Background
PCOS is one of the most common female reproductive disorders and affects 5–20% of reproductive-age women worldwide [
1,
2]. It is associated with a wide range of detrimental health effects, impacting reproductive, endocrine and metabolomic function [
3]. However, the etiology of PCOS is controversial, and the current treatment in clinical practice relies on empirical rather than etiology-specific therapies [
2]. Despite the great strides in our understanding of PCOS pathogenesis from genetic, neuroendocrine, and hormonal perspectives, we have yet to elucidate the underlying pathophysiological mechanisms. Only after understanding the initiation and development of PCOS can we then translate these findings into more effective therapeutics.
The gut microbiota is associated with a wide range of diseases, including reproductive and gynecological diseases [
4‐
6]. There is increasing evidence to indicate that the gut microbiota interacts with the host by regulating key biological processes, such as metabolic process, hormone secretion, and immune response [
7]. Indeed, dysbiosis of the gut microbiota has also been reported in microbiota-centric studies on PCOS. A recent study from Qi et al. reported that mice with fecal microbiota transplantation from patients with PCOS would develop a PCOS-like syndrome, indicating a causal role of gut microbiota in PCOS [
5]. This study further demonstrated that modification of the gut microbiota, alongside bacteria-related bile acid and immune changes, may be a novel treatment for PCOS. Thus, this is an important study that shed light on both the etiology and novel therapy in PCOS.
However, many studies that contain cohorts from a range of regions and ethnicities report inconsistent and, occasionally, contradictory results. Though this variance may be explained by limited sample size and different choices of sequencing platforms, the heterogeneity among studies should be addressed to identify disease-specific bacterial biomarkers across a population, which could deepen our understanding of disease pathogenesis and would enable the development of new and effective therapies. Therefore, there is a need to compare microbial perturbations and differentiate a set of homogeneous gut microbial features from different studies. Meta-analysis is an effective method for integrating existing knowledge to uncover commonalities across different research, and interestingly, there is currently a lack of published meta-analyses that explore bacterial characteristics in PCOS patients. The present meta-analysis fills this gap in the literature by evaluating alterations in the gut microbiota of patients with PCOS across multiple studies and aims to elucidate consistent gut microbiota profiles and features with therapeutic potential.
Discussion
This systematic review firstly assesses the gut microbiota dysbiosis in PCOS. We observed consistency in the pattern of gut microbial dysbiosis despite variations across studies, which suggests an important role of gut microbiota in PCOS. The main findings of this study include the following: (1) the alterations in alpha-diversity indices indicated the disruption of bacterial phylogenetic abundance and ecological evenness while the observed bacteria showed only a mild change; (2) although the compositional changes of gut microbiota had been reported in many studies, there was inconsistency in the reporting of beta-diversity across studies; (3) dysbiosis including depletion of SCFA-related beneficial bacteria and bile-acid-metabolizing bacteria and enrichment of pro-inflammatory bacteria were observed in PCOS patients.
A robust microbial diversity is essential to the gut microbiota’s resilience to stress and is a key indicator of good health. Reduced diversity may result from the enrichment of pathogenic microorganisms and indicates a less healthy state in general [
44]. In concordance with previous studies [
45], our meta-analysis also showed decreased Shannon index and phylogenetic diversity index in PCOS patients. For other measurements, there were variances across all studies, with overall no statistically significant results when the Chao1 index, Simpson index, and observed species were assessed. It is important to highlight that different alpha diversity indices indicate different microbial profiles. For instance, while the Chao 1 index and the observed species are based on the total number of bacteria within a community, the Shannon index and the phylogenetic diversity index additionally consider bacterial evenness and phylogenetic abundance, respectively. Therefore, though there was heterogeneity between the studies, including differences in region, ethnicity, and methodologies, the statistically significant difference between alpha-diversity indices suggests that dysbiosis was manifested through the reduction of phylogenetic abundance and disruption of bacterial evenness rather than a change in the number of bacteria. As pointed out by Shade [
44], the common assumption that “higher diversity is better” oversimplifies complex mechanisms, and understanding the mechanism behind these diversity indicators with contextual data would advance knowledge. Our analysis of alpha-diversity suggests that the dysbiotic microbial profile in PCOS patients would be the result of disproportional bacterial taxa rather than the changes of certain bacterial presence. These results indicated that the PCOS-related gut microbial profile might be featured by the changes of species within genera, which is out of the resolution of 16S sequencing and requires metagenomic sequencing to further evaluate and identify disease-specific biomarkers. Moreover, it is worth pointing out that the interstudy heterogeneity of alpha-diversity in pooled estimation decreased in certain subgroups; for instance, the heterogeneity of the Shannon index was lower within the normal, overweight, or obese group. To some extent, we also noted a decrease in other indices. This observation echoed the previous findings [
46] that metabolic dysfunction could be an important factor influencing both gut microbiota and PCOS. Similarly, for our analysis on beta-diversity, significantly inconsistent results were observed between 24 included studies with no potential confounding factors revealed by logistic regression analysis. A common phenomenon is that most studies do not conduct subgroup analysis according to the characteristics of samples when analyzing beta-diversity, which limits the discovery of confounding factors that affect beta-diversity. Nevertheless, further exploration is needed to confirm the link between alpha-diversity, beta-diversity, PCOS patients, and healthy controls.
There is evidence that gut microbial dysbiosis could play a causal role in PCOS [
5]. This provides a novel angle for researchers to propose gut bacteria as a potential etiology of PCOS. It has also been reported that PCOS patients exhibit increased pro-inflammatory state [
47], disrupted gut barrier [
48], and metabolic disorders [
49], all of which are also considered to be the result of interactions between the gut microbiota and the host [
50,
51]. In this way, a connection between the gut microbiota and PCOS could be established. In the present study, despite the variations and complexity of the gut microbiota, we observed several distinct microbial signatures in PCOS patients. In patients with PCOS, there were enrichment of
Fusobacterium,
Escherichia, and
Bacteroides at the genus level and
Bacteroides fragilis and
Escherichia coli at the species level. Interestingly,
Fusobacterium has also been observed to be enriched in the oral microbiota of PCOS patients [
52].
Fusobacterium is known to be an opportunistic pathogen and its pathogenic role has been suggested in a variety of diseases [
53,
54]. An abundance of
Fusobacterium is involved in promoting inflammation and increasing gut barrier permeability in metabolic disorders [
55]. These detrimental characteristics echo the manifestations of inflammation and metabolic disorders in PCOS [
49]. Similarly,
Escherichia could increase the virulence of commensal bacteria by enhancing mucosal attachment, invasion, and intracellular persistence, which leads to epithelial dysfunction and increased barrier permeability [
56]. The biological mechanism that underpins the role of the gut microbiota and its metabolites in linking gut permeability and PCOS has been previously postulated [
57].
Bacteroides are usually commensal in the gut, but some strains of
Bacteroides have been identified as opportunistic pathogens [
58]. The enterotoxigenic
Bacteroides fragilis, which has the toxic gene encoding fragilysin, has been associated with different diseases [
59,
60].
Bacteroides vulgatus has also been shown to be enriched in the gut microbiota of PCOS patients and induced PCOS-like symptoms in a murine model by altering bile acid metabolism and host immune response [
5], which is the only differential species whose effect on PCOS was validated in animal model. Other consistent microbes also worth to be investigated in further study.
Based on the bacterial taxonomy and function results, our findings indicated several patterns of microbial disorder in PCOS. The clusters of bacteria that harbor beneficial or adverse effects to the host were identified, though single bacteria were weakly reproduced among studies. Microbial patterns that predominated in PCOS were of SCFA-producing and bile-acid-metabolizing bacteria, which was also evidenced by the altered bacterial function of secondary bile acid biosynthesis [
5] and fatty acid biosynthesis [
22,
40].
Among the included studies, several SCFA-producing bacteria were consistently reported depleted in PCOS, including
Butyricimonas,
Blautia,
Coprococcus, and
Faecalibacterium prausnitzii. These bacteria are regarded as SCFA producers and have beneficial effects on hosts, which were discussed in the included studies. Specifically, the SCFA producers were identified in the 16S sequencing studies, which was further supported by the observation of the bacterial function of SCFA metabolism in gut metagenomics. SCFAs, mainly acetate, propionate, and butyrate, are generated by gut bacteria metabolizing dietary elements and prebiotics [
61]. Studies have shown that SCFAs play an important role in mitigating inflammation and maintaining gut barrier function through the stimulation, synthesis, and release of phagocytic molecules, anti-inflammatory cytokines, chemokines, and protective peptides [
62,
63]. Butylated starch could be metabolized by the gut microbiota to produce SCFAs, which could stimulate the peptide-tyrosine-tyrosine secretion and the hypothalamic-pituitary-ovarian axis to alleviate PCOS [
64]. In parallel with these research findings, it has been demonstrated that
F. prausnitzii has anti-inflammatory capacities and supports mucosal immune homeostasis [
65,
66], making it a next-generation probiotic with great therapeutic potential. One mechanism for these beneficial effects may be mediated by its ability to produce butyrate [
67]. Butyrate can reduce oxidative stress and pro-inflammatory activity by maintaining the integrity of the gut barrier and limiting the translocation of bacteria and bacterial components such as lipopolysaccharide into the systemic circulation [
68,
69]. In a recent study, Zhang and colleagues [
22] further demonstrated that the colonization of
Bifidobacterium lactis, an SCFA-producing probiotic, was related to the fluctuations in the levels of SCFAs, sex hormones, and signal peptides, which proposed a potential mechanism of how probiotics interact and regulate with the host. However, there was no report on SCFAs employing gut metabolomics in the included studies. Thus, the SCFAs profile in the gut would be of interest in future studies.
Bacteria involved in bile acid metabolism have also been associated with PCOS. Bile acids are produced in the liver by the oxidation of cholesterol which is catalyzed by a series of cytochromes P450 [
70]. After a meal, bile acids are released into the duodenum where it is conjugated, and then the conjugated bile acids are reabsorbed from the ileum to the liver through the portal vein [
71]. This cycle preserves more than 95% of the bile acid pool [
71]. A small proportion of bile acids are secreted into the colon where they are mainly bio-transformed by the gut microbiota or excreted into faces [
71,
72]. Most gram-positive bacteria, like
Ruminococcus and
Clostridium, and some gram-negative bacteria can metabolize bile acids [
73]. Indeed, Qi and colleagues reported that the alteration of gut microbiota was associated with the reduction of bile acid (glycodeoxycholic acid and tauroursodeoxycholic acid) and IL-22 secretion [
5]. Moreover, Yang and colleagues demonstrated that administration of chenodeoxycholic acid could improve PCOS phenotypes in mice experiments. Since bile acids play a crucial role in food digestion and energy metabolism, dysfunction in the secretion and reabsorption of bile acids could be a characteristic of insulin resistance, obesity, and type 2 diabetes [
74,
75]. Additionally, bile acid receptors, Farnesoid X receptor and G-protein coupled bile acid receptor 1, regulate various elements of glucose, lipid, and energy metabolism [
74]. While metabolic disorders, including hyperinsulinemia, insulin resistance and obesity, co-occur with PCOS [
49], further studies revealing the relationship between bile acid and PCOS are needed to determine the etiological role of gut microbiota-mediated regulation of bile acids in PCOS.
It is noteworthy that the estrobolome, which is defined as “the aggregate of enteric bacterial genes whose products are capable of metabolizing estrogens” by Plottel and Blaser [
76], has also been closely related to gynecological diseases [
73,
77,
78]. In a review by Kwa and colleagues [
79], 60 bacterial genera from human gut microbiota have the potential to encode β-glucuronidase and/or β-galactosidase using the data from Human Microbiome Project. This includes
Bacteroides,
Bifidobacterium,
Escherichia,
Faecalibacterium, and
Lactobacillus, which were altered in PCOS patients across studies within this meta-analysis and postulated to be capable of impacting endogenous estrogen metabolism [
76]. The enriched bacterial hydroxysteroid deconjugate activity may contribute to the modulation of the interconversion of conjugated forms of estrogens as well as androgenic molecules [
80], which could also play a role in hormonal dysregulation in PCOS. The astrobleme is under-studied in the context of PCOS, and therefore, further research on the astrobleme could provide novel insights into understanding the role of the gut microbiota in hormone-mediated conditions like PCOS.
We performed a comprehensive meta-analysis by examining 28 papers, with a combined total of 1022 patients, and demonstrated the impact of the gut microbiota on PCOS. Our findings support dysbiosis as a hypothesized cause of PCOS, with disease-specific changes in microbial composition. However, there were several limitations of this study: (1) the sample sizes of current studies on the role of gut microbiota in PCOS were relatively limited, and thus, our meta-analysis might be underpowered and future studies are required to validate our findings in a larger population; (2) though microbial patterns were observed, disease-specific bacteria varied between studies. This could be in part due to the heterogeneity of the gut microbiota between study cohorts and their complex interactions with physiological and environmental factors. In addition, differences in methodology, including wet and dry lab protocols, would widen this gap; (3) the studies included in our meta-analysis were mostly based on 16S rRNA gene sequencing, which limited the interpretation at a species level. Furthermore, sequencing datasets were rarely provided, which rendered it difficult for researchers to integrate data and perform bioinformatic analyses using the same pipeline; (4) significant heterogeneity was observed in the pooled analysis of Chao 1, observed species, and PD whole tree indices. Although we performed sub-group and meta-regression analyses, no confounding factors were discovered that might elucidate the source of heterogeneity; (5) publication bias was observed in studies that reported Chao 1, but trim-and-fill analysis revealed that our result was stable; (6) most of the studies did not discern between patients that were newly diagnosed or currently under therapies. Further studies should address this point as the type of medication provided is a major factor that could profoundly influence the gut microbiota.