The most common dysbiotic state in the vagina occurs with BV, especially in reproductive-aged women worldwide. Its risk for susceptibility to other obstetric and gynecological diseases, such as preterm birth and sexually transmitted infections (STIs), renders a high global economic burden fueling the impetus for screening and therapeutic studies (Peebles et al., 2019). Nugent scoring based on microscopic evaluation of Gram-stained vaginal smear samples provided standardized measures for assessing vaginal health (Nugent et al., 1991; Coleman and Gaydos, 2018). Although the Nugent scoring is proposed as a gold standard and allows comparability between ethnic groups, it requires time and expertise, for which the Amsel criteria is more preferred especially in resource-limited settings (Bhujel et al., 2021). Clinically, the Amsel criteria are more commonly used, where 3 out of the 4 conditions are met for a diagnosis: 1) thin, homogenous discharge; 2) pH of vaginal fluid above 4.5; 3) clue cells on microscopy (more than 20% of epithelial cells); 4) positive KOH test (Amsel et al., 1983; Donders, 2010). However, given the lower specificity and sensitivity of the Amsel criteria (37 to 70%) as compared to Nugent scoring (94-97%) (CDC, 2021), variations in clinical diagnosis can affect the understanding of the contribution of ethnic differences to the prevalence of the condition and the etiology of the disease relevant to the microbiome.

First-line treatment for BV is mainly clindamycin or metronidazole, with cure rates decreasing from >90% (Joesoef et al., 1999) to 50-80% (Eschenbach, 2007) in recent reports of known BV cases, accompanied by high recurrence rates (Bradshaw et al., 2006; Coudray and Madhivanan, 2020; Verwijs et al., 2020). The variability in response to antibiotic treatment may be due to differences in how efficient the inconsistency the BV-associated biofilm is disrupted (Bradshaw and Sobel, 2016; Wu et al., 2022; Sousa et al., 2023). Further, within biofilms, BV-associated bacteria may possess inherent antibiotic resistance mechanisms, such as pumping, chemical neutralization, and matrix barriers (Hardy et al., 2017; Nourbakhsh et al., 2022). This poor clearance can possibly lead to a high recurrence rate, where more than half of the women experience BV symptoms again within one year of the antibiotic treatment and likely enrichment of antibiotic-resistance genes in the vaginal environment (Beigi et al., 2004a; Bradshaw et al., 2006).

During BV, the vagina fosters a pro-inflammatory environment, which can be partly attributed to the immunomodulatory effects of the anaerobic bacteria and their metabolites. Notably, short-chain fatty acids (SCFAs) are elevated during BV and their abundance is associated with increased inflammation (Delgado-Diaz et al., 2020; Schwecht et al., 2023). The presence of SCFAs such as acetate, propionate, butyrate, and succinate combined with biogenic amine production in the absence of lactobacilli increases vaginal pH >4.5 (O’Hanlon et al., 2011), making it favorable for growing anaerobic bacteria that produce virulence factors that can degrade mucin, compromise the vaginal epithelial barrier integrity, and stimulate pro-inflammatory responses (Aldunate et al., 2015). Further, the recognition of bacterial cells by the toll-like receptors (TLRs) on the vaginal epithelial cells launches an inflammatory response (Aldunate et al., 2015; Delgado-Diaz et al., 2020). Aside from the production of amines, recent research found that specific amino acid metabolites secreted by BV-associated bacteria, such as imidazole propionate, can interfere with vaginal epithelial cell function and further contribute to inflammation via the mTOR signaling pathway (Berard et al., 2023).

Within the context of the vaginal microbiome, the molecular and pathophysiological mechanisms of BV development remain largely unknown. What is known so far is that women suffering from BV are characterized by a dysbiotic vaginal microbiome dominated by a diverse group of anaerobic bacteria, such as Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia, and Sneathia amnii (Fredricks et al., 2005; Muzny et al., 2019; Carter et al., 2023). The VMB is also depleted of Lactobacillus spp., except for L. iners, often found adapting and coexisting with BV-associated bacteria (Zheng et al., 2021). G. vaginalis and Prevotella have been touted as primary instigators (Machado et al., 2013; Muzny et al., 2018, 2019; van Teijlingen et al., 2024) of BV and biofilms are formed with other important anaerobic bacteria during pathogenesis (Bradshaw and Sobel, 2016; Hardy et al., 2017). Due to the nature of the polymicrobial infection, metabolic environmental shifts lead to continued host inflammatory responses and disrupted epithelial cell barrier, which become further aggravated, leading to secondary infections and adverse gynecological disease sequelae.

Aerobic vaginitis, due to its similarity to BV, can be misdiagnosed and incorrectly treated. It has been associated with complications such as vaginal inflammation, bacteriuria, lower urinary tract infections, and acute pyelonephritis (Kaambo et al., 2018). AV is characterized by an increase in enteric aerobic commensals not generally found in BV, such as group B Streptococcus agalactiae (GBS), Staphylococcus aureus, Escherichia coli, and enterococci, in the vaginal microbiome (Donders et al., 2017). Compared to the normal vaginal flora, these aerobic bacteria increase by 3- to 5-fold and are associated with vaginal mucosal inflammation due to the disruption of Lactobacillus dominance (Fan et al., 2013). The main symptoms are similar to those of BV but present as negative on the KOH test (Donders et al., 2015). Severe inflammation is apparent in vaginal smears, where an increase in intermediate and basal cells occurs. It is accompanied by increased turnover of the superficial epithelial cell layer and possibly epidermal desquamation, vaginal epithelial atrophy, small erosions, and ulceration (Petrova et al., 2015). Immune system dysregulation is the main factor contributing to AV pathogenesis (Oerlemans et al., 2020a). Similarly, reduced estrogen levels and localized immunity make AV prevalent in postmenopausal women (Stika, 2010). Pathogenic members of AV, such as GBS, are an essential focus, as they can occasionally cause morbidity in older adults, pregnant women, and patients with underlying medical conditions (Kwatra et al., 2014; Brigtsen et al., 2015). GBS colonization of pregnant women is considered a significant cause of severe neonatal infections, including neonatal sepsis, meningitis, and pneumonia (Krauss-Silva et al., 2014). In addition to GBS, Enterococcus faecalis has also been associated with preterm birth, low birth weight, and puerperal sepsis, resulting in significant maternal and neonatal morbidity and mortality (Cools et al., 2016; Kim et al., 2014).

As such, preferred treatments for AV are kanamycin and quinolones, which have intrinsic activity against disease-causing pathogenic bacteria while potentially minimizing interference with the vaginal microbiota (Tempera and Furneri, 2010). So far, the use of meclocycline and kanamycin for the treatment of AV allows a focus on Gram-negative bacilli rather than Gram-positive cocci, as these drugs are not absorbed and may leave extra vaginal lactobacilli even in patients with severe AV (Tempera and Furneri, 2010). Likewise, clindamycin for treating BV is also used for AV treatment due to its broad spectrum of activity against several aerobic Gram-positive coccal species; however, infection control with clindamycin is short-lived and may not cover all species associated with AV (Sousa et al., 2023). On the other hand, non-absorbable, broad-spectrum antibiotics such as carbapenems and clavulanic acid-beta-lactam combination (amoxiclav) are very effective for rapid, short-term improvement of severe symptoms from aerobic infections, especially deep cutaneous vulvovaginitis caused by GBS or methicillin-resistant S. aureus (MRSA) (Donders et al., 2015). Some beta-lactam antibiotics, however, are associated with less efficacy in eradicating E. coli vaginal colonization, leading to a high frequency of recurrent urinary tract infections (Stapleton, 2016). Nevertheless, due to significant inflammation in AV patients, including leukocyte and parabasal cell infiltration, frequent and prolonged use of antibiotics may result in adverse side effects. It may need to be accompanied by local administration of estrogens or a combination of probiotics and a very low dose of local estriol for postmenopausal or immunocompromised patients (Donders et al., 2015).

Like BV and AV, VVC is another significant public health concern. In the US alone, more than 70% of women experience fungal infection at least once in their life, with inflammation of the vagina and vulva as the primary concern (Nyirjesy et al., 2022). They are commonly colonized in the vagina by Candida albicans and non-albicans species such as C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis. Other risk factors for VVC include antibiotic usage, lifestyle choices such as sexual activity, serum glucose levels in diabetic and pre-diabetic females, estrogen balance, immunoregulatory deficiencies, allergies, and gene polymorphisms (Satora et al., 2023). Thus, VVC classifications help to form treatment modalities based on uncomplicated and complicated VVC. Those with uncomplicated VVC are infrequent, with mild to moderate symptoms. Candida albicans is usually the main culprit in uncomplicated VVC. On the other hand, non-albicans candidiasis mainly occurs in those who are immunocompromised, leading to more severe and recurrent VVC (Nyirjesy et al., 2022).

Immunity against candidiasis is complex and involves multiple processes, including pathogen recognition through receptors such as Toll-like receptors (TLR) and C-type lectin receptors (CLR), active innate and adaptive immune cells such as macrophages, dendritic cells (DC), and T cells, as well as the production of cytokines and chemokines such as IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α (Netea et al., 2015; Pathakumari et al., 2020; Zhou et al., 2021a). A prospective study found higher serum levels of cytokines, including IL-4, IL-6, and IL-10, and lower levels of IFN-γ, TNF-α, and IL-17F in patients with recurrent VVC compared to those with VVC. This indicates that Th1/Th2 immunity may play an essential role in VVC (Ge et al., 2022). The host immune response against candidiasis has been comprehensively summarized in other reviews and will not be discussed in detail in this review (Netea et al., 2015; Pathakumari et al., 2020; Zhou et al., 2021a).

However, Candida spp. has evolved mechanisms for immune evasion and dissemination by which it can hijack macrophages and instigate hyphal escape via pyroptosis (Uwamahoro et al., 2014) and macrophage extracellular trap formation (ETosis) (Netea et al., 2015; Olivier et al., 2022). To escape immune detection, Candida spp. executes morphological switching from non-infectious yeast to hyphae inside the macrophages. It instigates hyphal escape by producing pore-forming proteins such as candidalysin (Olivier et al., 2022), leading to inflammation and macrophage rupture, consequently releasing the pathogen. Further, Candida spp. can neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids that may promote evasion and survival, although regulated by mitochondrial respiration (Westman et al., 2018; Silao et al., 2024). Other non-canonical strategies promoting colonization and evasion include β-glucan masking, which is triggered by stressors, nutrients, antifungal drugs, and other factors such as nitrogen availability and quorum sensing molecules (Pradhan et al., 2019). Such adaptations may contribute to the possibility of recurrence aside from other factors that can promote or induce VVC.

Even in healthy females, Candida albicans can co-exist as commensal members of the Lactobacillus-dominated VMB. Vaginal lactobacilli are known to produce anti-Candida factors, specifically with growth inhibition and targeting of hyphal components. Nevertheless, it has been reported that candidiasis occurs more frequently with an L. iners-dominated microbiome (CST III) as compared to an L. crispatus-dominated flora (CST II) (Tortelli et al., 2020). L. crispatus can inhibit the growth of C. albicans through the production of lactic acid and other anti-Candida molecules (Jang et al., 2019) and has the potential to reduce hyphal morphogenesis (Wang et al., 2017). Lactobacilli can further inhibit C. albicans virulence by preventing fungal adhesion to vaginal epithelial cells and hindering biofilm formation (Takano et al., 2023). Candida has been found to co-occur with high abundance of lactobacilli and BV-associated bacteria (Liu et al., 2013). Candida colonization is more commonly asymptomatic and may not always progress to VVC (Tortelli et al., 2020). However, some studies have implicated lactobacilli-rich VMBs associated with symptomatic VVC (Beigi et al., 2004b; McClelland et al., 2009). Nevertheless, with focus on the species level of lactobacilli, it has been more linked to the presence of an L. iners-rich VMB than an L. crispatus-rich VMB (Tortelli et al., 2020). In this context, the exact mechanism leading to the switch between commensal and pathogenic forms of C. albicans is not fully understood, especially in the context of Lactobacillus-Candida interactions in the vaginal environment (Takano et al., 2023; Silao et al., 2024).

Azole antifungal agents are used as first-line treatment, leading to adverse consequences such as the rise in multidrug-resistant Candida spp. Thus, despite standard treatment, the high prevalence of VVC and yeast colonization still puts this high on the World Health Organization’s (WHO) “critical priority” group. A review of evidence conducted by Nyirjesy et al. (2022) identifies the need for novel treatment approaches for recurrent VVC where no alternative treatments, even with oral probiotics, have solid evidence for support yet. Nevertheless, oteseconazole, another antifungal drug that finished a phase 3 study, presents a new option for recurrent VVC with fewer adverse effects that was approved in the USA for women who are not of reproductive potential (Martens et al., 2022; Siddiqui et al., 2023).

BV contributes to the proinflammatory cytokines in the vaginal tract of HIV-infected women (Mitchell et al., 2008). However, with these inflammatory states and disrupted barriers, infectious particles such as HIV can traverse through the genital epithelium via tears in the squamous epithelium or transcytosis across the single cell layer of the endocervix, ultimately infecting underlying CD4+ target cells in the submucosa (Vyshenska et al., 2017). The net outcome of these interactions favors HIV infection and replication by attracting target cells, which will subsequently become infected and further propagate the infection.

Recent publications have demonstrated that women with non-Lactobacillus-dominant VMB were at a greater risk of HIV infection, where increased mucosal inflammation enhanced the rate of sexual transmission of HIV in the female genital tract (Vitali et al., 2017; Wessels et al., 2018; Wang et al., 2023). On the other hand, L. crispatus is associated with reduced inflammation due to an immunoregulatory environment, exclusion of BV-associated bacteria, and reduced HIV risk. However, L. iners has been associated with fewer immune benefits and lower HIV protection or exclusion of BV-associated bacteria, possibly providing a more intermediate transitional stage to dysbiosis (Armstrong and Kaul, 2021). It is possible that women can develop a persistent long-term state where BV is frequent and that this state is associated with more frequent HIV infection. BV is more prevalent and persistent among HIV-infected women, particularly among those who are immunocompromised (Jamieson et al., 2001; Schellenberg et al., 2012).

A dysbiotic VMB poses similar risks for the increase in HPV infection and persistence (Mitra et al., 2016; Vyshenska et al., 2017; Muzny et al., 2020). CST-III (L. iners-dominated) and CST-IV (dominated by facultative anaerobes) are suggested to be risk factors for persistent HPV infection (Mei et al., 2022) due to low protective immunity with the loss of healthy vaginal lactobacilli. In addition, HPV proteins E6 and E7 enhance IL-10 expression and macrophage type 2 production (Kremleva and Sgibnev, 2016). Cytokines such as IL-6, IL-8 and TGFβ-1 could also potentially influence the growth of different vaginal microbes (Kremleva and Sgibnev, 2016).

During this time, strict anaerobic bacteria start to colonize the vagina and destroy the protective barrier of the cervical epithelium. This event promotes HPV entry of host cells, paving the way for subsequent integration of the HPV virus into the host DNA and accompanying cell division for virus replication. Nevertheless, a Lactobacillus-dominated VMB, especially with L. crispatus (Cheng et al., 2020), protects against HPV and prevents HPV persistence and subsequent disease development (Zhang et al., 2018; Muzny et al., 2020). In another study, an L. gasseri-dominated microflora (CST II) is also associated with faster HPV clearance (Mei et al., 2022). Without these protective mechanisms in place, the immune system cannot cope with the strong immunological pressure brought about by dysbiosis, allowing tumor evasion strategies that lead to cervical intraepithelial neoplasia or cervical cancer development in unresolved HPV infections (Piersma, 2011; Zhou et al., 2021b).

Trichomonas vaginalis infection is associated with an increased risk of HIV infection and cervical and prostate cancer (Dessì et al., 2019; Margarita et al., 2020). The vaginal microbiota also plays a role in mediating susceptibility to T. vaginalis parasite infection, increasing the risk of trichomoniasis among women with BV or a Nugent score > 3 (Hinderfeld et al., 2019; Balkus et al., 2014; Margarita et al., 2020). Here, the loss of lactobacilli leads to an increased pH, creating an environment more favorable for T. vaginalis growth and pathogenicity (Margarita et al., 2020). Studies have also demonstrated an association between prevalent trichomoniasis and detection of Prevotella and Sneathia (non-amnii) species. These bacterial species may mediate susceptibility by producing nicotinamide metabolites that promote T. vaginalis infection (Jarrett et al., 2019). In contrast, suppression of L. iners in the initial interaction with T. vaginalis occurs but shows the possibility of adapting and surviving after longer exposure to T. vaginalis (Chiu et al., 2021). According to this relationship, interventions that decrease BV incidence and promote eubiosis could potentially contribute to reductions in trichomoniasis incidence (Balkus et al., 2014).

Lactobacilli display distinct strain differentiation in both genotypic and phenotypic characteristics within the same species. Within vaginal species, each genome encodes species-specific protein families. Among the most common vaginal lactobacilli—L. crispatus, L. gasseri, L. iners, and L. jensenii—L. iners exhibited the smallest average genome size at 1.3 Mbp, lacking integral membrane proteins, transcriptional regulators, acetyltransferase GNAT family members, and certain ABC transport proteins. However, they possessed unique ABC transporter permeases, thiol-activated cytolysin (inerolysin), and other protein families absent in other Lactobacillus species (Rampersaud et al., 2011; Macklaim et al., 2013). On the other hand, L. crispatus, known for its extensive genome size among vaginal lactobacilli, exhibited unique proteins such as DNA polymerase, bacteriocins, and toxin-antitoxin systems, absent in other vaginal strains. Generally, L. crispatus harbored all common protein families with the other Lactobacillus species, but no protein families were significantly more abundant in L. crispatus (Damelin et al., 2011). For L. gasseri, proteins related to copper resistance/homeostasis, toxin-antitoxin systems, and pediocin immunity were exclusive across all tested strains. In the case of L. jensenii, protein families involving families associated with ABC transport, transcriptional regulation, and a citrate transporter were unique. However, certain protein families such as the glucitol/sorbitol phosphotransferase system were present in other vaginal lactobacilli but were missing in L. jensenii (Merhej et al., 2009; Mendes-Soares et al., 2014).

Pan et al. (2020) also compared phenotypic assays of L. crispatus and L. gasseri in healthy vaginal microbiomes and human gastrointestinal tracts. Variations in the growth patterns and fermentative capacities are observed within the same species, where vaginal lactobacilli performed well in a simulated vaginal fluid, demonstrating higher lactic acid resistance at a later growth phase was necessary for the maintenance of low vaginal pH. Their findings underscored that the isolation source of a strain could influence its potential probiotic functionality, emphasizing its consideration in probiotic formulation (Pan et al., 2020). Similarly, clinical evidence shows that specific oral probiotic strains or their combinations may elevate vaginal lactobacilli counts in healthy women or women with BV and/or VVC and support natural VMB during/after recovery from antibiotics/antifungal treatment (De Alberti et al., 2015; Heczko et al., 2015; Mezzasalma et al., 2017). Nevertheless, current clinical research also shows that this has not always been the case in other BV, GBS infection and VVC cohorts, where there was no consistent improvement and vaginal colonization of orally administered probiotic strains (Hanson et al., 2014; Husain et al., 2020; Farr et al., 2020). As far, it seems that given the longer length, higher dosing, and necessity for lactobacilli to withstand gastric and bile acids for treatment via the oral route, the direct intravaginal route of antibiotic and LBP therapy is deemed more effective, relevant to the source of the strain.

Analysis of L. crispatus strains from various environments revealed genetic adaptations of strains to their ecological niches. Certain well-adapted metabolic and growth capacities to the vaginal tract can provide an advantageous basis for colonization and provide means for restoration of the natural vaginal lactobacilli. Notably, a vaginal isolate, L. crispatus PRL2021, demonstrates superior ecological competence in a simulated vaginal fluid compared to other vaginal Lactobacillus species, indicating its potential in higher growth performance in the vaginal microbiome and competitive mechanisms (Mancabelli et al., 2021). L. crispatus strains from Lactobacillus-dominated and dysbiotic vaginal microbiomes were also studied, but with no phenotypic distinction observed between strains, specifically with organic acid production (Abdelmaksoud et al., 2016; van der Veer et al., 2019). However, they observed a disproportionally higher abundance of gene fragments encoding for glycosyltransferases among strains isolated from dysbiotic microbiomes, suggesting a role for cell surface glycoconjugates in shaping vaginal microbiota-host interactions. Additionally, they noted the ability of L. crispatus to grow on glycogen, correlating with the presence of a full-length pullulanase type I gene (van der Veer et al., 2019). In all studies, glycogen utilization deemed favorable for selection as it is the most abundant carbohydrate source in the vaginal milieu, supporting both growth and lactic acid production.

Lactic acid has both antimicrobial and immune modulatory functions. It does not only acidify the vaginal environment but is also associated with lower levels of pro-inflammatory cytokines (e.g., IL-1α, IL-6, IL-8, and TNFα), which help to generate a homeostatic environment (Aldunate et al., 2015). Several in vitro studies have demonstrated that lactic acid is a potential antiviral and bactericidal compound, inhibiting replication/growth of genital STI pathogens and pathobionts, including C. trachomatis, N. gonorrhoeae, GBS, HPV and HIV (Muzny et al., 2020). In this context, acidity and protonated lactic acid are responsible for the anti-trichomonadal effects of L. gasseri (Pradines et al., 2022) alongside other lactobacillar bacteriostatic and bactericidal compounds in the vagina (Brotman et al., 2012). Other antimicrobial compounds such as hydrogen peroxide-producing enzymes are common among vaginal L. crispatus strains (van der Veer et al., 2019). The rationality of hydrogen peroxide production as a significant factor in vaginal health has been debated due to the lack of oxygen in the healthy vaginal tract (Tachedjian et al., 2018), and that physiological concentrations of hydrogen peroxide (<100 uM) do not affect BV bacteria (O’Hanlon et al., 2011). Nevertheless, given certain physiological factors causing vaginal dysbiosis such as in AV and VVC, it may still be relevant as the natural vaginal strains still maintain the core functional genes. In this context, vaginal L. crispatus supernatants are strongly inhibiting of Candida albicans growth, virulence gene expression and hyphal formation (Wang et al., 2017). Furthermore, bacteriocins have also been found in certain vaginal lactobacilli. Lactocin 160 from a vaginal L. rhamnosus, induces transient pore formation via disruption of the chemiosmotic potential on the cytoplasmic membrane of G. vaginalis (Turovskiy et al., 2009), whereas Gassericin E from L. gasseri EV1461 affects both related and non-related strains, inhibiting the growth of BV pathogens (Maldonado-Barragán et al., 2016).

Further, LBP host colonization and the inhibition of pathogen adhesion are important selective traits for maintaining mucosal barrier integrity (Boris et al., 1998; Osset et al., 2001; Delgado-Diaz et al., 2022). Lactobacilli can induce host defense against pathogens through the formation of microcolonies that attach to epithelial cell receptors and form a physical barrier to pathogen attachment (Lebeer et al., 2010; Petrova et al., 2013). For instance, L. gasseri inhibits T. vaginalis colonization of vaginal epithelial cells through the action of its cells and a cell-surface aggregation-promoting factor (Pradines et al., 2022). Similarly, genomic studies of L. crispatus also found genes encoding fibronectin-binding L. crispatus protein (LACT01268) and L. crispatus adhesins (LACT01712 and LACT02327) that have been found to interfere with fibronectin-binding and pilus components of G. vaginalis (Koumans et al., 2007; Yeoman et al., 2010). One such fibronectin-binding protein encoded by L. crispatus the LEA protein, which can potentially exert inhibitory effects by competing with the same attachment sites as the pili of G. vaginalis (Edelman et al., 2012; Ojala et al., 2014). Core proteins of L. crispatus may play a crucial role in protecting the vagina from pathogens and bacterial vaginosis and highlight intricate mechanisms through which L. crispatus maintains vaginal health.

At the same time, potential LBPs demonstrate the ability to improve host innate immune responses. TLRs on vaginal epithelial cells regulate the production of cytokines by responding to molecular patterns (MAMPs) present on the bacterial surface. For instance, Gardnerella can induce an epithelial cell response through nuclear factor kappa B (NF-κB) activation via a TLR2-dependent signaling pathway (Anton et al., 2022). In AV, inflammatory responses can also be induced through TLR4 activation of NF-κB (Mirmonsef et al., 2011; Rose et al., 2012). Given this, TLR2/TLR6 modulation has been suggested to play an anti-inflammatory role in certain situations (Li et al., 2013). To ameliorate the low activation of TLR2/TLR6 typically observed in an inflammatory state, Lactobacillus-dominated commensal communities may be an important part of maintaining homeostasis and inducing an immunomodulatory role to reduce vaginal inflammation (Nasu and Narahara, 2010; Hearps et al., 2017). Surface active molecules such as bacterial exopolysaccharides (EPS) and peptidoglycan on the cell surfaces are also present in L. crispatus (Ojala et al., 2014; Chee et al., 2020). EPS can enhance the ability of vaginal VK2 cells in producing anti-candidal human defensin-2 protein, and likewise improving colonization potential (Donnarumma et al., 2014). In vitro evidence also shows the ability of the vaginal L. crispatus peptidoglycan to stimulate CD207 expression in Langerhans cells, the antigen presenting dendritic cells in vagina, to reduce HIV receptor entry (Song et al., 2018).

Lastly, it has been demonstrated that certain Lactobacillus species such as L. delbrueckii and L. rhamnosus taken as oral probiotics may dampen lipopolysaccharide (LPS)-induced expression of HLA-DR, CD86, CD80, CD83, and IL-12 in human dendritic cells (Esmaeili et al., 2018; Odendaal et al., 2024). Although it is not yet well understood, it may be a process through which the microbiota modulates immunotolerance in early pregnancy (Inversetti et al., 2023; Odendaal et al., 2024). Nevertheless, further investigation is needed to explore the mechanisms associated with local immunomodulation within the cervicovaginal microbiome, and their relevance to the implantation regulation and pregnancy development.

In the US, alternative treatment options are classified only as vaginal probiotics, due to the more straightforward track for getting them out to the market. Currently, there are seven different vaginal probiotics available for BV support. The AZO Complete Feminine Balance™ and Jarro-Dophilus^® Women contain similar strains of L. crispatus LbV 88, L. jensenii LbV 116, L. gasseri LbV 150N, and L. rhamnosus LbV 96, which are a mix of the most common Lactobacillus spp. found in the vagina and has been documented to have increased recovery rates from BV (Laue et al., 2018). On the other hand, Bio-K+^® Women’s Health, Jarrow Formulas^® Fem-Dophilus^® 1 Billion, Jarrow Formulas^® Fem-Dophilus^® 5 Billion, RepHresh™ Pro-B™ Probiotic, and UltraFlora^® Women’s utilize strains isolated from the urogenital tract, such as L. reuteri RC-14 and L. rhamnosus GR-1, which did not improve BV cure rates as a dietary supplement, but can contribute to improved vaginal flora composition (Hummelen et al., 2010).

Moving forward, it is crucial to consistently differentiate between using Lactobacillus strains as a traditional probiotic and its use as an LBP. More relevant regulatory functions and expectations are necessary for the drug development track, considering that the target populations are diseased individuals who may be immune-sensitive (Cordaillat-Simmons et al., 2020). As far, Lactobacillus bacteremia is rare and mainly correlated to intestinal translocation in severely immunocompromised patients (i.e. uncontrolled diabetes mellitus, malignancy or have undergone organ transplantation), urogenital pathologies, congenital and valvular heart diseases or those who have undergone invasive procedures, that may be more susceptible to probiotic use (Salminen et al., 2004; Rossi et al., 2022; Salminen et al., 2006; Bapna et al., 2023; Kullar et al., 2023). There are currently no clinical reports of Lactobacillus bacteremia with intravaginally administered LBP strains, but vaginal lactobacilli, specifically L. jensenii, has been reported in polymicrobial bacteremia with Veillonella montpellierensis during pregnancy in an immunocompetent but anemic patient at 33-weeks of gestation (Toprak et al., 2022). Another recent case involves L. jensenii and P. bivia polymicrobial bacteremia leading to renal and perinephric abscesses with an otherwise immunocompetent patient but has undergone ureteral stent procedure (Mohan et al., 2020). Other case reports of L. jensenii bacteremia within obstetrics and gynecology that have occurred after vaginal delivery (Marciniak et al., 2014) and elective abortion using dilatation and curettage (Suárez-García et al., 2012) with patients who presented with infective endocarditis.

Nevertheless, the use of lactobacilli as a biotherapeutic drug is still justifiable due to its lower risk of use as a “generally regarded as safe” (GRAS) microorganism and its positive benefit-risk ratio compared to antibiotic usage in patients suffering from recurrent vaginal infections. Likewise, such side effects and contraindications can be averted with a well-characterized LBP strain lacking pathogenic factors and a defined antimicrobial sensitivity spectrum. These LBP candidates should be further explored within the ongoing clinical trials. Nevertheless, same as other drugs, high doses should also be used with caution, and monitored carefully by the administering physician along with any presenting underlying illnesses.

Thus, vaginal lactobacilli developed as intravaginally administered LBPs are meant to undergo further trait selection and safety testing, including considerations of the proper dosing, delivery form and optimal administration frequency and route, therapeutic indications, and expected side effects. Further, strain-specific properties and treatments prior to LBP administration is crucial (Wu et al., 2022). Based on this, various treatment modalities have been considered, such as the use of single lactobacilli strains, multi-strain lactobacilli combinations, and vaginal microbiome transplantation (VMT), with varying levels of efficacy specifically against BV, with or without antibiotic therapy prior to LBP treatment.

Substantial research has explored LBPs for treating recurrent BV and VVC, but they are not yet a standard clinical approach due to differences in trial size, methods, and uses. Recurrent BV has been the most impactful in the health and economic sectors, especially with its associations with diseases such as HPV, HIV and Trichomonas infection. Thus, clinical trials utilizing strains native to vaginal microbiome are on their way for recurrent infections. Those with clinical trial IDs, are intravaginally administered, and have published results are summarized in Table 1 .

Early phase 1 trials have shown that vaginal administration of L. rhamnosus GR1 and L. reuteri RC-14 can increase vaginal Lactobacillus and modulate the immune response via TLR2 (Bisanz et al., 2014). EcoVag^® with L. gasseri DSM 14869 and L. rhamnosus DSM 14870 showed possibility for adjunct therapy during three menstrual cycles, demonstrating a higher BV-free rate (64.90%) in the lactobacilli group compared to placebo group (46.2%) (Larsson et al., 2008). EcoVag^® also shows strong potential in recurrent VVC alongside fluconazole treatment, but with only a 6- or 12-month cure rate of 50 and 67%, respectively, alongside clindamycin/metronidazole treatment for BV, respectively (Pendharkar et al., 2015). L. plantarum WCFS1, L. pentosus KCA1 and L. rhamnosus GG administered as a probiotic gel against VVC also achieved 45% symptom relief in their interventional therapy (Oerlemans et al., 2020b). Lastly in a phase 2 trial, L. rhamnosus BMX54 showed long-term use benefits for HPV clearance, in the context of vaginal dysbiosis (Palma et al., 2018).

As far, it is still debatable if the effect may be dependent on Lactobacillus species or their natural ecological niche, as similar reference strain standards have been utilized. However, given that most studies are interventional with BV recurrence, larger clinical cohorts are still lacking. Further, long-term effects are only found in a few of these studies, and few have been done without the use of antibiotics; thus, the effect on varying vaginal microbiomes, retention, and mechanisms of action of these probiotic strains developed as potential LBP candidates may still need elucidation as the LBP drug functionality is still lacking in most studies.

Nevertheless, it has generally been accepted that native strains are more competitive and well-adapted to their ecological niche, such as in the use of glycogen in the vaginal microenvironment as a carbon source. Most lactobacilli are not capable of utilizing glycogen directly, but native vaginal strains have a generally efficient acquisition of glycogen-derived resources released by α-amylase production that break down glycogen in the vaginal tract (Nunn and Forney, 2016). Notably, LBPs containing native vaginal microbiome strains also stand out for their potential benefits in biofilm disruption and immunomodulation (Łaniewski and Herbst-Kralovetz, 2021). Exciting new prospectives for using natural, non-antibiotic pre-treatment alternatives, potentially in combination with LBPs, lie ahead (Latham-Cork et al., 2021).

Extensive research highlights L. crispatus as advantageous, providing protection against various pathogens and correlating with positive health and pregnancy outcomes (Ghartey et al., 2014; Argentini et al., 2022; Baud et al., 2023). Furthermore, compared to other dominant lactobacilli strains, pangenomic gene analyses have shown greater enrichment and evolutionary acquisition of key beneficial and protective functional genes (Bhattacharya et al., 2023). Moreover, although bacteriocin and organic acid biosynthesis are still strain-specific, L. crispatus seem to more ubiquitously carry key functionalities as compared to the heterogeneity found in either L. iners, L. gasseri or L. jensenii (Bhattacharya et al., 2023).

L. crispatus strains DSM32717, DSM32720, DSM32718 and DSM32716 which come from a collection of reproductive tract microorganisms, have been phenotyped and observed to be effective in reducing BV and VVC symptoms using both oral and vaginal capsules (Mändar et al., 2023). However, as far, Lactin-V based on the single strain L. crispatus CTV-05, has been the only one that has reached clinical phase 3, recently showing relative success in their clinical phase 2 trials for recurrent urinary tract infections in non-pregnant women (Stapleton et al., 2011), and in their phase 2a, and phase 2b trials for recurrent BV applications (Hemmerling et al., 2010; Cohen et al., 2020). In the studies, a 78% colonization rate in women treated for BV, and a lower BV recurrence after 12 weeks with Lactin-V has been demonstrated (Hemmerling et al., 2010; Cohen et al., 2020). Furthermore, Lactin-V has recently shown to be generally safe and well tolerated in pregnant women (Bayar et al., 2023), and is currently being investigated for use in reducing pre-term birth in women at high risk and enhancing in vitro fertilization success rates.