increase in human and mouse sperm was mediated by the TLR4 receptor. Future experiments will be conducted to define the mechanisms by which this occurs. VLY is a cytolysin that forms pores in the cell membrane ( Ragaliauskas et al ., 2019 ). In addition to causing cells to leak their intracellular contents ( Randis et al ., 2013 ; Morrill et al ., 2023 ) the disruptions to the membrane would likely also allow extracellular Ca 2+ to enter the cytoplasm, as we saw in both mouse and human sperm. Because VLY embeds in the human cell membrane, it is expected to be irreversible. Thus, it was not surprising that we were unable to wash out the effects of VLY in sperm. In conclusion, our in vitro results show that the BV-associated toxins LPS and VLY modulate sperm Ca 2+ entry, leading to dysregulated hyperactivation, inhibition of acrosomal exocytosis, and impaired fertility. If these effects occur in vivo , then BV toxins in the vagina could impair key events necessary to prepare sperm for fertilization. This mechanism may contribute to infertility in patients with BV. Author’s role S.B. was involved in study design, data collection, analysis, and manuscript preparation. C.S., A.L., and W.L. provided their expertise and were responsible for the study design, conceptualization, supervision, and review of the manuscript. S.B., L.A., R.M., J.F., C.S., A.L., and W.L. contributed to the methodology, data collection, analysis, and investigation. Data visualization was performed by S.B., P.L., E.L., S.S, and A.G. Initial standardisation on VLY cloning and expression was done by V.R. LPS testing of reagents was done by C.A. VLY protein was prepared by S.M. and H.Z. All authors reviewed and contributed to the final manuscript. Funding This work was supported by the National Institutes of Health (grant #R01 HD069631 to CS). 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