Study the impact of Bacterial Vaginosis toxins on mouse and human sperm function - PROJECT SUMMARY/ABSTRACT Bacterial vaginosis (BV) is a common, recurrent vaginal dysbiosis affecting nearly 30% of reproductive-aged women in the U.S. It involves a loss of protective Lactobacillus and an overgrowth of anaerobes such as Gardnerella and Prevotella, leading to elevated levels of BV-associated toxins with major implications for gynecological health and fertility. Notably, 37.4% of women with unexplained infertility are diagnosed with BV, yet the mechanisms remain poorly understood. The female reproductive tract fluid is essential for triggering sperm capacitation, a key maturation step for fertilization. However, the impact of BV on this process has been largely overlooked. My recent findings (Bhagwat et al., 2025, in press) reveal a novel mechanism by which BV toxins impair sperm function. Specifically, lipopolysaccharide (LPS, a toxin made by Prevotella) and vaginolysin (VLY, a Gardnerella toxin) interfere with sperm capacitation and reduce sperm fertility in vitro. Both toxins impair sperm hyperactivated motility, and acrosomal exocytosis (AE) in mouse and human sperm, and trigger premature and irreversible Ca2+ influx, which may result in sperm damage. This proposal aims to define the molecular mechanisms of BV-induced sperm dysfunction and assess the in vivo consequences of BV toxins on fertility, aligning with NICHD’s 2025 mission to improve reproductive health. I hypothesize that BV toxins impair sperm capacitation by dysregulating Ca2+ signalling, leading to impaired sperm hyperactivation and AE, disrupting sperm migration in the female tract and thereby impairing sperm fertilizing ability. During the K99 phase, I will: (1) Elucidate the mechanisms by which LPS and VLY induce Ca2+ influx. Since my preliminary data indicate that LPS-mediated Ca2+ influx requires both the TLR4 receptor and the TRPV4 Ca2+ channel, I will determine the contribution of these signalling receptors to the LPS response, by measuring single-cell Ca2+ dynamics, cAMP, and phosphorylation of PKA-PI3K-GSK3α pathway proteins in sperm from TLR4 and TRPV4 knockout mice. (2) Elucidate the mechanism of Ca2+ entry by VLY using scanning electron microscopy to examine VLY-induced pore formation and test the roles of membrane cholesterol and CD59 in this process. (3) Assess in vivo sperm function in a mouse BV model and wild-type mouse sperm exposed to physiologically relevant levels of LPS and VLY, by quantifying sperm migration, motility, AE, and fertility in the female reproductive tract. (4) Quantify LPS and VLY in vaginal specimens from women with and without BV and test their effects on human sperm motility, hyperactivation, and AE. During the R00 phase, I will model BV-associated infertility using a human vagina- and cervix-chip system. These chips will be colonized with Gardnerella and Prevotella, and incubated with sperm to evaluate motility, hyperactivation, AE, and mucus penetration under BV-mimicking conditions. This research will define how BV impairs sperm function, provide mechanistic insight into BV-associated infertility, and establish a high-throughput platform for evaluating human sperm responses to dysbiotic reproductive tract conditions.
