The VMB plays a crucial role in female reproductive tract health and disease (15). A Lactobacillus-dominated VMB is generally considered indicative of a healthy vaginal environment, whereas an unhealthy VMB is characterized by high microbial diversity and heterogeneity, with reduced Lactobacillus abundance and increased loads of anaerobic bacteria, including Gardnerella, Atopobium, Mobiluncus, Prevotella, Streptococcus, Chlamydia and Mycoplasma (16). Following menarche, elevated circulating estrogen promotes vaginal epithelial proliferation and glycogen deposition; the breakdown products of this glycogen serve as preferential carbon sources for Lactobacillus spp., reinforcing their dominance and lowering vaginal pH (17). This acidic milieu, together with bacteriocins and other antimicrobial compounds produced by lactobacilli, establishes a mucosal microenvironment that is inhospitable to most other microorganisms but favorable to Lactobacillus spp. Consequently, Lactobacillus spp. are regarded as keystone members of the VMB during the reproductive years and contribute to protection against BV, pelvic inflammatory disease, candidiasis, sexually transmitted infections and oncogenic human papillomavirus infection (11,18). Ravel et al (18) characterized the VMB of reproductive-age women across four ethnic groups using 16S rRNA gene sequencing and defined five CSTs based on dominant genera and species (as shown in Fig. 1). CSTs I-III and V are LDOM communities (>90% Lactobacillus spp.), dominated respectively by L. crispatus, L. gasseri, L. iners and L. jensenii. By contrast, CST IV is called the Lactobacillus-depleted state and is dominated by strict and facultative anaerobes. It contains almost no Lactobacillus and shows high microbial diversity (19).

Throughout a woman's life span, both intrinsic and extrinsic factors modulate VMB composition. Prior to menarche, low estrogen levels correlate with a high-diversity microbiota comprising aerobic, anaerobic and enteric bacteria (20). Although some women maintain VMB stability across the menstrual cycle, the endocrine withdrawal of estrogen and progesterone during menses, alongside endometrial shedding and vaginal bleeding, induces transient shifts marked by decreased Lactobacillus abundance that typically rebounds in the follicular phase (21). During pregnancy, hormonal stability, markedly elevated estrogen concentrations and the absence of menses favor a Lactobacillus dominated (LDOM) VMB (22). Estrogen is the principal host factor sustaining the VMB, promoting epithelial thickening and intracellular glycogen synthesis (23). Glycogen is hydrolyzed by host α-amylase into glucose and maltose, substrates fermented by Lactobacillus spp. into lactic acid, thereby reducing local pH (24). Moreover, various vaginal bacteria encode amylase-like enzymes capable of glycogen metabolism (25,26). Glycogen may also originate from epithelial-cell lysis induced by high lactate concentrations and cytolysins secreted by Lactobacilli, a process potentially mediated by hyaluronidase-1 and matrix metalloproteinase (MMP)-8 (27). With the onset of puberty and rising estrogen, vaginal pH acidifies and meta-analyses have demonstrated enhanced VMB stability and Lactobacillus predominance (28,29). Similarly, cyclical and pregnancy-related fluctuations in VMB stability parallel estrogen levels; elevated estrogen is consistently associated with increased VMB stability, which is beneficial for vaginal health. Conversely, decreased estrogen post-pregnancy and in menopause is linked to reduced VMB stability; however, estrogen therapy in postmenopausal women restores a pronounced LDOM state (30,31). Notably, estrogen-containing contraceptives increase Lactobacillus abundance in reproductive-aged women and decrease BV incidence (32,33), further supporting a causal role for estrogen in VMB regulation.

During pregnancy, the VMB is characterized by decreased richness and diversity, with Lactobacillus spp. emerging as the dominant genus. This maintenance of microbial homeostasis is closely linked to rising estrogen levels (as shown in Fig. 2), which not only stimulate glycogen synthesis but also promote increased lactic acid production by Lactobacillus. As gestation advances into the late trimester, Lactobacillus proliferation is often accompanied by a marked decline in dysbiosis-associated taxa such as G. vaginalis, A. vaginae and P. bivia (34). Studies show that during pregnancy, vaginal microbiota communities dominated by Lactobacillus usually remain stable or slowly change into other community types still dominated by Lactobacillus (8,35,36). Communities showing dysbiosis in mid-gestation often become dominated by Lactobacillus. When Gardnerella vaginalis dominates, its communities are relatively unstable and usually shift to a Lactobacillus iners community (35,36). These dynamic shifts align with the overall trend toward an optimized vaginal microenvironment during pregnancy. As white women generally possess higher baseline Lactobacillus levels prior to conception (36), black women demonstrate a more pronounced transition toward Lactobacillus dominance and enhanced VMB stability throughout pregnancy (35,36), suggesting a stronger modulatory effect of gestation on the VMB in this population. At parturition, the precipitous decline in estrogen precipitates a significant destabilization of the VMB (37,38), leading to community restructuring: Lactobacillus proportions decrease while anaerobic genera such as Peptoniphilus, Prevotella and Anaerococcus proliferate; taxa whose overgrowth is often linked to adverse outcomes (8,37). Moreover, short interpregnancy intervals (<1 year) have been shown to markedly increase the risk of PTB and other obstetric complications (39-41).

Intra-amniotic infection is a major trigger of PTB (42), particularly in early gestation (43). Most intra-amniotic infections originate from ascending colonization of the lower genital tract by bacteria present before or during pregnancy (44) and cultures of preterm fetal membranes have confirmed the involvement of BV-associated species (45). Moreover, asymptomatic BV in pregnant women confers more than a two-fold increased risk of PTB compared with BV-negative women (46); BV diagnosed before 20 weeks' gestation is associated with a five-fold higher risk (47) and diagnosis before 16 weeks with a seven-fold increase (48). Clinical trials have investigated antibiotic treatment of asymptomatic BV to prevent PTB (49,50) and several studies suggest efficacy in high-risk populations (51-53). In addition, BV has been identified as an independent risk factor for PTB (54). The global prevalence of BV ranges from 23-29% (55), characterized by replacement of Lactobacilli with anaerobic Gram-negative bacteria in the VMB (56). A cross-sectional study of 50 BV patients using denaturing gradient gel electrophoresis fingerprinting detected significant enrichment of Firmicutes, Fusobacteria, Bacteroidetes and Acinetobacter (57). In BV-associated VMBs, Gardnerella, Atopobium, Prevotella, Porphyromonas, Sneathia, Mobiluncus, Mycoplasma BVAB1, BVAB2, Mageeibacillus indolicus (BVAB3) and Peptostreptococcus are frequently enriched (56). Moreover, G. vaginalis has been reported in the majority of clinically diagnosed BV cases (58). Numerous G. vaginalis isolates produce vaginolysin, a cholesterol-dependent cytolysin that disrupts mammalian cell membranes and induces cell death. Amino-acid sequence variations among vaginolysin toxins from different G. vaginalis strains underlie differences in pathogenicity (59). G. vaginalis comprises four clades, G. vaginalis, G. leopoldii, G. piotii and G. swidsinskii (60,61), all of which can occur at low abundance in asymptomatic women, often co-existing within the same VMB (62). Overgrowth of these taxa leads to Lactobacillus depletion and disruption of the natural lactic acid defense mechanism, causing vaginal pH to rise above 4.5 (63,64). BV-associated bacteria damage the vaginal mucosal and epithelial barriers by producing biofilms, sialidases and cytolysins, elevating pH and secreting colonization-enhancing enzymes. High expression of cholesterol-dependent cytolysin genes in L. iners and G. vaginalis correlates with reduced Lactobacillus abundance (65). Epithelial cell models indicate that pore forming toxins such as vaginolysin from G. vaginalis (66) and putative inerolysin from L. iners (67) interact with lipid rafts to lyse epithelial cells, suggesting a mechanism for BV pathogenesis. Vaginal biofilms resist clearance of BV associated bacteria by acidic environments, lactobacilli derived antimicrobials, host immunity and antibiotics (56,68). Although their precise role remains unclear, these biofilms are considered critical for BV onset and recurrence (68,69). In vitro studies show that initial attachment of G. vaginalis may be mediated by L. iners or Peptoniphilus spp., while biofilm maturation is enhanced by Atopobium vaginae, Prevotella bivia, Fusobacterium nucleatum and Mobiluncus spp (68,70), indicating that interspecies interactions promote biofilm formation.

Abnormal VMB is recognized as a potential risk factor for PTB. Clinical studies show that bacteria isolated from the placenta and fetal membranes of preterm deliveries closely match those in the vagina, supporting the idea that ascending vaginal colonization by pathogens frequently leads to intra-amniotic infection and PTB (71) and can be reproduced in various animal models of ascending infection (72,73). Romero et al (74) compared the VMB of 18 women who delivered preterm with 70 term controls and found no significant differences in bacterial taxa, relative abundances, or CST distributions when stratified by gestational age. A Peruvian cohort likewise reported no association between vaginal CST and PTB (5). These discrepancies may reflect differences in maternal ethnicity, geographic location, and gestational age at sampling. Fettweis et al (35) profiled the VMB and cytokine milieu of 45 African-American women with PTB vs. 90 term-matched controls. Multi-omics analyses showed markedly lower levels of L. crispatus and higher abundances of BVAB1, Sneathia amnii, TM7-H1 and a subset of Prevotella in the PTB group. Term deliveries were more likely to harbor L. crispatus and exhibit reduced prevalence of A. vaginae and G. vaginalis. A small study of nulliparous African-American women noted a nonsignificant trend toward reduced diversity in spontaneous PTB (75), and another black cohort found that although specific Lactobacillus abundances were not linked to PTB risk, lower overall diversity associated with PTB among African-American women (76). In a UK study, L. iners dominance at 16 weeks was markedly associated with cervical shortening and PTB before 34 weeks, whereas L. crispatus dominance strongly predicted term birth; high-diversity communities were not linked to cervical shortening or PTB, but Lactobacillus depletion was relatively uncommon in that cohort (77). The role of L. iners remains ambiguous, as it appears in both healthy and dysbiotic VMBs. Unlike the exclusionary L. crispatus, L. iners has a reduced genome lacking key metabolic functions (78), suggesting that L. iners has evolved to tolerate other vaginal microbes, including pathogens, and is often found in transitional microbiota states. Consequently, L. iners may be unable to prevent pathogen overgrowth during pregnancy, indirectly fostering high-risk VMB profiles. Srinivasan et al (79) reported that high levels of L. iners can be detected in women who are BV positive or BV negative and across a broad range of vaginal pH values. Although this phenomenon has been extensively documented under BV conditions (80), it does not provide direct evidence that the species is pathogenic during dysbiosis. Of women ~50% are thought to harbor L. iners (23), so its persistent detection during BV is unsurprising. Another study showed that the detection rate of L. iners is similar among women with normal, intermediate and BV Nugent scores (81); however, relative abundance may be more decisive and further work is required to quantify both relative and absolute levels under diverse conditions. Shipitsyna et al (82) observed a decline in L. iners during active BV and therefore suggested that it is unlikely to be a primary pathogen; by contrast, other investigators argue that communities dominated by L. iners are more prone to shift toward dysbiosis, whereas those dominated by L. crispatus appear more stable (83). A semi-quantitative qPCR study that sampled Belgian women (predominantly Caucasian) throughout the menstrual cycle found that during active BV, menstruation and after intercourse, the previously dominant L. crispatus sharply decreased, while L. iners became predominant, often accompanied by a concomitant rise in Gardnerella vaginalis (84), indicating greater adaptability of L. iners to fluctuations in the vaginal environment such as BV. Macklaim et al (85) further demonstrated that genes involved in mannose and maltose uptake and glycogen degradation are markedly upregulated in L. iners under BV. Additional studies show that both the abundance and gene expression of L. iners vary with the phase of the menstrual cycle (86); it is also possible that host physiological changes first trigger BV and that L. iners subsequently adapts. Clarifying this temporal sequence will be critical for determining whether, and to what extent, L. iners is harmful to the host.

In a cohort of 111 pregnant women, those harboring only a single Lactobacillus species exhibited higher PTB risk than women with multiple Lactobacillus spp. In addition, this study also indicated that L. iners was the dominant species in PTB cases (87). Tabatabaei et al (7) similarly reported that Lactobacillus spp. may reduce PTB risk, whereas BV-associated CSTs increase it. Another study confirms that the Lactobacillus depleted CST (CST IV), marked by elevated Gardnerella or Mycoplasma, is linked to greater PTB risk (8), underscoring that Lactobacillus species diversity is a key determinant of pregnancy outcome. Four studies of PPROM cases revealed that ~50% of women exhibited moderate or low Lactobacillus dominance and high diversity (88). In individual PPROM patients, ~50% of those with Lactobacillus dominated VMBs prior to membrane rupture developed dysbiosis afterwards and erythromycin treatment exacerbated reductions in Lactobacillus alongside enrichment of potential pathogens (89). In a prospective cohort, 25% of women who later experienced PPROM had low Lactobacillus abundance and high microbial diversity before membrane rupture, compared with just 3% of women who delivered at term with intact membranes (90). Enhanced vaginal microbial diversity, particularly during the mid-gestational period, demonstrated a significant correlation with PPROM, while vaginal microbiota compositions lacking Lactobacillus predominance emerged as a consistent predictive biomarker for PPROM across all gestational age intervals.

Multiple studies have demonstrated significant population specific differences in vaginal microbiome composition and their associations with PTB risk. DiGiulio et al (91) identified ethnicity as a strong determinant of VMB composition and PTB risk. In a predominantly white cohort, CSTs characterized by Lactobacillus depletion, especially those enriched for Gardnerella or Ureaplasma, were associated with shorter gestational duration and higher PTB risk. A subsequent comparison of Californian white and Alabaman black women revealed more frequent Lactobacillus depletion and Gardnerella enrichment among black women, yet these features predicted PTB only in white women; in both groups, L. crispatus dominance conferred protection against early delivery (92). Hyman et al (93) reported greater bacterial diversity among white women who delivered preterm, while overall species diversity was higher in African-American women. A more recent investigation comparing black and non-black American women confirmed that Lactobacillus depletion was more prevalent in black women but increased PTB risk only in white women; it also identified distinct taxa markedly associated with spontaneous PTB, with stronger effects in the black cohort (94). The protective role of L. crispatus has also been validated in European, Middle Eastern and Asian populations, where L. iners dominance associated with higher PTB risk and L. crispatus dominance with term delivery (87). Sun et al (95) showed in stratified analyses that African-American women have increased abundance of Lactobacillus iners and decreased abundance of Lactobacillus crispatus and that lower levels of L. crispatus are associated with increased risk of spontaneous PTB. Another study of Asian and Myanmar/Karen minority women combined microbiome profiling with cytokine analysis and found that signatures composed of specific taxa and inflammatory cytokines predict PTB more accurately than single markers, thus providing a framework for biomarker development in resource limited settings (96). Fettweis et al (35) employed multi omics approaches to identify a marked reduction in L. crispatus and increases in pathogenic taxa such as BVAB1 and Sneathia amnii alongside elevated levels of pro inflammatory cytokines in cases of PTB, highlighting these features as candidate biomarkers across populations. These findings indicate that intervention strategies should prioritize probiotic strains that enhance or maintain protective Lactobacilli colonization in specific populations and that biomarker discovery should integrate microbial and host inflammatory multi omics signatures to achieve precise cross population applicability.