Composition of a healthy vaginal microbiome

The vaginal microbiome (VM) is a diverse and dynamic microbial community, a composition that fluctuates throughout different stages of a woman’s life (Ref. Reference Auriemma, Scairati, Del Vecchio, Liccardi, Verde, Pirchio, Pivonello, Ercolini and Colao13). Lactobacillus, first discovered in 1892, was thought to be the sole microbial agent of the VM (Ref. Reference Amabebe and Anumba14). However, it was later established that VM is a complex structure, comprising various microbial community state types (CSTs) that alternate between different life periods, depending on different menstrual cycle stages (Refs Reference García-Velasco, Menabrito and Catalán15, Reference Ravel, Gajer, Abdo, Schneider, Koenig, McCulle, Karlebach, Gorle, Russell, Tacket, Brotman, Davis, Ault, Peralta and Forney16). Until now, five major CSTs have been identified, including CST-I, -II, -III and -V, dominated by L. crispatus, L. gasseri, L. iners, and L. jensenii, respectively, and non-Lactobacillus anaerobic microorganisms, including Gardnerella, Prevotella and Atopobium, comprise CST-IV (Table 1) (Refs Reference Dong, Wang, Wang, Xu, Liu, Li, Lv, Pan, Xiong, Liu and Zhu17, Reference Tsamir-Rimon and Borenstein18, Reference Molina, Andralojc, Huynen, Leenders and Melchers19).

Table 1.

Overview of CSTs and their bacterial composition in the vaginal microbiome

Table 1. Long description

The table is organized into ten columns representing different C S T categories and sub-types.

* C S T Roman numeral I: Divided into C S T-I A and C S T-I B. Both are dominated by Lactobacillus crispatus. C S T-I A has a lower abundance of Lactobacillus acidophilus and Lactobacillus iners with lower diversity. C S T-I B has a higher abundance of L. acidophilus and higher diversity.

* C S T Roman numeral II: Dominated by Lactobacillus gasseri.

* C S T Roman numeral III: Divided into C S T-III A and C S T-III B. Both are dominated by Lactobacillus iners. C S T-III B is specifically noted for being co-dominated by L. acidophilus and non-Lactobacillus species.

* C S T Roman numeral IV: Divided into four sub-groups. Sub-group A is dominated by Candidatus Lachnocurva vaginae with moderate Gardnerella vaginalis. Sub-group B is dominated by Gardnerella vaginalis and moderate Atopobium vaginae. Sub-group C 0 is dominated by Prevotella. Sub-group C 1, C 2, C 3, C 4 contains high levels of Streptococcus species, Enterococcus species, Bifidobacterium species, or Staphylococcus species isolates.

* C S T Roman numeral V: Dominated by Lactobacillus jensenii.

CSTs: community state types.

In post-menopausal women, a shift from Lactobacilli-dominant CSTs to CST-IV is observed, and the VM composition of post-menopausal women is predominantly inhabited by Streptococcus spp. (CST-IV C1, C2, C3, and C4) and Prevotella spp. (CST-IV C0), while Lactobacillus species (CSTs-I to III) are the dominant species in premenopausal women, in contrast to Atopobium species (CSTs-IV A and B), which are the predominant bacteria in perimenopausal women (Refs Reference Neumann, Pausan, Haid, Weiss, Kolovetsiou-Kreiner, Amtmann, Winkler, Mahnert, Jantscher-Krenn and Moissl-Eichinger20, Reference Brotman, He, Gajer, Fadrosh, Sharma, Mongodin, Ravel, Glover and Rath21). Moreover, although the VM in most healthy women of reproductive age consists of CST-I to CST-III, approximately 25% of women of child-bearing age do not possess a Lactobacillus-dominant vaginal composition, as observed in African and Hispanic women, probably due to cultural, behavioural and genetic differences (Ref. Reference Anahtar, Byrne, Doherty, Bowman, Yamamoto, Soumillon, Padavattan, Ismail, Moodley, Sabatini, Ghebremichael, Nusbaum, Huttenhower, Virgin, Ndung’u, Dong, Walker, Fichorova and Kwon22).

Throughout the pregnancy, the abundance of Faecalibacterium declines, and an increase in Proteobacteria spp., Streptococci and some Lactobacilli species, including L. vaginalis, L. crispatus, L. gasseri, and L. jensenii, is more prominent, particularly within the third trimester, resulting in a less diverse microbial community, with Lactobacillus species being the dominant group (Refs Reference Romero, Theis, Gomez-Lopez, Winters, Panzer, Lin, Galaz, Greenberg, Shaffer, Kracht, Chaiworapongsa, Jung, Gotsch, Ravel, Peddada and Tarca23, Reference Saraf, Sheikh, Ahmad, Gillevet, Bokhari and Javed24). Additionally, one longitudinal study of British women with uncomplicated pregnancies reported CST-I in 40%, CST-III in 27%, CST-V in 13%, CST-II in 9% and CST-IV in 8%. Nevertheless, not all women experience such alterations, as elevated levels of proteobacteria in the third trimester are not always documented in pregnant participants, possibly due to differences in gene expression profiles (Ref. Reference MacIntyre, Chandiramani, Lee, Kindinger, Smith, Angelopoulos, Lehne, Arulkumaran, Brown, Teoh, Holmes, Nicoholson, Marchesi and Bennett25).

Protective mechanisms of the vaginal microbiota

While CST-IV presence in a proportion of reproductive women is considered healthy, it has been associated with various vaginal-related infections (Ref. Reference Valeriano, Lahtinen, Hwang, Zhang, Du and Schuppe-Koistinen26). To maintain VM composition, various defence mechanisms within the vaginal cavity have evolved (Figure 1).

Figure 1.

A. Dysbiosis of vaginal microbiota and mechanisms of pathogens’ virulence. B. Normal vaginal microbiota and the Lactobacillus mechanism of action.

Figure 1. Long description

The diagram is divided into two main sections under a central illustration of the female reproductive system.

Panel A, titled Dysbiosis of vaginal microbiota, is on the left. At the top, a blue downward arrow indicates a decrease in antimicrobial byproducts and Lactic acid. This leads to Pathogens invasion and an Inflammatory response, resulting in an Imbalance V M. Below this, C S T I I I / I V diverse microbial communities are shown. A dashed arrow indicates mucus degradation and barrier disruption. The vaginal epithelium is labeled as Disrupted vaginal epithelium, covered by a layer of Thin mucus. Pathogens are shown forming a Polymicrobial biofilm on the surface. Blue and red arrows indicate a decrease in Estrogen and Glycogen, and an increase in Vaginal discharge and p H greater than 4.5.

Panel B, titled Normal vaginal microbiota, is on the right. At the top, a red upward arrow indicates an increase in Antimicrobial byproducts such as Bacteriocin and H sub 2 O sub 2. These byproducts are shown inhibiting pathogens. Below this, the Lactobacillus Domaine C S T I / I I / V is shown. Metabolic pathways show Pyruvate and Maltose converting to Lactic Acid, which is marked with a red upward arrow. The epithelium is labeled as Thick vaginal epithelium, covered by Thick mucus. Blue and red arrows indicate an increase in Estrogen and Glycogen, and a decrease in p H to less than 4.5.

Lactic acid production

Lactic acid (LA), produced by L. acidophilus, contains two isoforms, including dextro and levo, and is capable of inhibiting several pathological microorganisms’ growth, including chlamydial growth inhibition at LA concentrations of 48–167 mM (Ref. Reference Pessione27). Gong et al. speculated that the inactivation of responsible particles for cell attachment and the reduction of disulfide in the pathogen’s membrane, leading to membrane disruption, were the primary mechanisms by which LA exerted its effects (Ref. Reference Gong, Luna, Yu and Fan28). Most recently, another study documented suppression of tumour necrosis factor-α (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and enhancement of vaginal epithelial barrier integrity due to increased cell–cell adhesion protein expression in a dose- and pH-dependent manner at 100 mM concentrations of LA, proposing that there may be more to the role of LA in vaginal microbiome community balance and defence against microbiome dysbiosis (Ref. Reference Schwecht, Nazli, Gill and Kaushic29). Additionally, LA appears to irreversibly disrupt HIV infectivity at concentrations of 0.5% or higher, probably through unfolding of HIV glycoprotein (gp) 120 envelope protein (Ref. Reference Aldunate, Tyssen, Johnson, Zakir, Sonza, Moench, Cone and Tachedjian30). Moreover, similar results to HIV have been demonstrated regarding herpes simplex virus (HSV), C. trachomatis and N. gonorrhoeae (Ref. Reference Chen, Min, Wang, Zhao, Luo, Lei, Wen, Luo, Zhou, Peng and Li31). Furthermore, an increase in LA results in a lower vaginal pH, providing a suitable medium for Lactobacillus species development, thereby increasing H₂O₂ and bacteriocin production, which are known for their antimicrobial properties (Ref. Reference Jin, Tao, Pavlova, So, Kiwanuka, Namukwaya, Saberbein and Wawer32).

Hydrogen peroxide

H₂O₂ is an oxidizing, antimicrobial byproduct of vaginal microorganisms that is involved in various cell functions, including proliferation promotion and protein regulation (Ref. Reference Imlay33). This water-soluble ubiquitous molecule is synthesized within the vaginal cavity by anaerobic microorganisms, including Lactobacilli, which colonize as high as 10⁶–10⁷ organisms per mL (Ref. Reference Hillier, Ma, Klebanoff and Eschenbach34). Additionally, the NADH-flavin reductase has been hypothesized to have a role in H₂O₂ production in several Lactobacillus species deficient in H₂O₂-scavenging enzymes (Ref. Reference Hertzberger, Arents, Dekker, Pridmore, Gysler, Kleerebezem and de Mattos35). The oxidizing property of this molecule increases radical OH production, which in turn induces DNA damage in the invading pathogens (Ref. Reference Martín and Suárez36). Although H₂O₂ is efficient in the elimination of pathogenic microorganisms at physiological levels, higher concentrations induce Lactobacillus species destruction (Ref. Reference O’Hanlon, Moench and Cone37). Moreover, evidence suggests that H₂O₂ functions as an immunomodulator within the vaginal environment rather than as an antimicrobial agent, as it promotes immune cell recruitment, suppresses inflammasome activation and regulates immune responses (Ref. Reference Erttmann and Gekara38). Moreover, despite speculations, physiological and supra-physiological levels of H₂O₂ have been reported to have no antimicrobial effects on pathogens responsible for BV, corroborated by Mijacˇ et al., who observed no protective effects in the presence of H₂O₂-producing Lactobacilli, as the abundance of such microorganisms was similar in women with vulvovaginal candidiasis (VVC) and women with normal flora (Refs Reference O’Hanlon, Moench and Cone37, Reference Tachedjian, O’Hanlon and Ravel39). Conversely, another study reported declining rates of Candida spp isolations following exposure to H₂O₂-producing Lactobacilli (Ref. Reference Hillier, Ma, Klebanoff and Eschenbach34). Altogether, the data support the hypothesis that H₂O₂ may exert antibacterial effects in the presence of other antimicrobial agents, whereas in isolation, its antibacterial properties are limited.

Bacteriocin secretion

Bacteriocins are ribosomally synthesized, protease-sensitive peptides or small proteins produced by Gram-positive and Gram-negative bacteria, exhibiting both bactericidal and bacteriostatic activities, which, based on structural features, heat stability and post-translational modifications, are categorized into three major classes: (1) Class I comprises post-translationally modified bacteriocins, including lantibiotics (Ia), labyrinthopeptins (Ib) and sanctibiotics (Ic); (2) Class II consists of unmodified, heat-stable bacteriocins, divided into pediocin-like (IIa), two-peptide (IIb), circular (IIc) and linear (IId) types; and (3) Class III bacteriocins are heat sensitive, such as colicin from Escherichia coli, helveticin M from L. crispatus, helveticin J from L. helveticus and enterolysin A from Enterococcus faecalis (Ref. Reference Yang, Lin, Sung and Fang40). Following their synthesis, bacteriocins attach to specific receptors on pathogenic microorganisms, permeabilize the bacterial cell wall and induce pore formation, eventually leading to cell leakage and intracellular adenosine triphosphate (ATP) depletion (Ref. Reference Cotter, Hill and Ross41). Additional effects include inhibiting intracellular enzymes by microcins and colicins, and cell wall degradation, particularly by lantibiotics and class IIa bacteriocins (Ref. Reference Liang, Zhou, Peng, Liang, Liu, Zhu and Mou42). Moreover, class I imposes its antibacterial effect by inhibiting cell membrane synthesis, while class II exerts this effect through pore formation (Ref. Reference Zheng, Gänzle, Lin, Ruan and Sun43).

Bacteriocins have also been reported to influence pathogenic bacteria within the VM community through mechanisms with inhibitory spectra ranging from broad to narrow and by preventing invasion by other species. One recent study reported in vitro identification of a bacteriocin produced by L. acidophilus KS400, which imposed its antimicrobial effects against Gardnerella vaginalis, Streptococcus agalactiae and Pseudomonas aeruginosa, with a loss in the antibacterial properties 32 hours post-fermentation, probably due to proteolytic degradation (Ref. Reference Gaspar, Donders, Palmeira-de-Oliveira, Queiroz, Tomaz, Martinez-de-Oliveira and Palmeira-de-Oliveira44). Moreover, bacteriocins produced by L. salivarius, L. fermentum and L. paragasseri are effective against E. faecalis, Enterococcus faecium, N. gonorrhoeae, and two Candida species (Refs Reference Pascual, Daniele, Giordano, Pájaro and Barberis45, Reference Nilsen, Swedek, Lagenaur and Parks46, Reference Vera Pingitore, Hébert, Nader-Macías and Sesma47).

Competitive exclusion and biofilm formation

Although it has been speculated that the composition of biofilm microorganisms is formed through the cooperation of different species, it is now evident that such a structure is accomplished through competition within biofilms, which are a matrix assembly of living microorganisms (Ref. Reference Foster and Bell48). In biofilms, microorganisms cooperate in synchrony against external threats via the Allee effect, which predicts a positive correlation between group density and composition, underscoring the benefits of grouping (Ref. Reference Allee and Bowen49). Although beneficial, such a composition increases the concentrations of antagonist metabolites, thereby increasing competition between microorganisms within the same matrix through suffocation, secretion of toxins into adjacent cells and antibiotic secretions against other species or even their kin, also known as kin competitors, which is observed in P. aeruginosa that exerts its competitive behaviour through two distinct strategies, bacteriophage utilization and changes in cyclic dimeric guanosine monophosphate (c-di-GMP), aiding surface adhesion (Refs Reference Tang, Yang, Zhuang, Han, Sun, Li, Fan, Cai, Yang, Yu, Yang and Li50, Reference Julou, Mora, Guillon, Croquette, Schalk, Bensimon and Desprat51). Similar interactions within the vaginal microbiome community can be observed in both pathogenic and protective biofilms (formed by Lactobacillus species), including G. vaginalis, which is categorized into four subgroups (A, B, C and D), often found together within a biofilm and resulting in increased kin competition through contact-dependent ways, which involves direct inoculation of bacterial toxins into their competitors (Ref. Reference Vaneechoutte, Guschin, Van Simaey, Gansemans, Van Nieuwerburgh and Cools52). Although contact-independent interactions have been speculated to exist among G. vaginalis subgroups, recent studies do not support this notion and instead attribute G. vaginalis kin competition to contact-dependent mechanisms (Ref. Reference Khan, Voordouw and Hill53). Moreover, G. vaginalis species appear to compete in a non-interfering, exploitative manner, known as scramble competition, in which the dominant subgroup (subgroup D) has the greatest ability to exploit a shared environment (Ref. Reference Khan, Voordouw and Hill53). Additionally, Lactobacilli species, the critical strains in the vaginal microbiome, exert their competitive traits through competition for nutrients, production of bacteriocins, exopolysaccharides and H₂O₂ and co-aggregation, ultimately leading to biofilm composition domination and hindering pathogenic microorganism growth, similarly observed within one study, where L. crispatus and L. plantarum strains were reported to produce highly potent anti-Candida cell-free supernatants (CFSs), further emphasizing the competitive nature of Lactobacilli species due to the competition for nutrients and inhibition of C. albicans adhesion and biofilm formation (Ref. Reference Takano, Kudo, Eguchi, Matsumoto, Oka, Yamasaki, Takahashi, Koshikawa, Takemura, Yamagishi, Mikamo and Kunishima54).

Immune modulation and barrier reinforcement

Lactobacilli strains may serve as probiotics, which are living microorganisms that ‘confer a health benefit to the host’, and have been reported to significantly mitigate inflammatory responses by reducing TNF-α and IL-1β and increasing IL-10. This anti-inflammatory cytokine effectively regulates host immune responses, thereby reducing unnecessary inflammation (Ref. Reference Steen, Wang, Balaji, Butte, Bollyky and Keswani55). L. crispatus, a key component of CST-I, has recently been demonstrated to decrease Helicobacter pylori adhesion to gastric epithelial cells and downregulate gene expression of several pro-inflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, in addition to upregulation of IL-10, and TGF-ß and avoidance of innate immune responses stimulation via inhibition of toll-like receptor 2 (TLR2) ligand activation by masking this ligand with S-layer proteins, which are known modulators of immune system and inflammatory responses, resulting in decreased TLR2-dependent pro-inflammatory pathway activation, and thereby modulating immune responses and protecting vaginal barrier against pathogen colonization (Refs Reference Fakharian, Sadeghi, Pouresmaeili, Soleimani and Yadegar56, Reference Decout, Krasias, Roberts, Gimeno Molina, Charenton, Brown Romero, Tee, Marchesi, Ng, Sykes, Bennett and MacIntyre57). Additionally, L. crispatus reduces C. albicans virulence and modulates cytokine production and cell adherence in vitro (Ref. Reference Niu, Li, Zhang, Wang and Liu58). L. gasseri, similar to L. crispatus, suppresses disintegrin and metalloprotease 17 (also known as TNF-α-converting enzyme) expression, regulates the NF-κB pathway, a known factor of inflammation response induction, and inhibits pro-inflammatory cytokine production, such as IL-6, against H. pylori (Refs Reference Gebremariam, Qazi, Somiah, Pathak, Sjölinder, Sverremark Ekström and Jonsson59, Reference Gao, Fan, Luo, Zheng, Zhang, Cao, Zhang, Li, Huang, Zhang, Huang, Xiao and Qiu60). Consistently, this strain exerts immunomodulatory effects within the vaginal environment through indole-3-lactic acid production, which activates an aryl hydrocarbon receptor-mediated immune response (Ref. Reference Zhang, Zheng, Zhang, Cao, Lin, Sun and Qiu61). Moreover, L. iners is primarily known for its immunomodulatory and barrier-enhancing effects due to its role in increased TNF mRNA expression and activation of TLR signalling pathways under stress, and similarly, L. jensenii inhibits NF-κB and MAPK signalling pathways and reduces IL-6, IL-1α and IL-8 production, in addition to increasing IL-1β, IL-12 and IL-10 production (Refs Reference Gao, Fan, Luo, Zheng, Zhang, Cao, Zhang, Li, Huang, Zhang, Huang, Xiao and Qiu60, Reference Villa, Cipolla, D’Ippolito, Amar, Shachor, Ingravalle, Scaldaferri, Puca, di Simone and Scambia62, Reference Nori, McGuire, Lawton, McAuliffe, Sinderen, Walsh, Cotter and Feehily63). Additionally, increased mucus viscosity induced by Lactobacillus confers anti-DNA damage effects and viral trapping, thereby enhancing the vaginal barrier (Refs Reference Han, Liu and Chen6, Reference Delgado-Diaz, Jesaveluk, Hayward, Tyssen, Alisoltani, Potgieter, Bell, Ross, Iranzadeh, Allali, Dabee, Barnabas, Gamieldien, Blackburn, Mulder, Smith, Edwards, Burgener, Bekker, Ravel, Passmore, Masson, Hearps and Tachedjian64, Reference Wagner, Sobierajska, Kania, Paradowska and Ciszewski65).

Vaginal microbiome dysbiosis

Vaginal dysbiosis (VD) is defined as any imbalance within the vaginal microbiome that results in disruption of normal Lactobacillus dominance, the hallmark of vaginal health, and overgrowth of pathogenic microorganisms, such as anaerobic and facultative bacteria, including C. albicans, T. vaginalis, N. gonorrhoeae, Mycoplasma genitalium, C. trachomatis and viral pathogens, with a global prevalence of 18%, most commonly presenting as BV, which has been associated with various other infections, including HPV, HIV, C. trachomatis, and N. gonorrhoeae infections (Refs Reference Mei, Wang, Chen, Wei, Zhang, Cui, Meng, Zhang, Liu, Ding and Niu66, Reference Maksimovic Celicanin, Haahr, Humaidan and Skafte-Holm67). VD increases the risk of gynaecological non-infectious disorders as well, including cervical cancer, preterm birth and infertility, miscarriages, intrauterine adhesions, polycystic ovary syndrome and uterine fibroids (Refs Reference Hong, Qin, Huang, Ding, Ma, Xuan, Zhu, Peng and Wang68, Reference Grewal, Lee, Smith, Brosens, Bourne, Al-Memar, Kundu, MacIntyre and Bennett69). Known factors contributing to VD are sexual activity, antibiotic use, hormonal fluctuations, immunosuppression, lack of personal hygiene, ageing and lifestyle factors (Refs Reference Saraf, Sheikh, Ahmad, Gillevet, Bokhari and Javed24, Reference Valeriano, Lahtinen, Hwang, Zhang, Du and Schuppe-Koistinen26).

In cases of vaginal microbiota disturbances, reduced Lactobacilli species result in alkaline pH (>4.5) and create an optimal environment for overgrowth of pathological microorganisms, resulting in biofilm formation and activation of the mammalian target of rapamycin (mTOR) pathway (Refs Reference Kairys, Carlson and Garg70, Reference Berard, Brubaker, Birse, Lamont, Mackelprang, Noël-Romas, Perner, Hou, Irungu, Mugo, Knodel, Muwonge, Katabira, Hughes, Levy, Calienes, Lauffenburger, Baeten, Celum, Hladik, Lingappa and Burgener71). The mTOR pathway disrupts the function of epithelial cells and increases mucosal inflammation by promoting the production of pro-inflammatory cytokines, recruiting immune cells to the site of infection, and ultimately increasing vulnerability and the risk of persistent infection (Refs Reference Mafi, Mansoori, Taeb, Sadeghi, Abbasi, Cho and Rostamzadeh72, Reference Teufel, Horvath, Peter, Ercan, Piscuoglio, Hall, Finke and Lehmann73). Additionally, C. albicans and T. vaginalis can enter the superficial layer of the vaginal lining and trigger an inflammatory response, which is characterized by polymorphonuclear cells and macrophages, inducing candidiasis presentations and dysbiosis (Ref. Reference Jeanmonod, Chippa and Jeanmonod74).

Human immunodeficiency virus (HIV)

Researchers have speculated that the inflammation and decreased epithelial integrity induced by BV can facilitate HIV entry into the tissue and increase NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation and pro-inflammatory cytokines, thereby increasing intracellular HIV expression (Ref. Reference Loussert-Ajaka, Mandelbrot, Delmas, Bastian, Benifla, Farfara, de Vincenzi, Matheron, Simon and Brun-Vezinet75). G. vaginalis isolates, induced by BV, adhere to the vaginal epithelial cells and form biofilm and vaginolysin. This bacterial cholesterol-dependent toxin forms pores in the epithelium and triggers signalling cascades, thereby increasing lytic cell death (Ref. Reference Kairys, Carlson and Garg70). Several BV-associated metabolites reduce anti-inflammatory cytokine production, including IL-8 and IL-1α, and increase the activation of the NLRP3 inflammasome, which in turn activates caspase-1 and triggers pyroptosis and production of pro-inflammatory cytokines, including IL-1β and IL-18, further amplified by the production of lipopolysaccharides via anaerobes (Ref. Reference Blevins, Xu, Biby and Zhang76). One study specifically aimed to evaluate whether there is a difference between BV-associated bacterial species regarding genital HIV shedding and reported that women with lower Lactobacilli spp. and higher M. hominis isolates were at a 100-fold higher risk of genital HIV shedding (Ref. Reference Mirmonsef, Krass, Landay and Spear77). Moreover, BV-positive women aged 35 years or older appear to be at higher risk of HIV seroconversion (Ref. Reference Apalata, Nojaholo, Seipone and Nxasana78).

Reduced Lactobacillus species abundance increases recruitment of immune cells, including CD4+ T-cells, which are HIV’s primary targets (Ref. Reference Wall, Kilembe, Vwalika, Haddad, Hunter, Lakhi, Chavuma, Htee Khu, Brill, Vwalika, Mwananyanda, Chomba, Mulenga, Tichacek and Allen79). Interestingly, studies suggest that CD4+ T-cells and T-helper cells (Th) with higher CCR5 and α4β7 expression are more affected by HIV (Refs Reference McKinnon, Nyanga, Chege, Izulla, Kimani, Huibner, Gelmon, Block, Cicala, Anzala, Arthos, Kimani and Kaul80, Reference Iyer, Sabula, Mehta, Haddad, Brown, Amara, Ofotokun and Sheth81). Notably, within the vaginal microbiome, Th17 CD4+ T-cells with the capability to produce IL-17A highly express both CCR5 and α4β7, which turns them into optimal targets for HIV (Ref. Reference McKinnon, Nyanga, Chege, Izulla, Kimani, Huibner, Gelmon, Block, Cicala, Anzala, Arthos, Kimani and Kaul80). Moreover, tissue-resident memory CD4+ T cells (T_RM), which usually reside in the lower female genital tract, are the main reservoirs for this viral pathogen (Ref. Reference Cantero-Pérez, Grau-Expósito, Serra-Peinado, Rosero, Luque-Ballesteros, Astorga-Gamaza, Castellví, Sanhueza, Tapia, Lloveras, Fernández, Prado, Solé-Sedeno, Tarrats, Lecumberri, Mañalich-Barrachina, Centeno-Mediavilla, Falcó, Buzon and Genescà82). Both of these CD4+ T-cell types are recruited in VD and potentially increase HIV acquisition in individuals with BV (Ref. Reference Schellekens, Schmidt, Morré, van Esch and de Vos van Steenwijk83).

Human papillomavirus (HPV)

HPVs are intracellular pathogens that infect the primitive keratinocytes of basal cells and enter the phase of viral DNA replication, leading to an increased number of viral copies per cell (50 to 100 copies per cell), and then enter deeper levels of the epithelial cell, where high-level viral gene expression occurs (Refs Reference Chow, Broker and Steinberg99, Reference Doorbar100). Moreover, HPV-positive individuals possess either CST-III VM, dominated by L. iners (CST-III), or lower Lactobacilli isolates compared to HPV-negative women, and BV has been associated with persistent HPV infection, as persistent inflammation chronically increases pro-inflammatory cytokines and reduces immune system’s ability to recognize and disable HPV, further increasing viral replication and invasion and thereby persistent HPV infection in the setting of vaginal dysbiosis (Refs Reference Sharifian, Shoja and Jalilvand101, Reference Loonen, Verhagen, Luijten-de Vrije, Lentjes-Beer, Huijsmans and van den Brule102, Reference Gillet, Meys, Verstraelen, Verhelst, De Sutter, Temmerman and Broeck103, Reference Zeng, Li, Jiao, Cai, Yao, Xu, Huang, Zhang and Chen104).

The vaginal epithelium is a stratified squamous epithelium, comprising various cells, including keratinocytes, one of the primary target cells in HPV infections that aid modulation of cell stress and identification of pathogens (Ref. Reference Nestle, Di Meglio, Qin and Nickoloff105). Particularly, such regulation is exerted through the expression of TLRs following TLR3 activation, including TLR7, and the initiation of the production of type I interferons (IFN), such as IFN-α and IFN-β, that possess antimicrobial properties and amplify Th1-type cytotoxic responses (Ref. Reference Nasu and Narahara106). High-risk HPV species downregulate the production of IFNs via interfering with IFN gene expression and hinder activation of IFN-α-mediated signalling (Ref. Reference Shreya, Laura and Kast107). Additionally, stimulation of vaginal epithelial keratinocytes due to injury or infection increases NLRP3 inflammasome, which triggers pro-inflammatory cytokine production, including IL-1β, IL-1α, and IL-1, a pivotal cytokine in innate immune system activation that is responsible for activation of T helpers and dendritic cells and promotion of B cell maturation (Ref. Reference Martinon, Mayor and Tschopp108). Moreover, recent research suggests that HPV downregulates pro-inflammatory cytokine gene expression, particularly IL-1β and IL-6, ultimately leading to the absence of signals necessary for recruitment of immune cells to the site of infection and resulting in long periods of unrecognized viral replication (Ref. Reference Karim, Meyers, Backendorf, Ludigs, Offringa, van Ommen, Melief, van der Burg and Boer109). Expectedly, the high rates of uninterrupted viral replication can increase the risk of genomic instability, DNA damage, and neoplastic transformations (Ref. Reference Katerji and Duerksen-Hughes110). Additionally, during the initial phases of HPV infection, low concentrations of antigens are secreted, which prevent the immune system from detecting the infection (Ref. Reference Zhou, Tuong and Frazer111). These mechanisms all lead to reduced vaginal defence against HPV and increase the rates of HPV escape from immune system recognition (Ref. Reference Britto, Goes, Sivro, Policarpo, Meirelles, Furtado, Almeida, Arthos, Cicala, Soares, Machado and Giannini112).

T. vaginalis

One of the most prevalent forms of STIs is trichomoniasis, which accounts for 250 million cases globally and is caused by an anaerobic extracellular protozoan parasite known as T. vaginalis (Ref. Reference Leitsch113). Recent studies depict that T. vaginalis may have an association with vaginal microbiome, implicating that 72% of women with T. vaginalis possess CST-IV, particularly due to higher vaginal pH in the presence of CST-IV, and that 30% of T. vaginalis-infected women are co-infected with BV, with no apparent explanation on whether BV contributes to T. vaginalis pathogenesis or vice versa (Ref. Reference Brotman, Bradford, Conrad, Gajer, Ault, Peralta, Forney, Carlton, Abdo and Ravel114). Additionally, the concurrent existence of CST-IV and T. vaginalis appears to disrupt epithelial tight junctions and increase epithelial permeability (Ref. Reference Hinderfeld, Phukan, Bär, Roberton and Simoes-Barbosa115).

Upon adherence to vaginal epithelial cells via parasite surface lipoglycan and other membrane proteins, T. vaginalis induces the production of pro-inflammatory cytokines, including IL-1, IL-6, IL-8, and IL-17 (Ref. Reference Han, Kim, Kim, Ahn and Ryu116). IL-1, in particular, can activate the NLRP3 inflammasome, resulting in an activated NF-κB pathway and increased pyroptosis, thereby creating a vicious cycle of vaginal dysbiosis and increased inflammation that is triggered via reductions in Lactobacilli species abundance, resulting in increased vaginal pH, elevated pro-inflammatory cytokine production, degradation of vaginal mucosa, increased cell death and increased growth of anaerobic bacteria, which in turn further increase the inflammation and promote growth of T. vaginalis (Ref. Reference Adapen, Réot and Menu117). Moreover, existing literature suggests that C. albicans and M. hominis can amplify the cytopathic properties of T. vaginalis in cases of co-infection (Refs Reference Ozçelik, Sümer, Değerli, Malatyalı and Sümer118, Reference Yuan, Chen, Qin, Shen, Qiao and Kong119). A recent systematic review and meta-analysis indicated an odds ratio (OR) of 1.87 and a hazard ratio (HR) of 2.08 regarding the association between BV and T. vaginalis co-infection. This is further corroborated by another study that found a two-fold increased risk of T. vaginalis infection acquisition in women with BV, suggesting a potential link to the diminished abundance of Lactobacilli caused by BV (Ref. Reference Schwebke, Nyirjesy, Dsouza and Getman120).

Hormonal contraception and menopause

VM colonization is traditionally thought to occur at birth when newborns inherit either maternal or environmental microbiota through vaginal delivery or C-section. However, recent studies suggest that colonization may initiate during the foetal stage, as few studies have reported isolating microorganisms from amniotic fluid and other foetal tissues (Ref. Reference Coscia, Bardanzellu, Caboni, Fanos and Peroni121). From birth until around 10 years of age, when the ovaries begin to produce oestrogen, a low abundance of Lactobacilli species has been reported (Ref. Reference Song, Acharya, Zhu, Deveney, Walther-Antonio, Tetel and Chia122). Following increased oestrogen production, an increase in glycogen and thickness of epithelium is observed. Furthermore, increased progesterone production during the luteal phase can promote epithelial turnover and shedding, leading to the release of glycogen from desquamated epithelial cells into the vaginal lumen. This free glycogen serves as a primary substrate for vaginal microbes, which metabolize it into glucose and subsequently ferment it into lactic acid, resulting in a reduced vaginal pH, suggesting that hormonal contraceptives may also affect Lactobacillus colonization (Ref. Reference Fosch, Ficoseco, Marchesi, Cocucci, Nader-Macias and Perazzi123). However, despite the protective role of lactic acid, menstruation can transiently increase the presence of anaerobic bacteria associated with BV, likely because menstrual bleeding alters the vaginal environment (e.g., increasing microbial diversity and reducing Lactobacillus spp.) (Ref. Reference Zhang, Bai, Zong, Li and Liu124).

Hormonal contraceptives, including the combined oral contraceptive pill (COCP) and the hormonal intrauterine device (IUD), function by continuously releasing synthetic oestrogen and/or progestin throughout the menstrual cycle. These hormones primarily inhibit ovulation and increase the viscosity of cervical mucus, thereby reducing the likelihood of sperm penetration and fertilization, and can also affect the VM composition, as one recent systematic review and meta-analysis revealed a strong negative correlation between the use of any hormonal contraceptive (excluding IUDs) and the prevalence, incidence or recurrence of BV (Refs Reference Casado-Espada, de Alarcón, de La Iglesia-Larrad, Bote-Bonaechea and Montejo125, Reference Vodstrcil, Hocking, Law, Walker, Tabrizi, Fairley and Bradshaw126). Menopause marks the permanent end of menstruation in women and is clinically diagnosed after 12 consecutive months without a menstrual period. It most commonly occurs around the median age of 51.4 years. It is associated with substantial hormonal shifts, most notably a marked decrease in oestrogen levels, which results in the thinning of the vaginal epithelium, decreased glycogen levels, and an increase in vaginal cavity pH, ultimately leading to a reduction in Lactobacillus spp. population (Ref. Reference Lehtoranta, Ala-Jaakkola, Laitila and Maukonen127).

Antibiotic use and over-the-counter hygiene products

Antibiotics are among the most frequently used medications for treating bacterial infections. In the study conducted by Oh et al., oral antibiotic administration in mice led to dysbiosis and an alteration in the composition of vaginal commensal bacteria, thereby enhancing the mice’s susceptibility to mucosal HSV-2 infection (Ref. Reference Oh, Kim, Chang, Kwon, Lee, Kang, Kim, Hwang, Yu, Nakae and Lee128). Karpinets et al. found that oral treatment with broad-spectrum antibiotics in mice altered both the composition and diversity of the gut and vaginal microbiomes, which subsequently influenced cervical cancer progression in an orthotopic model. While antibiotics reduced microbial richness and diversity in the gut, they increased both in the vagina. Beneficial taxa such as Bacteroides, Ruminococcaceae and Lachnospiraceae were enriched vaginally. In contrast, potential pathogens like Proteobacteria declined (Ref. Reference Karpinets, Solley, Mikkelson, Dorta-Estremera, Nookala, Medrano, Petrosino, Mezzari, Zhang, Futreal, Sastry, Colbert and Klopp129). While antibiotic therapy effectively decreases the prevalence of G vaginalis and other species associated with BV, the post-treatment microbiota is predominantly composed of L. iners, rather than the more protective species such as L. crispatus and L. jensenii (Ref. Reference Srinivasan, Liu, Mitchell, Fiedler, Thomas, Agnew, Marrazzo and Fredricks130). Ahrens et al. tracked the vaginal microbiota over 26 days in women treated for STIs or BV, comparing them to those from healthy controls. Treatment with metronidazole shifted the microbiota from CST-IV to CST-III in 50% of patients, while azithromycin and tetracyclines had minimal effect on the microbiota structure. G. vaginalis persisted in 30% of BV cases, and Ureaplasma parvum was often detected even after azithromycin treatment (Ref. Reference Ahrens, Andersen, Lilje, Johannesen, Dahl, Baig, Jensen and Falk131). On the other hand, Mayer et al. reported a rapid and significant shift in the vaginal microbiota after treatment for BV using specific qPCRs. They observed that Leptotrichia/Sneathia and Megasphaera were quickly cleared, and a substantial reduction in G. vaginalis was noted in all patients. However, since most women were treated with local metronidazole, the higher local concentrations of the drug may account for the observed differences (Ref. Reference Mayer, Srinivasan, Fiedler, Marrazzo, Fredricks and Schiffer132).

Practices related to personal hygiene, such as vaginal douching or other intravaginal activities (e.g. cleansing or the insertion of products), have the potential to affect the balance and stability of the vaginal microbiota. Various factors, including religious beliefs, cultural norms and the level of education or awareness regarding health often shape the choices individuals make regarding these practices (Ref. Reference Morsli, Gimenez, Magnan, Salipante, Huberlant, Letouzey and Lavigne133). Certain over-the-counter vaginal products can influence the vaginal epithelium and may have a cytotoxic effect on the survival of beneficial Lactobacillus species in the vaginal environment (Ref. Reference Łaniewski, Owen, Khnanisho, Brotman and Herbst-Kralovetz134). For instance, using soap or shampoo for vaginal cleansing could disrupt the vaginal microbiota, which may increase the risk of vaginal symptoms compared to simply using water for washing (Ref. Reference Baker, Padhi, Torondel, Das, Dutta, Sahoo, Das, Dreibelbis, Caruso, Freeman, Sager and Panigrahi135). In a study involving 25 healthy Dutch women with a median age of 24 years, 60% of whom used combined oral contraceptives, the effect of an over-the-counter lactic acid-containing intravaginal douching product on the vaginal microbiota was assessed. While routine vaginal douching did not significantly alter the composition of the vaginal microbiota or pH, its use during menstruation was associated with a 2.6-fold increased risk of vaginal dysbiosis (OR = 2.6; 95% CI: 1.0–6.5; p = 0.099). Additionally, menstruation alone was linked to a higher presence of anaerobes associated with BV (OR = 1.7; 95% CI: 1.0–2.8), and douching appeared to promote the occurrence of C. albicans infections (Ref. Reference Van Der Veer, Bruisten, Van Houdt, Matser, Tachedjian, Van De Wijgert, Vries and Van Der Helm136). A recent observational study by Brown et al. involving 33 sexually active women found that simply discontinuing vaginal douching did not result in significant alterations to the vaginal microbiota or an increased risk of BV. The study suggested that other external influences, including antibiotic use, lubricants, dietary factors, smoking cessation, and condom use, were more strongly associated with changes in the vaginal microbiota (Ref. Reference Brown, He, Shardell, Ravel, Ghanem, Zenilman and Brotman137).

Sexual activity and semen exposure

Recent studies have explored the potential connection between sexual activity and the vaginal microbiota, revealing that sexual behaviour can affect both its composition and diversity, while also significantly contributing to lower genital tract inflammation and the risk of STIs (Ref. Reference Brown, He, Shardell, Ravel, Ghanem, Zenilman and Brotman137). Moreover, various sexual behaviours, such as penile-vaginal intercourse, receptive oral sex, the utilization of sex toys and condom application, may significantly impact the composition of the vaginal microbiota (Ref. Reference Brown, Schwartz, Robinson, O’Hanlon, Bradford, He, Mark, Bruno, Ravel and Brotman138). In studies examining the effects of sexual intercourse with a stable partner on VM composition, no significant differences were found between women with stable partners and those with multiple partners (Ref. Reference Marconi, El-Zein, Ravel, Ma, Lima, Carvalho, Alves Rosane, Parada Cristina, Leite Sandra, Giraldo Paulo, Franco, Eduardo and Silva Marcia139). However, women who engaged in penile-vaginal intercourse were more likely to be colonized by a type III community state, with G. vaginalis present in a minority of cases (Ref. Reference Marconi, El-Zein, Ravel, Ma, Lima, Carvalho, Alves Rosane, Parada Cristina, Leite Sandra, Giraldo Paulo, Franco, Eduardo and Silva Marcia139). The results suggest that CST type III, which is dominated by L. iners, may not offer protective benefits and could serve as a risk factor for the development of STIs. Additionally, studies propose that G. vaginalis is acquired from the penile skin microbiota of a sexual partner, supporting the theory that G. vaginalis can be transmitted sexually during unprotected intercourse (Ref. Reference Novak, Ravel, Ma, Ferreira, da Rocha Tristão, Silva and Marconi140). Additionally, prostitution is a notable risk factor for alterations in the vaginal microbiota. It is frequently linked to various sexual practices, multiple partners, inconsistent condom use, and high rates of contraceptive use (Ref. Reference Novak, Ravel, Ma, Ferreira, da Rocha Tristão, Silva and Marconi140). The VM of sex workers typically lacks Lactobacillus, with bacterial communities characterized by CST-IV, significantly increasing the risk of acquiring STIs, particularly HIV (Ref. Reference Novak, Ravel, Ma, Ferreira, da Rocha Tristão, Silva and Marconi140).

Exposure to semen during unprotected sexual intercourse, as well as inconsistent or incorrect use of condoms, can contribute to the heterosexual transmission of HIV. This may occur by influencing the inflammatory response and disrupting the natural balance of the vaginal microbiota in the female genital tract, which could increase the risk of infection (Ref. Reference Schjenken and Robertson141). Moreover, semen can influence various physiological and pathological processes in the female genital tract. This includes changes in tissue structure, immune reactions to foreign antigens present in seminal fluid, and an increased risk of bacterial and viral infections, such as HIV (Ref. Reference Schjenken and Robertson141). The presence of microbial communities in semen can trigger immune and inflammatory responses by influencing and potentially disrupting the composition of the vaginal microbiota (Ref. Reference Mändar, Türk, Korrovits, Ausmees and Punab142). Following unprotected sexual intercourse, the microbial content of semen has been linked to a notable reduction in the levels of naturally dominant Lactobacillus species, along with an increase in various bacterial species commonly associated with BV (Ref. Reference Mändar, Türk, Korrovits, Ausmees and Punab142).

Modulating the vaginal microbiome to prevent STI-targeted therapies

Emerging evidence suggests that modulation of the vaginal microbiome toward a Lactobacillus-dominant state may be associated with improved gynaecological and reproductive health, including reduced risk of vaginitis (such as BV), cervical intraepithelial neoplasia, STIs and preterm birth, highlighting its potential as a therapeutic target in women’s health (Ref. Reference Vieira-Baptista, De Seta, Verstraelen, Ventolini, Lonnee-Hoffmann and Lev-Sagie143). Conventional therapeutic approaches, such as antibiotics and antifungal agents, frequently demonstrate limited efficacy and are associated with substantial rates of disease recurrence (Ref. Reference van de Wijgert and Verwijs144). Potential mechanisms through which exogenous Lactobacillus strains may influence the VM include vaginal recolonization, enhanced production or release of lactic acid and other antimicrobial substances, as well as modulation of the local mucosal immune response (Ref. Reference van de Wijgert and Verwijs144). Antibiotics often fail to penetrate BV-associated biofilms effectively, and prolonged use of these agents for recurrent BV has been associated with an increased risk of adverse reactions, antimicrobial resistance and secondary fungal infections, such as candidiasis (Ref. Reference Muzny, Łaniewski, Schwebke and Herbst-Kralovetz145). Multiple prospective studies have identified a link between disrupted VM, particularly BV and reduced levels of Lactobacillus species, and a heightened risk of acquiring STIs (Ref. Reference Bertini146). Women with BV have been reported to experience higher rates of infections such as HPV, HIV, HSV and pelvic inflammatory disease (PID). Accordingly, BV, a prevalent vaginal infection that is often asymptomatic, may serve as a predisposing factor for the acquisition of STIs. Therefore, eliminating this widespread condition in both developed and developing countries could be considered a key strategy in the primary prevention of STIs (Ref. Reference Bertini146). Various strategies have been proposed to therapeutically modulate the vaginal microbiome, including probiotics, prebiotics, synbiotics and VMT (Ref. Reference Xavier-Santos, Bedani, de Almeida Vieira, Padilha, Lima, Silva, Ferreira, Giraldo, Pagnossa, Sivieri, Antunes and Sant’Ana147).

Probiotics

Probiotics refer to live microorganisms that offer health benefits when administered in adequate amounts (Ref. Reference Hill, Guarner, Reid, Gibson, Merenstein, Pot, Morelli, Canani, Flint, Salminen, Calder and Sanders148). In the context of vaginal health, they are commonly applied either as preventive or therapeutic agents for vaginal disorders, and often serve as complementary treatments alongside conventional antibiotic or antifungal regimens. While generally regarded as safe, probiotics may occasionally lead to adverse effects, ranging from mild gastrointestinal symptoms to more serious complications such as sepsis or the horizontal transfer of antibiotic resistance genes to host microbiota (Ref. Reference Vieira-Baptista, De Seta, Verstraelen, Ventolini, Lonnee-Hoffmann and Lev-Sagie143). Research on probiotic therapy targeting the vaginal microbiota has predominantly focused on BV, with limited investigation into candidiasis and other forms of dysbiosis (Ref. Reference Vieira-Baptista, De Seta, Verstraelen, Ventolini, Lonnee-Hoffmann and Lev-Sagie143). Several factors complicate the interpretation of studies on probiotic therapy for vaginal health. First, probiotic formulations vary widely in their composition, often including different Lactobacillus and non-Lactobacillus species. Second, there is limited evidence supporting successful vaginal colonization by administered strains, and many studies fail to distinguish between endogenous and probiotic lactobacilli. Third, inconsistent manufacturing standards and insufficient quality control raise concerns about the purity and potency of commercial probiotic products (Ref. Reference Hillier149). Various vaginal probiotic formulations containing Lactobacillus species have been developed and evaluated, but their outcomes have generally been inconsistent (Ref. Reference France, Alizadeh, Brown, Ma and Ravel150). Several factors may account for the limited effectiveness of these probiotic treatments. In certain instances, the formulations included bacterial strains not typically found in the human vaginal microbiota, as they were developed initially for gastrointestinal applications (Ref. Reference Oerlemans, Bellen, Claes, Henkens, Allonsius, Wittouck, van den Broek, Wuyts, Kiekens, Donders and Lebeer151). Additionally, some probiotics were administered orally with the assumption that systemic physiological changes would promote a vaginal environment conducive to Lactobacillus colonization (Ref. Reference Oerlemans, Bellen, Claes, Henkens, Allonsius, Wittouck, van den Broek, Wuyts, Kiekens, Donders and Lebeer151). A recent randomized, double-blind, placebo-controlled clinical trial evaluated the effectiveness of Lactin-V, a vaginally administered probiotic containing L. crispatus. Administered to women with BV following metronidazole therapy, the probiotic was associated with a 15% reduction in BV recurrence compared to placebo, with recurrence rates of 30% and 45%, respectively (Ref. Reference Cohen, Wierzbicki, French, Morris, Newmann, Reno, Green, Miller, Powell, Parks and Hemmerling152). Due to the crucial role lactobacilli play in preventing infections such as HPV, HIV and HSV – as demonstrated in laboratory and animal studies – there has been increasing interest in developing lactobacilli-based probiotics as potential therapeutic agents for STIs in women. As a result, several investigations and clinical trials have been conducted to assess their efficacy (Ref. Reference Avitabile, Menotti, Croatti, Giordani, Parolin and Vitali11). Palma et al. have further demonstrated the significance of reestablishing eubiosis in managing HPV infections. In their investigation, 117 women with coexisting HPV and either BV or bacterial vaginitis – who had previously received antimicrobial therapy (metronidazole or fluconazole) – were administered L. rhamnosus BMX 54 (NORMOGIN®) as vaginal tablets for either a short duration (3 months) or a prolonged period (6 months). Women who underwent the extended probiotic regimen experienced markedly fewer recurrences of bacterial and vaginal infections than those on the shorter course. Additionally, long-term use of the probiotic was associated with improvements in HPV outcomes, including higher rates of resolution of HPV-related cytological abnormalities and increased overall clearance of the virus (Ref. Reference Palma, Recine, Domenici, Giorgini, Pierangeli and Panici153). Given that L. crispatus M247 is a well-characterized probiotic strain known for its ability to establish itself in the vaginal environment after gastrointestinal transit, it has been suggested that restoring vaginal eubiosis with this strain may enhance host defences against HPV, thereby promoting viral clearance. Supporting this hypothesis, a separate clinical trial demonstrated that a 90-day oral administration of L. crispatus M247 led to a shift toward a CST I profile (characterized by L. crispatus dominance) in the majority of HPV-positive participants, which correlated with a higher HPV clearance rate. By the conclusion of the trial, participants whose vaginal microbiota remained in CST III or CST IV continued to test positive for high-risk or low-risk HPV. In contrast, only 20% of those with CST I microbiota remained HPV-positive (Ref. Reference Di Pierro, Criscuolo, Dei Giudici, Senatori, Sesti, Ciotti and Piccione154). Table 2 summarizes the current clinical trials on probiotics, prebiotics, and synbiotics for vaginal flora restoration.

Table 2.

Summary of clinical trials evaluating probiotic, prebiotic and synbiotic interventions for vaginal health

Table 2. Long description

The table consists of six columns: Intervention, Conditions, Enrollment, Phase, Findings, and N C T dot Id. It lists 19 separate clinical trials.

* Row 1: Lactobacillus crispatus M 2 4 7 for vaginal flora imbalance and H P V. Enrollment 90. Phase 4. Study in progress.

* Row 2: L. crispatus M 2 4 7 (Crispact) for H P V. Enrollment 150. Not applicable phase. Not yet recruiting.

* Row 3: L. crispatus M 2 4 7 (Crispact) for H P V. Enrollment 66. Not applicable phase. Completed in 2024, results pending.

* Row 4: Monnalisa touch laser therapy plus L. crispatus M 2 4 7 for atrophic vulvovaginitis. Enrollment 75. Not applicable phase. Not yet recruiting.

* Row 5: S T P 4 multi-strain probiotic for vaginal diseases. Enrollment 80. Not applicable phase. Recruitment completed, results pending.

* Row 6: Metronidazole plus pr O Vag for B V and bacterial vaginitis. Enrollment 594. Not applicable phase. Showed significant delay in B V recurrence and improved Nugent scores.

* Row 7: Umeta-Miyue (L. rhamnosus G R-1 and L. reuteri R C-14) plus metronidazole for B V. Enrollment 126. Phase 4. Demonstrated improved microbiota composition.

* Row 8: Oral probiotic product for vaginal health. Enrollment 37. Not applicable phase. Results not publicly available.

* Row 9: Metronidazole and L. crispatus C T V-05 (L A C T I N-V) for B V. Enrollment 228. Phase 2. Significantly reduced B V recurrence (30 percent versus 45 percent).

* Row 10: L A C T I N-V for B V and H I V infections. Enrollment 45. Phase 2. Safe and reduced activated H I V target cells.

* Row 11: L A C T I N-V applicator (escalating doses) for B V. Enrollment 12. Phase 1. Safe and well tolerated at doses up to 2 times 10 super 9 C F U.

* Row 12: L A C T I N-V for B V. Enrollment 40. Phase 2. Safely colonized 61 to 78 percent of users.

* Row 13: Microbial product F B 1 0 1 for vaginal flora imbalance. Enrollment 34. Not applicable phase. Shifted microbiome toward Lactobacillus-dominated state for up to 8 weeks.

* Row 14: L. rhamnosus G R-1 and L. reuteri R C-14 for B V in postmenopausal women. Enrollment 14. Early Phase 1. Increased colonization and reduced Atopobium.

* Row 15: Multi-strain Lactobacillus probiotics for breast cancer patients. Enrollment 27. Not applicable phase. 63 percent showed improved microbiota versus 36 percent in placebo.

* Row 16: L. rhamnosus B M X 54 for vaginal infection and H P V. Enrollment 117. Phase 2. Long-term use (6 months) showed 79.4 percent resolution of cytological abnormalities.

* Row 17: V H-01 synbiotic suppository for vaginal personal care. Enrollment 200. Not applicable phase. 90 percent of users achieved optimal microbiome within 21 days.

* Row 18: Prebiotic plus probiotic supplement for B V and yeast infection. Enrollment 120. Not applicable phase. Recruitment not yet commenced.

* Row 19: O M N i-B i O T i C F L O R A plus for vaginal microbiome in infertile women. Enrollment 80. Not applicable phase. Significant increase in beneficial Lactobacillus after 1 month.

Note: For information about the status of R&D of probiotics, prebiotics and synbiotics, please see www.clincialtrials.gov.

Prebiotics and synbiotics

From a microbiome perspective, prebiotics are defined as nutraceutical compounds that stimulate the growth or activity of probiotic or beneficial endogenous microorganisms (Ref. Reference Gibson, Hutkins, Sanders, Prescott, Reimer, Salminen, Scott, Stanton, Swanson, Cani, Verbeke and Reid155). Due to the established effectiveness of prebiotics in enhancing intestinal health, studies have explored whether these compounds can also promote the growth of vaginal lactobacilli (Ref. Reference Collins, McMillan, Seney, van der Veer, Kort, Sumarah and Reid156). Monocultures of various Lactobacillus species (L. crispatus, L. vaginalis, L. gasseri, Lactobacillus johnsonii, L. jensenii, and L. iners), along with BV-associated bacteria and C. albicans, were examined in vitro to evaluate their ability to utilize prebiotics, including lactitol, lactulose, raffinose, and oligofructose. Among these prebiotics, lactulose was found to selectively stimulate the growth of vaginal lactobacilli, particularly L. crispatus, while showing no significant effect on BV-associated bacteria or C. albicans (Ref. Reference Collins, McMillan, Seney, van der Veer, Kort, Sumarah and Reid156). A randomized clinical trial with 39 participants compared galacto-oligosaccharides and Trifolium pratense extract to a placebo, following metronidazole treatment for BV. At 16 days, the treatment group showed consistently low Nugent scores (≤3), while 24% of the placebo group had scores >3 (p = 0.016). However, recurrence rates at day 84 were similar between groups (11% in treatment versus 19% in placebo). No adverse effects were reported (Ref. Reference Coste, Judlin, Lepargneur and Bou-Antoun157). The method of administering prebiotics remains controversial, with vaginal applications, including pessaries, creams, and douches, being the most commonly utilized. Prebiotics are generally considered safe, and any adverse effects, such as diarrhoea, bloating and flatulence, are typically attributed to their osmotic effects in the intestines (Ref. Reference Davani-Davari, Negahdaripour, Karimzadeh, Seifan, Mohkam, Masoumi, Berenjian and Ghasemi158).

A limitation of prebiotic use is its reliance on the presence of lactobacilli, which are often absent or significantly reduced in conditions of dysbiosis. Synbiotics, a combination of prebiotics and probiotics, are based on the concept that prebiotics can enhance the growth and functionality of probiotics (Ref. Reference Swanson, Gibson, Hutkins, Reimer, Reid, Verbeke, Scott, Holscher, Azad, Delzenne and Sanders159). In a randomized controlled study involving 48 women with recurrent BV, the addition of a probiotic formulation – comprising L. acidophilus GLA-14 and L. rhamnosus HN001 – along with bovine lactoferrin as an adjunct to metronidazole therapy was found to significantly reduce the recurrence rate of the condition (Ref. Reference Russo, Karadja and De Seta160). In another randomized, double-blind clinical trial, administering a combination of micronutrients and L. rhamnosus CAN-1 to 21 women undergoing HIV treatment resulted in increased CD4+ cell counts, fewer hospitalizations, and an overall improvement in quality of life (Ref. Reference Hemsworth, Hekmat and Reid161). In a double-blind, placebo-controlled trial, a synbiotic regimen containing L. plantarum and Pediococcus acidilactici was found to be safe and produced a modest increase in the CD4+/CD8+ ratio among HIV-positive participants (Ref. Reference Blázquez-Bondia, Parera, Català-Moll, Casadellà, Elizalde-Torrent, Aguiló, Espadaler-Mazo, Santos, Paredes and Noguera-Julian162). In contrast, supplementation with Lactobacillus casei Shirota over 12 weeks did not yield significant benefits in individuals living with HIV (Ref. Reference de Barros Tenore, Avelino-Silva, Costa, Franco, Sabino, Kalil, Barros, Zelinda and Georges163).

Vaginal microbiota transplantation: emerging data and ethical considerations

VMT involves the transfer of vaginal fluid from healthy donors to recipients, aiming to restore microbial balance (eubiosis) by introducing an entire exogenous bacterial community. In contrast to probiotics, which typically deliver selected bacterial strains, VMT provides a complete and diverse microbiome (Ref. Reference Abbe and Mitchell164) (Figure 2). A 2019 case series was the first to evaluate VMT in five women with recurrent BV unresponsive to antibiotics. Four achieved sustained remission—two after one transplant, two after multiple – with no adverse effects. Successful cases showed L. crispatus dominance, while the partial responder had L. gasseri, suggesting L. crispatus may be key to effective microbiota restoration (Ref. Reference Lev-Sagie, Goldman-Wohl, Cohen, Dori-Bachash, Leshem, Mor, Strahilevitz, Moses, Shapiro, Yagel and Elinav165). In a recent case study, a woman with a complex obstetric history and pronounced symptomatic vaginal dysbiosis dominated by Gardnerella species was successfully managed using VMT without the need for prior antibiotic treatment. Notably, the procedure resulted in the effective engraftment of donor strains, highlighting the potential of antibiotic-free VMT as a promising strategy for restoring vaginal microbial balance in challenging clinical scenarios (Ref. Reference Wrønding, Vomstein, Bosma, Mortensen, Westh, Heintz, Mollerup, Petersen, Ensign, DeLong, van Hylckama Vlieg, Thomsen and Nielsen166). In a double-blinded randomized trial, Wroending et al. showed that VMT without antibiotics effectively restored a healthy VM in women with dysbiosis. Donor microbiota successfully engrafted, the procedure was safe, and no severe adverse effects were observed, supporting VMT as a promising alternative to antibiotics (Ref. Reference Wroending, Vomstein, Lundgaard, Delong, Mollerup, Mortensen, Bosma, Hellerung, Engel, Wiil, Heintz, Halkjær, Hugerth, Hartwig, Petersen, Thomsen, Westergaard, Cour Freiesleben, Westh, Hylckama Vlieg, Ensign and Nielsen167). The primary risks associated with VMT include the potential transmission of pathogens from donor to recipient, notably antimicrobial-resistant microorganisms that may evade current detection methods. Additionally, the unintentional transfer of sperm present in vaginal fluid poses a risk of unintended pregnancy. Given that the long-term effects of VMT are still unknown, rigorous donor selection criteria and comprehensive screening protocols are essential to ensure safety. A standardized approach for screening universal VMT donors has been proposed to address these concerns (Ref. Reference DeLong, Bensouda, Zulfiqar, Zierden, Hoang, Abraham, Coleman, Cone, Gravitt, Hendrix, Fuchs, Gaydos, Weld and Ensign168). Future research involving larger cohorts and randomized, placebo-controlled trials is essential to evaluate the efficacy and long-term stability of VMT. Another emerging theoretical approach within microbiome transplantation involves the genetic engineering or manipulation of microorganisms to endow them with targeted properties, such as the ability to produce therapeutic compounds or exert specific health benefits (Ref. Reference Vargason and Anselmo169). Table 3 highlights the current clinical trials on VMT for vaginal flora restoration.

Figure 2.

Vaginal microbiota transplantation is an emerging therapeutic strategy aimed at reestablishing a balanced vaginal microbial community and mitigating BV. The primary objective of this intervention is to restore the disrupted microbial homeostasis commonly associated with BV. To begin with, suitable donors undergo comprehensive screening, which involves testing their blood, CVF, and urine. Following this, CVF samples are obtained and prepared under controlled conditions. Finally, the processed CVF, containing a balanced and healthy vaginal microbiota, is introduced into recipients to reestablish a stable vaginal microbial ecosystem and promote normal physiological function. BV: bacterial vaginosis; CVF: cervicovaginal fluid; VM: vaginal microbiome.

Figure 2. Long description

The diagram is organized into three main stages across the top and a processing workflow at the bottom.

Top-Left: Healthy donor. An anatomical illustration shows a healthy reproductive system. A circular inset labeled Eubiotic V M displays a balanced mix of green and blue rod-shaped bacteria. A blue arrow points down to a collection of Urine forward slash Blood forward slash C V F samples in a cup and test tubes.

Bottom Row: The samples move right into a Screening box containing lab equipment, a petri dish, and a microscope. This leads to an Assessment and sample collection stage, followed by a Sample processing stage where a final purified blue liquid is shown in a conical tube.

Center-Top: Recipient patient. The anatomical illustration shows yellow-shaded areas indicating infection. A circular inset labeled Dysbiotic V M shows a mix of pink rods and purple spheres. A large blue L-shaped arrow labeled Vaginal microbiota transplantation points from the processed sample tube up toward this patient.

Top-Right: Recovered patient. A black arrow points from the recipient to the final stage. The anatomical illustration shows a healthy state, and the circular inset labeled Restored V M shows the return of the green and blue rod-shaped bacteria.

Table 3.

Overview of clinical trials on vaginal microbiota transplantation (VMT) for vaginal flora restoration

Table 3. Long description

The table consists of six columns and four rows of data.

* Row 1: Intervention is Biological vaginal flora transplant. Condition is Recurrent bacterial vaginitis. Enrollment is 10 estimated. Phase is Not applicable. Findings state that in a pilot study of five women with recurrent B V, four showed complete symptom resolution and restoration of Lactobacillus-dominant microbiota with no adverse events. N C T Id is N C T 0 2 2 3 6 4 2 9.

* Row 2: Interventions include Biological vaginal microbiome transplant, Drug oral metronidazole, and Other sterile saline. Condition is Recurrent bacterial vaginitis. Enrollment is 62 estimated. Phase is Phase 1 and Phase 2. Findings indicate this randomized placebo-controlled trial showed good safety and promising colonization results for restoring Lactobacillus dominance. N C T Id is N C T 0 4 0 4 6 9 0 0.

* Row 3: Interventions are Biological vaginal microbiome transplantation and Biological placebo. Condition is B V. Enrollment is 100 estimated. Phase is Not applicable. Findings describe an ongoing trial where earlier pilot work showed V M T can restore healthy microbiota and reduce recurrence, though final results are not yet published. N C T Id is N C T 0 4 5 1 7 4 8 7.

* Row 4: Interventions include Biological vaginal microbiome transplant, Biological placebo, and Other no intervention. Condition is Vaginal dysbiosis and Vaginal microbiome. Enrollment is 302 actual. Phase is Not applicable. Findings note an ongoing trial where preliminary data indicate improvement in microbiome composition and clinical outcomes. N C T Id is N C T 0 4 8 5 5 0 0 6.

Note: For information about the status of R&D of VMT, please see www.clinicaltrials.gov.