Genetic heritage plays a role determining VMB composition as the CSTs are known to vary among ethnic groups. In general, lactobacilli dominate the VMB among the Asian and White women, whereas Black or African-American and Hispanic women are more likely to be dominated with diverse VMB, containing several BV-associated species, such as Gardnerella vaginalis, BVAB1, BVAB2, Atopobium vaginae, Megasphaera spp., Sneathia spp., and Prevotella spp. (Ravel et al., 2011; Fettweis et al., 2014; Borgdorff et al., 2017; Gupta et al., 2017) and CST IV-A or B (France et al., 2020). Women with black ethnicity are considerably more likely to be diagnosed with BV and PTB (Fettweis et al., 2014). Interestingly, L. iners-dominated VMB (CST III) is prevalent in VMB of women in all ethnic groups [reviewed by Petrova et al. (2017)]. Limited evidence exists for vaginal Candida species and their prevalence among ethnic groups (Wei et al., 2010).

Reproductive hormones and especially estrogen play a key role in shaping the VMB composition throughout life from the onset of puberty, during reproductive years toward menopause, and postmenopause when the estrogen levels decline [reviewed by Moosa et al. (2020)].

In the vaginal epithelial cells, estrogen increases glycogen storage, which is an important energy source for vaginal lactobacilli affecting their ability to colonize the vaginal tract and produce lactic acid, and which further promotes the ability to grow and thrive in acidic conditions. Therefore, it is evident that estrogen fluctuations throughout menstrual cycle modulates VMB composition (Eschenbach et al., 2000; Srinivasan et al., 2010; Gajer et al., 2012). The highest estrogen levels occur in the follicular phase, which correlates with dominance of lactobacilli and low bacterial diversity (Kaur et al., 2020). Gajer et al. (2012) investigated temporal dynamics of the vaginal bacterial communities in 32 women at reproductive age over 16 weeks (including 4–5 menstrual cycles). The study revealed variation across women in terms of how communities changed over short periods of time. For instance, women with CST IV-B often transitioned to CST III. Likewise, women with CST I (L. crispatus) most often transitioned to CST III (L. iners), or CST IV-A. Interestingly, CST II (L. gasseri) rarely underwent transitioned to other CSTs. The bacterial communities seemed to be less stable during menses and to have low abundance of lactobacilli, although diversity was not necessarily increased. Similarly, in another study with 14 healthy women, Srinivasan et al. (2010) showed that vaginal lactobacilli fluctuated over time. However, at the onset and during menses, the dominance of L. crispatus and L. jensenii significantly decreased, whereas G. vaginalis and L. iners increased and subsequently decreased with the end of menstruation. This finding may be potentially explained by the fact that G. vaginalis requires iron for growth and could acquire iron from menstrual blood by lysing erythrocytes with vaginolysin (Jarosik et al., 1998; Srinivasan et al., 2010). L. iners is also able to grow well in blood agar (Falsen et al., 1999), but not in MRS or Rogosa agar, indicating the potential requirement of iron of this species for growth and thus may explain why the strains favor co-occurrence during menses. Another longitudinal study investigating VMB composition throughout two menstrual cycles reported that L. crispatus was the least affected by the menses (Lopes Dos Santos Santiago et al., 2011). With regard to the effects of menstrual cycle on the mycobiota, studies suggest that the estrogen peak during ovulation stimulates the growth of C. albicans resulting in higher occurrence of symptoms of C. albicans later in the luteal phase (Eschenbach et al., 2000).

Despite the widespread use of hormonal contraceptives during reproductive years (United Nations, Department of Economic and Social Affairs, Population Division, 2019), their effects on VMB communities and the dynamics have not been fully elucidated (Achilles and Hillier, 2013; Anahtar et al., 2018) and there are inconsistent results due to variable contraceptive methods used. However, it seems that there is an association between oral contraceptives and reduced BV (Achilles and Hillier, 2013; Brooks et al., 2017; Anahtar et al., 2018). For yeasts, there is no consensus whether hormonal contraceptives increase or reduce vaginal yeast colonization or their infections (Achilles and Hillier, 2013). A study by Song et al. showed that healthy women not using hormonal contraceptives and women using combined contraceptives had similar periodic fluctuations of VMB that corresponded to stages of the menstrual cycle and high Lactobacillus abundance. Then, women with progestin-only contraceptives showed altered periodic fluctuations of VMB and low average abundance of Lactobacillus (Song et al., 2020).

Similar to the gut microbiota, different lifestyle factors influence VMB composition [reviewed by Bradford and Ravel (2017); Moosa et al. (2020)]. Here we focus on the most documented factors as well as emerging factors influencing VMB that are a direct result of modernization of the society. Intuitively, personal hygiene practices (i.e., vaginal douching, use of soaps, type of underwear, menstrual protection, and sprays) are the most direct ways to affect VMB composition. Among these, research suggests that vaginal douching seems to be most strongly associated with increased risk of BV/vaginal dysbiosis (Klebanoff et al., 2010; Lewis et al., 2017). In contrast, for the occurrence of Candida infection type in VVC, vaginal douching seems to have a low impact (Shaaban et al., 2015). Regarding sexual habits, multiple sexual partners are also a known risk factor for BV/lactobacilli depletion (Beigi et al., 2005; Schwebke and Desmond, 2005).

In addition, smoking is a well-known factor to increase the risk of vaginal dysbiosis and BV (Hellberg et al., 2000; Cherpes et al., 2008; Brotman et al., 2014a) by, e.g., affecting estrogen production (Westhoff et al., 1996) and altering vaginal metabolite production profile, particularly by increasing levels of nicotine and derivatives as well as biogenic amines (Nelson et al., 2018). Similarly, alcohol use is associated with increased BV cases (Francis et al., 2016).

Interestingly, new emerging research indicates that the modernization of the society related to, e.g., increased psychological stress, consumption of processed food rich in fat and carbohydrates, and urbanization has an impact on VMB [Neggers et al., 2007; Thoma et al., 2011b; reviewed by Amabebe and Anumba (2018a); Vargas-Robles et al. (2020)]. Women experience more stress than men (American Psychological Association, 2018) and the effects of stress seem to extend to the vaginal tract. More specifically, research implies that chronic psychosocial stress can influence the balance in the vaginal lactobacilli, potentially through dysregulated immune system and elevated cortisol levels, which further correlate with reduced vaginal glycogen, lower abundance of lactobacilli, elevated vaginal pH, and increased proinflammatory response [reviewed by Amabebe and Anumba (2018a)].

Diet does not only shape the gut microbiota, but the effects are known to extend to the vaginal tract. More specifically, research suggests that healthy diet rich in nutrients and with low glycemic index and lower fat intake could reduce the risk of BV (Neggers et al., 2007; Thoma et al., 2011b). Furthermore, micronutrient intake, particularly increased folate, vitamin A, and calcium could decrease BV risk (Neggers et al., 2007). In addition, diet rich in betaine is associated with higher vaginal Lactobacillus spp. abundance (Tuddenham et al., 2019). Interestingly, a recent study by Song et al. (2020) reported that overall vaginal microbial diversity was higher among vegetarian women than non-vegetarians, although the sample size was small. Brookheart et al. (2019) reported that independent of ethnicity, overweight and obese women suffer more frequently from BV than lean women. Potential underlying reasons include obesity-associated dysfunction/disturbances in the host metabolism, hormonal, and immune system regulation which may affect vaginal environment. Furthermore, obesity is associated with dysbiotic gut microbiota composition which can further affect VMB composition by serving as an “extravaginal source” for vaginal bacteria (Marrazzo et al., 2012). However, larger studies exploring the role of various diets and/or body mass index (BMI) on specific CSTs are lacking.

Urbanization may also play a role in affecting the VMB composition by increasing diversity, but the data is yet limited to a few ethnic groups (Vargas-Robles et al., 2020). Among the socioeconomic factors, there is an indication that the education level seems to be associated with VMB composition. A study by Virtanen et al. (2019) showed that Finnish women aged 25–45 with higher education level had more often Lactobacillus-dominated VMB community (especially L. crispatus).

Vaginal mucosal immune system both interacts with and regulates VMB composition [e.g., reviewed by Smith and Ravel (2017); Villa et al. (2020)]. The interplay between the immune system and the VMB is complex and involves variable factors such as epithelial and immune cells, antimicrobial peptides, pro/anti-inflammatory cytokines/chemokines, as well as secretory antibodies (Wira et al., 2010; Yarbrough et al., 2015). Epithelial and immune cells (dendritic cells) in the cervicovaginal mucosa maintain homeostasis with the VMB and simultaneously survey for pathogens. These cells detect microbial structures (antigens) via pattern recognition receptors such as Toll-like receptors (TLRs), which induces production of antimicrobial peptides as well immunomodulatory cytokines/chemokines. Dendritic cells further function as an integral link between innate and adaptive immune system by presenting antigens to other immune cells such as macrophages, NK-cells, neutrophils, and T- and B-cells (White et al., 1997; Duluc et al., 2013). In general, when epithelial cells encounter endogenous vaginal lactobacilli, epithelial cells produce low levels of antimicrobial peptides/cytokines resulting in mucosal homeostasis. Depletion of the endogenous lactobacilli and the dominance of BV-associated community results in higher levels and a different secretion profile of antimicrobial peptides as well as inflammatory cytokines/chemokines [reviewed by Smith and Ravel (2017), Yarbrough et al. (2015)]. Specifically, there are indications that vaginal lactobacilli assigned to CSTs affect differently the type and magnitude of innate immune response. For instance, there are reports showing that CST-IV induces higher pro-inflammatory response (IL-1α, IL-1β, TNF- α, IFN-γ, IL-4, IL-8, IL-12p70) than CST-I or CST-II, whereas CST-III induces an intermediate response (IL-8) (Rose et al., 2012; Anahtar et al., 2015). However, more studies are needed to understand in-depth how different CSTs regulate the immune system in a healthy state and transition into dysbiosis.

Bacterial vaginosis is the most common vaginal disorder/dysbiosis of women affecting at least 30% of women in reproductive age annually (Koumans et al., 2007; Bradshaw et al., 2013). BV is characterized by a decrease in lactobacilli and increase in atypical anaerobic bacteria (van de Wijgert and Jespers, 2017). BV increases the risk of the acquisition of sexually transmitted diseases, such as Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, herpes simplex virus type 2 (HSV-2), HPV, and HIV (Cherpes et al., 2003; Wiesenfeld et al., 2003; Myer et al., 2005; Allsworth et al., 2008; Brotman et al., 2010; Liang et al., 2019). BV is also a risk factor for other infections such as pelvic inflammatory disease, endometritis, chorioamnionitis, and amniotic fluid infection (Hay et al., 1994; Hillier et al., 1995; Koumans et al., 1999). C. albicans-associated VVC is one of the most common mucosal infections in the female genital tract (De Bernardis et al., 2018). Estimations show that majority of women (>75%) suffer from Candida infections at least once in their lifetime (Bongomin et al., 2017). Some women are more susceptible to VVC and recurrence of VVC is common (Blostein et al., 2017). C. glabrata is the second most common type causing 15% of the infections (Shaaban et al., 2013).

The major modulators of VMB composition are antibiotics and antifungal medicines targeted against vaginal infections. Metronidazole and clindamycin are the first-line antibiotic regimens for BV. The short-term cure rates are approximately 80%, with 50% recurrence rate within 6–12 months (Bradshaw and Sobel, 2016). Biofilm formation as well as antibiotic resistance of BV-associated bacteria such as G. vaginalis may be determinant factors for persistence and recurrence (Bradshaw and Sobel, 2016; Verwijs et al., 2020). The same applies for C. albicans which is effective in biofilm formation (Chandra et al., 2001). The hyphal (mycelial) form contributes to adherence and mucosal invasion is characteristic of symptomatic disease (Pappas et al., 2018). Biofilm provides increased virulence as well as resistance to host immune responses’ antimicrobial agents and may lead to recurrent candidiasis and reduced impact of antifungal treatments (Bradford and Ravel, 2017; Kalia et al., 2020). With regard to endogenous lactobacilli, some trials show that use of metronizadole, doxycycline, azithromycin, clotrimazole, and fluconazole has no substantial impact on overall vaginal Lactobacillus spp. colonization (Agnew and Hillier, 1995; Nyirjesy et al., 2006). Specific vaginal lactobacilli tolerate metronidazole up to 1000 μg/ml in vitro (Simoes et al., 2001). Furthermore, in women with diagnosed BV, few studies show that VMB seems to recover from metronidazole treatment (metronidazole) within a few days (Mayer et al., 2015; Lehtoranta et al., 2020), and several studies show that L. iners dominates the community in transition to the recovery (Macklaim et al., 2015; Mayer et al., 2015; Deng et al., 2018; Lehtoranta et al., 2020).

Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). As probiotics are commonly lactobacilli, their role in vaginal health has been extensively investigated especially in the context of vaginal infections in premenopausal women (Borges et al., 2014; Petrova et al., 2015). Increasing evidence show that specific probiotic strains or their combinations 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 (Xie et al., 2017; Li et al., 2019). However, there is high heterogeneity among the studies due to variation in study designs and probiotic strains used. Probiotics may elicit beneficial effects in the vaginal tract in several ways, i.e., by producing lactic acid and hydrogen peroxide to lower the vaginal pH. Furthermore, probiotics may produce antimicrobial compounds and stimulate the immune system to help to maintain bacterial balance in the vaginal tract. By adhering to vaginal epithelia, probiotics may inhibit attachment of pathogenic bacteria and utilize the same nutrients as pathogens and thereby restrict their growth (Petrova et al., 2015; Chee et al., 2020).

Probiotics targeted for vaginal health are widely available as dietary supplements or vaginal capsules/suppositories. In vaginal applications, probiotics are applied directly at the site of action, whereas orally supplemented probiotics need to first passage through the gastrointestinal tract before migrating to the vaginal tract. Interestingly, research shows that both application routes are efficacious (Li et al., 2019; Wang et al., 2019). Orally administered probiotics, however, may provide additional beneficial effects to vaginal health via so called “gut-vagina axis” by balancing gut microbiota and inhibiting/preventing ascension of urogenital pathogens from the rectum to vaginal tract as well as stimulating the gut and systemic immune system.

Thus far, no reports exist on probiotics and their impact on VMB in pre-puberty or puberty. However, it is important to acknowledge the role of estrogen in driving the VMB composition in pre-puberty and puberty, and this should be taken into consideration when designing probiotic studies in this age group for supporting vaginal health.

Infertility is a global concern affecting millions of people of reproductive age (Mascarenhas et al., 2012). In women, multiple factors can cause infertility, such as disorders concerning fallopian tubes, uterus, ovaries, as well as the endocrine system (Walker and Tobler, 2021). Knowledge on the role of VMB in fertility is increasing. BV affects, on average 30% of women in reproductive age and is a known risk factor for infertility [Koumans et al., 2007; reviewed by van Oostrum et al. (2013)]. Research shows that, particularly, the depletion of vaginal lactobacilli coupled with increased presence of G. vaginalis, A. vaginae, Ureaplasma parvum, Ureaplasma urealyticum, as well as Candida spp. are associated with issues in fertility [reviewed by Koedooder et al. (2019a)]. Vaginal dysbiosis is also linked with increased risk of sexually transmitted diseases (STDs) and may promote ascension of bacterial pathogens to fallopian tubes affecting reproduction (García-Velasco et al., 2017, 2020). Low abundance of vaginal lactobacilli is associated with infertility and vice versa (García-Velasco et al., 2020; Hong et al., 2020). Furthermore, composition of VMB can impact the success of fertility treatment/artificial reproductive technologies. For instance, high dominance of vaginal lactobacilli and in particular L. crispatus seems to affect positively on a successful outcome of the in vitro fertilization/embryo transfer (Hyman et al., 2012; Koedooder et al., 2019b).

In normal healthy pregnancy, studies comparing VMB composition between pregnant and non-pregnant women show that lactobacilli dominance increases throughout pregnancy along with increased stability and decreased dominance of bacteria associated with BV (e.g., Romero et al., 2014; Serrano et al., 2019). Notably, VMB change and increase in lactobacilli, especially L. iners, throughout pregnancy is most pronounced in women of African ancestry (Serrano et al., 2019). Among dominating lactobacilli, L. crispatus, seems to be the most stable species across pregnancy (Serrano et al., 2019). These findings are supported by several studies [reviewed by Anahtar et al. (2018)]. After delivery in the postpartum period, the composition of VMB shifts to less diverse and is characterized by lower abundance of Lactobacillus spp. (CST I, II, CST V) and significantly higher proportions of Streptococcus anginosus, P. bivia, and L. iners (Nunn et al., 2021).

It is likely that both estrogen and progesterone contribute to the increased dominance of lactobacilli during pregnancy, which stimulate glycogen accumulation in the vaginal epithelial cells favoring Lactobacillus spp. colonization (Anahtar et al., 2018). During pregnancy, the function of the mucosal barrier decreases due to congestion and edema of vaginal mucosa. Furthermore, pregnancy increases immune system modulation, which may lead to differential responses against pathogens (Mor and Cardenas, 2010). Microbial dysbiosis is linked with negative reproductive outcomes, such as prelabor rupture of membranes and PTB (Elovitz et al., 2019). Fettweis and colleagues assessed how VMB changes across pregnancies correlate with the risk of PTB by comparing data from 45 preterm and 90 term birth controls in a cohort of women of predominantly African ancestry (Fettweis et al., 2019). Those women who delivered preterm significantly exhibited lower vaginal levels of L. crispatus and, e.g., higher levels of BVAB1, Sneathia amnii, and Prevotella spp. Early pregnancy, such as the first 8 weeks of gestation, is a critical time for successful pregnancy as most miscarriages occur early in the first trimester (Preisler et al., 2015). A study by Al-Memar et al. (2020) investigating 161 pregnancies reported that in miscarriages occurring during the first trimester, the vaginal bacterial composition was less abundant with Lactobacillus spp., and a significantly higher proportion of these cases were dominated by CST IV.

As mentioned above, Nugent score is one of the key diagnostic tools for BV. Intermediate Nugent score (4–6) is of particular interest as it is seen as a risk for developing BV (Guedou et al., 2012; Amegashie et al., 2017). A study by Petricevic et al. (2008) showed that VMB can be balanced in postmenopausal women (n = 72) with intermediate Nugent score by 14-day supplementation of probiotics L. rhamnosus GR-1 and L. reuteri RC-14. In this clinical trial, Nugent score improved in 60% of the women in the probiotic group, whereas the improvement was only 16% in the placebo group. Another study with the same strains demonstrated that 3-day vaginal administration of the probiotics did not change the Nugent score in postmenopausal women (n = 14) with intermediate Nugent scores (Bisanz et al., 2014). However, the 16S sequencing analyses showed that there was a significant increase in the vaginal abundance of Lactobacillus spp. in the probiotic group, indicating a potential health benefit from the microbiota perspective.

Promising results have been received from a clinical trial performed in 22 menopausal breast cancer patients under chemotherapy (Marschalek et al., 2017). The study participants had vaginal atrophy and intermediate Nugent Score at the baseline and received either combination of four probiotics (L. crispatus LbV 88, L. rhamnosus LbV 96, L. jensenii LbV 116, L. gasseri LbV 150N) or placebo twice a day for 2 weeks. Vaginal swabs were collected at baseline, 1 day after the treatment, and 1 week after the treatment. Improvement toward healthy Nugent Score (0–3) was detected in 63% women in the probiotic group and in 36% in the placebo group. Nugent Score was reduced at Day 1 and after 1 week in the probiotic group. In addition, a pilot placebo-controlled clinical trial by Kwak et al. (2017) investigated the vaginal colonization of topically applied L. gasseri LN40, Limosilactobacillus fermentum LN99, and L. rhamnosus LN113 product for 10 days in postmenopausal women (n = 18). Potential vaginal persistence was shown for LN99 and LN113 either at the end or after the intervention, but not for LN40. Another study in postmenopausal women with symptoms of vaginal atrophy and taking vaginal tablets containing low dose of estriol (0.03 mg) and L. acidophilus KS400 showed benefits in improving vaginal maturation index and related clinical symptoms (Jaisamrarn et al., 2013).

Furthermore, there are multiple studies on BV and probiotic benefits on VMB, including women with the age range varying from 18 to 70 years [reviewed by Hanson et al. (2016); Li et al. (2019), Wang et al. (2019); van de Wijgert and Verwijs (2020)]. In most of the studies there is no clear differentiation between pre- and postmenopausal women. An observational pilot study in women with history of recurrent BV and undergoing surgical menopause showed that when L. rhamnosus BMX 54 was administered as vaginal tablets altogether for 6 months, women experienced less BV recurrences (Parma et al., 2013). However, due to relatively low number of studies focusing on menopausal/postmenopausal population only, more studies on probiotics’ effects on VMB focusing on this population are warranted.