Gut microbial plays a vital role in regulating energy storage and human metabolism. As such, substantial focus has been directed to microbiota-targeted agents as a new target for the treatment of polycystic ovary syndrome (PCOS) and related metabolic diseases. Several studies have found variations in gut microbiota composition between PCOS patients and healthy people. Besides, obese PCOS patients have been shown to exhibit more severe gut dysbiosis. However, the precise mechanism underlying the relationship between gut microbiota and the occurrence and development of PCOS has not been fully revealed. Insulin resistance (IR) is considered the important pathological basis of reproductive dysfunction in patients with PCOS [4-7]. 50% of patients with PCOS have IR, independent of obesity[31, 32]. However, there is limited information on the roles played by intestinal flora in the development of IR and its link with PCOS. Herein, our results revealed that gut dysbiosis was more severe in PCOS patients with insulin resistance than in the PCOS-NIR and HC groups. Furthermore, several taxa at the phylum level were related to the clinical characteristics of PCOS and were significantly correlated with metabolic biomarkers, including HOMA-IR, abdominal obesity, free fatty acids, and other indicators.
Moreover, the results of the study showed that the composition of gut microbiota of PCOS patients with normal BMI was changed, but there was no significant difference in α-diversity among the three groups. It is noteworthy that several studies have reported conflicting results regarding the composition and function of the intestinal flora in PCOS patients. According to a recent meta-analysis, decreased intestinal microbiome diversity and changes in diversity are closely associated with obesity in humans [33, 34]. Previous studies have reported a significant decrease in gut microbiota diversity in PCOS patients or letrozole-induced mouse models[19, 20, 35]. Liu et al. found that obese PCOS patients had the lowest diversity of gut microbiota [19]. Meanwhile, studies have shown that sex hormones are related to changes in gut microbiom[36, 37]. Torres et al. found that PCOS patients exhibited a lower diversity of gut microbiota than healthy controls, and that total testosterone levels were associated with reduced diversity[20]. However, Insener et al. did not find a decrease in the diversity of gut microbiota in all PCOS patients with hyperandrogenemia, whether obese or not. and diversity may be different from PCOS diagnostic criteria[38]. Similarly, in this study, β- diversity of gut microbiota did not differ significantly among the three groups of samples according to weighted and unweighted clustering analysis. In a recent study, weighted UniFrac range-based hierarchical clustering and PCoA analysis indicated a clear distinction between the HC group and the IR group, whereas the NIR group could not be distinguished from the HC and IR groups[39]. Although there is no agreed standard for choosing sample size in microbiome studies, a study estimated that a sample size of 10 subjects per unweighted group (total sample size of 30) and 20 subjects per weighted group (total sample size of 60) may provide accurate statistical results for weighted analysis[40]. The α and β diversity of intestinal flora may be influenced by sex, sex hormones, and obesity. In this study, the BMI of all the subjects was within the normal range, and thus the effect of obesity itself on the gut microbiota composition of the PCOS patients could not be considered. Maybe due to the small sample size, a more definite answer should be given after increasing the sample size in the future.
According to the analysis of the structural composition of gut microbiota conducted in this study, the three groups were mainly composed of Bacteroidetes and Firmicutes at the phylum level. At the genus level, the relative abundance of Enterococcus in the PCOS group increased significantly (P<0.05) and was highest in the IR group. Enterococcus is a common gram-positive bacteria and can be divided into five categories according to phylogenetic similarity. Among them, Enterococcus faecalis and Enterococcus faecium are the major pathogenic bacteria to humans[41, 42]. Although the causal role of genus Enterococcus in the occurrence and development of metabolic diseases has not been fully revealed, previous studies have found that Enterococcus is more abundant in the gut microbiota of obese children and adolescents [43], as well as in mice under a high-fat/high-sugar "Western" diet[44]. A recent study found that Enterococcus can regulate the level of incretin hormone glucagon-like peptide-1 (GLP-1). GeIE secreted by E. fasecalis can degrade GLP-1 (GLP-1), leading to abnormal insulin secretion[45]. At the same time, GeIE can degrade intestinal gastric inhibitory peptides (just like leptin), and thereby interfere with the metabolism of the host. Similarly, in our study, Enterococcus was the most abundant genera in the PCOS-IR group, and its abundance was positively correlated with insulin resistance index. GLP-1 plays a role in regulating glucose homeostasis and reducing appetite in the body. Considering the vital role GLP-1 plays in the development of type 2 diabetes and other metabolic diseases, we speculated that Enterococcus could influence the occurrence and development of PCOS by regulating the GLP-1 signaling pathway, especially in patients with IR. Studies have shown that after oral glucose tolerance test, GLP-1 activity in lean PCOS patients is usually lower than in healthy women [46]. The use of GLP-1 receptor agonists in the treatment of PCOS patients can improve symptoms and reduce metabolic complications by reducing weight and insulin resistance[47, 48]. Insulin secretion and gastric emptying are affected by intestinal flora environment and intestinal flora imbalance. Estelle et al. proved the important role of intestinal flora in controlling GLP-1-induced insulin secretion and gastric emptying in mice[49]. Therefore, the role of Enterococcus in the regulation of GLP-1 level in PCOS patients should be investigated further.
The critical bacterial genus in the intestinal tract of patients with PCOS was identified using LEfSe multilevel species discrimination and LDA. According to the results, Rothia played vital roles in the PCOS-IR group, whereas Prevotella was the dominant bacterial group in the HC group. Rats fed a high-fat diet have been shown to exhibit significantly increased fat, reduced insulin sensitivity, increased abundance of esophageal Rothia, and Rothia were associated with fasting blood glucose and insulin. These observations were associated with the expression of inflammatory genes and fatty acid transport and metabolism in the esophagus[50]. Changes in Rothia flora in the adolescent oral cavity are associated with obesity[51]. Women with gestational diabetes have also shown an increase in Rothia abundance, relative to women with normal blood sugar[52]. In this study, Rothia had significant advantages in the PCOS-IR group, and its abundance was positively correlated with waist circumference and free fatty acid (FFA). Previous studies have shown that non-obese PCOS patients with insulin resistance have a more obvious centrality distribution of fat [37]. In our study, PCOS patients with normal BMI had abdominal fat accumulation. Besides, Rothia abundance in the PCOS-IR group was related to abdominal obesity. Intestinal flora may participate in the process of visceral fat metabolism, releasing too much free fatty acids, increasing lipotoxicity, and reducing insulin sensitivity. Prevotella is a bacterium that produces short-chain fatty acids (SCFA), regulates the uptake of nutrients and hormone levels in the gut. Also, it participates in energy metabolism, and its decreased abundance is significantly associated with increased testosterone and pro-inflammatory cytokines[39]. In this study, the HC group had the highest abundance of Prevotella, maintaining the balance of intestinal flora, whereas the NIR group and IR group had a reduced abundance of Prevotella. However, we did not observe a correlation between the decreased abundance of the Prevotella and the sex hormones and biochemical indicators, which could be due to a small sample size used herein. The relationship between PCOS and intestinal flora is complex and could be related to genetic, lifestyle, and environmental factors [53, 54]. In this study, we excluded the influence of smoking, taking antibiotics and probiotics on intestinal flora in the included subjects. However, different backgrounds (such as region, race, lifestyle, and diet habit) of the subjects could be responsible for the differences in the PCOS-related intestinal flora[55].