4.2. Predisposing Conditions for BV Development
Predisposing factors for the development of BV remain relatively unclear, but some of them include the course of menstrual cycles, sexual activity, or even personal hygiene [
26]. Increasing attention is therefore being paid to the importance of the composition of the microbiome in the vaginal environment and its interactions in preventing colonization by pathogenic microorganisms [
2]. The importance of the dominance of
Lactobacillus spp. (>70%) in the vaginal microbiome is emphasized due to their production of compounds with antimicrobial activity, e.g., hydrogen peroxide or bacteriocins, for benefits related to improving sexual and reproductive health and preventing the growth of pathogenic microorganisms [
19,
26]. Studies of the microbiome of the vagina, but also of the gut and oral cavity, suggest that, compared to healthy women, PCOS patients are more likely to experience inflammation, leading to destabilization of such a microenvironment [
19,
44,
45]. Patients with PCOS have chronic low-grade inflammation. According to a new microbiological hypothesis, a clear correlation of pathological changes, dysbiosis and hyperandrogenemia, or insulin resistance, and the role of gut–brain axis mediators in the gut microbiome are emphasized. In the case of women with PCOS, the impacts of factors faced by patients, such as high triglyceride, fasting glucose, and insulin levels, metabolic disorders, or diabetes, on the composition and changes in the microbiome and the role of
Lactobacillus spp. in infection control and metabolic balancing processes, are highlighted [
44,
45,
46,
47].
For women with PCOS, additional inflammatory factors may therefore exacerbate the complications associated with this syndrome. Therefore, in the case of PCOS patients, the importance of achieving a balanced microbiome, facilitating control of the symptoms, is emphasized. This is confirmed by studies conducted on animal models, but also based on the results of analyses using probiotic/synbiotic therapies in women with PCOS. Beneficial effects of
Lactobacillus spp. have been demonstrated, e.g., on the regulation of intestinal microflora related to sex hormones [
48,
49]. A person’s microbiome plays an important role and is considered their “second genome” [
2]. The vaginal microbiome is a dynamic system that is influenced by cyclic hormonal changes (mainly estrogen and the menstrual cycle), glycogen levels, changes in the vaginal epithelium, or sexual activity, among other factors. To date, many hypotheses have been developed regarding the dynamics of changes in the vaginal microbiome community, but they do not provide a clear answer. Hence, especially in the case of patients with PCOS, it is worth paying attention to the impact of this condition on the interactions taking place within the vaginal microbiome and its imbalance [
26,
50], especially since currently the data on the vaginal microbiome for patients with PCOS are quite limited.
4.3. Impact of Hormones
In a woman’s lifetime, the vaginal microbiome is modulated by hormonal changes, from prepubertal to postmenopausal, as well as bimonthly hormonal fluctuations. In addition, age, immune system status, and even ethnicity, lifestyle, and diet, and the use of medications such as antibiotics, as well as probiotic/synbiotic supplementation, also have an impact [
51]. Sex hormones affect the mechanisms of antigen presentation, cytokine production, and immunoglobulin production and transport, but also the mucosa of the reproductive system, its immunity, and the stability and balance of the vaginal microbiome [
6]. In the case of PCOS, endocrine disorders, hormonal changes, and, of course, the consequences of these processes, may play an important role in shaping the vaginal microbiome. Many studies in PCOS patients emphasize the presence of chronic low-grade inflammation pointing to important inflammatory factors, including those such as IL-18, associated with insulin resistance, obesity, or metabolic syndrome, and even predicting long-term cardiovascular mortality. In addition, it is important to pay attention to the effects of systemic inflammation when the vaginal microbiome is disrupted. Studies show that in such cases there is an increase in IL-8 and tumor necrosis factor alpha, which affect the hypothalamic–pituitary–ovarian axis through the blood and lymph [
6,
52,
53,
54]. An important element, therefore, is the prevention of BV and its complications, which is not easy due to the not-fully-explained interactions of the reproductive tract mucosa with the vaginal microbiome, as well as the multifactorial and dynamic changes in this process, both from a microbiological and immunological point of view [
55]. In women with PCOS, hormonal disorders (including estrogen and progesterone) or irregular menstrual cycles, affecting periodic changes in the epithelium of the reproductive tract, play a particular role in the maintenance of the vaginal microecosystem [
2]. Moreover, androgens have been shown to modulate the gut microbiome, which is associated with intestinal dysbiosis in women with PCOS and hyperandrogenism [
49,
56,
57]. Women with high testosterone levels were found to have higher levels of gut microbiome diversity, similar to PCOS, and its additional impact on the vaginal microbiome. Moreover, a study by Hong et al. (2021) found that patients with PCOS, regardless of testosterone levels, had a higher prevalence of Gardnerella, the microbiome most associated with BV development, compared to healthy women without PCOS [
6]. This may also suggest the possibility of a higher prevalence of BV in a group of women with PCOS. As an example, a study by Hong et al. (2021) in women with PCOS found an effect of testosterone levels on
L. crispatus and
L. iners populations, and a study by Lu et al. (2021) found a correlation between the number of
Lactobacillus spp. and FSH levels. A high percentage of these microorganisms is maintained at normal levels, and when it decreases, an increase in the percentage of opportunistic microorganisms in the vaginal microbiome is found [
6,
44]. A decrease in the proportion of
Lactobacillus spp., especially
L. crispatus, in the vaginal microbiome of women with PCOS is accompanied by a significant increase in pathogenic microorganisms such as
Gardnerella vaginalis and
Prevotella sp., but also Mycoplasma and Chlamydia trachomatis. An example is the study by Gu et al. (2022) in PCOS patients of the effect of the length of menstrual cycles on the reduction in the percentage of
Lactobacillus sp., while inoculating with pathogenic microorganisms [
2,
6,
7,
58].
PCOS syndrome causes the imbalance of the vaginal microbiome due to the elimination of
Lactobacillus spp. and facilitates the growth of anaerobic and pathogenic microorganisms. Hence, PCOS syndrome can predispose, for example, to BV and resulting reproductive failure, preventing embryo implantation or fetal growth [
7].
4.4. Impact of Immune Response
In the present study, the imbalance of the bacterial microflora of the analyzed patients was confirmed by the increasing percentage of patients with the elimination of lactic acid bacteria in the vaginal secretions with an increase in pH, in which an increase in the number of clue cells was also found. In the PCOS patients analyzed, there was a correlation with an increased number of leukocytes in the vagina along with an increase in pH. It is also worth noting that in addition to the involvement of microorganisms in the development of BV, the importance of the immune response also plays an important role [
59]. In the development of BV and homeostasis of the vaginal mucosa, the role of cervical and vaginal immunity is indicated, which is influenced by many diverse factors, such as genetics, reproductive hormones, psychosomatic stress, diet, and even physical activity [
59]. Thus, in the case of a condition such as PCOS syndrome, this aspect assumes particular importance.
It is suggested that in women with BV, the number of neutrophils in the vagina usually does not differ from that in healthy women. The immune response shows a significant increase in the vaginal interleukin IL-1beta, as well as a suppression of the pro-inflammatory response by hydrolytic enzymes produced by microorganisms, resulting in an impaired increase in IL-8 and precisely the absence of neutrophils in most BV patients [
60], which has not been confirmed by our analysis. In the case of the PCOS patient group in our analysis, the increase in neutrophils could therefore be explained by the potential development of infections caused by non-culturable microorganisms that were not identified during the evaluation of the vaginal microbiome, which were predisposed by the state of developed or developing BV, and would require further investigation in patients. Interleukin testing is not included in routine analyses of the vaginal microbiome and possible identification of BV; hence, it was not performed in the presented PCOS patients. Currently, little research has emerged on the vaginal microbiome in patients with PCOS. However, it is worth paying special attention to the disorders associated with this syndrome both in terms of immunity, physiological homeostatic processes, and pathological dysbiosis, including the vaginal microbiome, which is potentially more likely to result in the development of BV.
Despite the prevalence and knowledge of BV pathophysiology, due to the etiology and pathogenesis of this process, some areas are unclear. Therefore, the need to develop metabolic biomarkers that are specific and unique to BV is emphasized. Studies by Aldunate et al. (2015) point to changes in metabolic pathways, redox homeostasis, and inflammatory pathways in the vaginal ecosystem due to the production of short-chain fatty acids (SCFAs) by bacteria associated with the development of bacterial vaginitis (BVAB), leading to vaginal dysbiosis. It is therefore crucial to comprehensively understand and clarify the mode of action of BVAB (bacteria associated with bacterial vaginosis) and host factors that lead to disruption of the vaginal microbiome and loss of lactic acid-producing microorganisms [
26]. The introduction of more precise and detailed diagnostic tests, available on a wider scale, would enable faster, unambiguous diagnosis, especially in the case of early development of BV, which is particularly important in patients with PCOS.
4.5. Impact of Diagnostics Methods for the BV Recognition
In the analysis we presented, in some patients with PCOS, despite their reported alarming symptoms, the diagnosis of BV was not confirmed on the basis of microbiological examination as the relevant accepted criteria were not met. It can be suspected that the process was at such an early stage that it could not be detected by routine methods commonly used in assessing the vaginal microbiome. It may be worth expanding the diagnostic criteria for women with PCOS, given the potentially higher likelihood of developing BV and dysbiosis of the vaginal microbiome. In addition, the introduction of molecular testing into the routine diagnosis of the BV microbiome especially in PCOS patients could yield more conclusive results and provide a different perspective on vaginal dysbiosis and the potential development of BV, also in the context of diagnosing infertility in PCOS patients.
To date, much attention has been paid to the diversity of the gut microbiome in women with PCOS. It is worth noting the diversity and disruption of the vaginal microbiome and the potential implications these may have for patients with PCOS, both from the point of view of the impact of this syndrome on changes in the diversity of microbial composition, and the extent to which a microbiome with this localization affects other mechanisms and processes, e.g., chronic inflammation. Recently, through extensive molecular analyses, it has been pointed out that, contrary to what was previously thought, the microbial community in the vaginal microbiome is definitely more complex and unique to each woman. Moreover, multifactorial influences on its composition are now being emphasized. In addition to hormones, age, host immunity, or genetic predisposition, the primary factors shaping the profile of microorganisms in the vagina also include, for example, diet or lifestyle, which become crucial in the case of PCOS patients for the composition of the microbiome, for example, the gut [
51,
61]. For women with PCOS, a number of studies have shown the beneficial effects of probiotics on improving metabolic rates and the gut microbiome. Given the potential impact of the gut microbiome on the vaginal microbiome, it is worth considering probiotics, including oral probiotics, in women with PCOS to balance the vaginal microbiome. In PCOS syndrome, dysbiosis of the microbiome of various locations, and thus also the possibility of developing, for example, bacterial vaginosis, is more common than in healthy patients [
62].
The importance of oral probiotic therapy on increasing the number of
Lactobacillus spp. assessed by the Nugent scale in the vaginal microbiome was demonstrated in a study by Petricevic et al. (2008) conducted in a group of postmenopausal women. Initial scores on the Nugent scale ranged from 4 to 6, and after probiotic therapy (with freeze-dried strains of
L. rhamnosus GR-1 and
L. reuteri RC-14), 60% of patients showed an improvement of at least two steps on the scale [
63].
4.6. Lactobacillus spp. Contribution in Vaginal Microbiome
Women with PCOS are indicated to have a lower proportion of
Lactobacillus spp. in the vaginal microbiome compared to healthy women (Tu et al. 2020). Thus, women with PCOS are potentially more likely to have an imbalanced vaginal microbiome and a higher incidence of BV. In addition, a dysregulated lower genital tract microbiome is associated with fertility and menstrual cycle disorders, which PCOS patients often face (Tu et al., 2020) [
7].
Lactobacillus spp. in the vaginal microbiome serve as a barrier to infections and have beneficial effects on the innate immune system and maintenance of vaginal homeostasis. Hence, the diversity of the vaginal microbiome should be rather low with the dominance of
Lactobacillus spp. [
51,
64]. However, women with PCOS exhibit a highly heterogeneous composition of the vaginal microbiome, with a reduced percentage of
Lactobacillus spp., and at the same time an increased proportion of potentially pathogenic microorganisms, such as
Gardnerella vaginalis or
Prevotella spp. Moreover, in the genital tract of PCOS patients, favorable conditions for such microorganisms are provided by an excessive number of amino acid metabolism pathways, oxidative phosphorylation, and N-glycan biosynthesis [
7]. In our analysis, even in PCOS patients without BV found according to routinely used criteria, the vast majority of PCOS patients had
Gardnerella vaginalis or an elevated pH of the vaginal environment, which in the case of these patients becomes particularly important in terms of more favorable conditions for the development of BV.
In the vaginal environment, the importance of
Lactobacillus spp. is particularly important due to their action as a barrier to infection due to the glycogen metabolism processes carried out, the production of lactic acid, and the maintenance of a low pH ≤ 4.5. Elimination of
Lactobacillus spp. results in an increase in vaginal pH and further, among other things, excessive proliferation of anaerobic bacteria and the development of BV, leading, for example, to fertility disorders or premature births [
65]. Currently, however, there is growing evidence of heterogeneous effects on the vaginal microbiome within the genus
Lactobacillus, depending on the characteristics of each species [
64]. According to a study by Ravel et al. (2011), five types of community status (CST) are found in women of childbearing age, depending on the proportion and species diversity of
Lactobacillus spp.: CST I—with
Lactobacillus crispatus predominating, II—with
L. gasseri, and V—with
L. jensenii, all most associated with good health. CST III (with
L. iners) is ambiguous and can show BV, dysbiosis, or a physiological condition [
50]. The clinical picture of BV is mainly characterized by CST IV status, with the predominance of microorganisms such as Gardnerella, Mobiluncus, or Prevotella and the elimination of
Lactobacillus spp. Sometimes more detailed breakdowns are made for CST IV, including subgroups: A—with
L. iners and with strict anaerobes; B—with bacterial species related to BV (BVAB); and C—with many various strict and facultative anaerobes [
51]. Hence, the mere presence of
Lactobacillus spp. does not guarantee the stability of the vaginal environment. Therefore, the routinely used assessment of the vaginal microbiome and determination of BV by standard methods should be revised. While microbiological analysis of vaginal secretions with assessment of the presence of
Lactobacillus spp., or additionally using the Nugent scale, provides some picture of the quantitative presence of
Lactobacillus spp., it does not allow for its qualitative assessment with consideration of the species of these bacilli. Such tests are also not commonly used in routine diagnostics. Therefore, when analyzing the results of PCOS patients in the present study, it was not possible to determine which
Lactobacillus species were detected. This fact is important because of the different properties and functions of individual
Lactobacillus species. Some patients, despite the presence of acceptable amounts of
Lactobacillus spp., reported symptoms suggestive of BV development. However, this was not reflected in routine diagnostic testing. Some studies (Ravel et al., 2011) indicate cases in which routinely used methods of assessing BV, such as high pH and high Nugent scores, do not provide a clear answer as to the composition of the vaginal microbiome. It turns out that some desirable microorganisms, such as
Lactobacillus spp., may still be present. An example is
L. iners, which is probably a species with lower protective capacities in maintaining the stability of the vaginal microbiome [
50,
66]. In patients with PCOS (Hong et al., 2021), a relationship between testosterone levels and the diversity of the β vaginal microbiome is indicated, in addition to the presence, proportion, and species diversity of
Lactobacillus, such as
L. crispatus and
L. iners [
6]. Hence, due to the protective potential for the vaginal environment of the genus
Lactobacillus, the importance of species identification is emphasized [
51]. Such detailed analyses, however, require more sophisticated techniques, such as molecular biology methods, than those used in routine diagnostic microbiological tests to assess the vaginal microbiome of patients, in which precise species identification can be made. However, they are currently rather used in scientific research.
Moreover, as in the case of the group of PCOS patients presented in our analysis, an imbalance of the vaginal microbiome, dysbiosis or already-developed BV may not always produce unambiguous and characteristic symptoms, which generates additional difficulties in assessing and implementing prompt and effective therapy. The individual needs of the vaginal ecosystem depending on a woman’s life stage (premenopausal, perimenopausal, postmenopausal) are also pointed out, resulting in a need for individual selection of probiotic therapies [
66]. Due to the growing need to identify species of
Lactobacillus, detailed diagnostics based on molecular tests may be more and more often included in routine tests, and not only at the level of scientific research. An example of the need for such opportunities is
L. iners in the vaginal environment. Although they are classified as probiotic microorganisms, attention is drawn to the differences in their activity in maintaining a healthy vaginal microbiome in relation to other dominant
Lactobacillus species (e.g.,
L. crispatus,
L. gasseri,
L. jensenii). The differences concern, for example, the ability to produce lactic acid in the vaginal microbiome.
L. iners, due to the lack of the gene encoding D-lactate dehydrogenase, produces only L-lactic acid as a result of fermentation of glycogen, and not both D- and L-lactic acid isomers that positively affect the immune system (Witkin et al., 2013; Pramanick et al., 2018) [
67,
68] and prevent the possibility of developing upper genital tract infections (Beghini et al., 2015) [
69]. The ability of
L. iners to produce only L-lactic acid therefore limits its effectiveness in the fight against pathogens in the vagina [
70]. A greater inhibitory potential of the D-isomer of lactic acid for exogenous bacterial infections has been demonstrated than in the case of its L-isomer. [
71,
72]. In the case of
L. iners, but also
G. vaginalis, low levels of or no D-isomer of lactic acid are found in BV, which may translate into an imbalance of both isomers and induction of proteins responsible for the ability of the bacteria to pass through the cervix and initiate infections of the upper reproductive tract [
67].
L. iners often shows high abundance even in the case of BV [
72]. This explains the nature of this microorganism and its limited possibilities of counteracting the development of pathogenic microorganisms such as
Candida spp. [
73]. or
Gardnerella vaginalis, by lowering the pH of the vaginal environment.
L. iners occurs less frequently at a low physiological pH of the vagina, when other species of the
Lactobacillus genus dominate. However, analogically, its presence is found when the pH increases, in the presence of
G. vaginalis, in pH typical of BV, and it can survive in vaginal dysbiosis conditions [
74] when the remaining
Lactobacillus spp. are substantially absent [
75,
76]. In addition, the low potential of
L. iners in preventing the colonization of the vaginal environment by anaerobes is indicated due to its low capacity to produce pyruvate H
2O
2 through oxidation [
72,
77,
78,
79].
Species of Lactobacillus were not identified in the PCOS patients analyzed in our study, as such diagnostics do not include routine vaginal microbiome testing available to the physician. According to routine diagnostics, the presented group of PCOS patients may not have been diagnosed with BV, although clinically it may have been initially suspected; however, the number of Lactobacillus spp. was high and therefore did not meet the criterion for a BV diagnosis, although perhaps the presence of Lactobacillus spp. alone did not fulfill its beneficial function.
4.7. Candida spp. Contribution in Vaginal Microbiome
In addition,
Candida spp. and
Gardnerella vaginalis were also found in the presented group of patients independently of the presence of
Lactobacillus spp., and some studies suggest that the microbiome with a dominance of
L. iners may be a specific “refuge” for
Candida spp. in the vagina (for example, [
73]). It is also worth noting that as a result of an imbalance of the vaginal microbiome, or dysbiosis, an environment is created that is conducive to the growth of other pathogenic microorganisms and opportunistic fungi, especially Candida (e.g.,
C. albicans,
C. glabrata,
C. krusei,
C. parapsilosis) [
51,
80]. BV can therefore be accompanied by vaginal candidiasis, generating additional symptoms observed by the patient, which distorts the picture of the original source of the patient’s clinical condition and results only in the empirical implementation of antifungal therapy. Recently, the high value of NAAT-based methods has been pointed out as facilitating both the diagnosis of BV and candidiasis. However, like other molecular tests, they are costly and are currently not widely used in the routine diagnosis of BV [
28]. In addition, there is the possibility of BV with associated candidiasis being a result of, for example, antibiotic therapy. A consequence of antibiotic therapy, especially broad-spectrum antibiotics, is the elimination of
Lactobacillus spp. from the vaginal microbiome, which in turn leads to its imbalance [
51]. In the case of our work, in the analyzed results of patients with PCOS, no correlation was observed between the number of patients with
Candida sp. present in vaginal secretions and an increase in pH or BV (with clue cells). Moreover, in the analyzed PCOS patients, the identified presence of
Candida spp. in the vaginal microbiome did not lead to automatic elimination of LAB, and in some patients both types of microorganisms were detected.