Self-Control Study on the Impact of Buzhong Yiqi Prescription on the Gut Microbiota of Obese Patients with PCOS and Phlegm-Dampness Syndrome Caused by Spleen Deciency

Background: Gut microbiota disorders have been closely related to polycystic ovarian syndrome (PCOS). Buzhong Yiqi prescription (BZYQ) has a great clinical effect on the treatment of obese patients with PCOS and phlegm-dampness syndrome caused by spleen deciency (SPSD). This study was performed to explore the alterations in the gut microbiota and fecal metabolites in obese patients with PCOS and SPSD who received BZYQ treatment. Methods: A total of 50 obese patients with PCOS and SPSD were recruited from the Changhai Hospital in Shanghai and accepted three months of BZYQ treatment. Sex hormone were detected and oral glucose tolerance was tested in the outpatient laboratory before and after the BZYQ treatment. Fecal samples were detected by 16S rRNA high-throughput sequencing and nontargeted metabolomic methods to determine the structure of the gut microbiota and metabolites, respectively. Results: BZYQ could signicantly alleviate the serum DHEAS (p<0.001) and T level (p<0.001) in obese patients with PCOS and SPSD. The structure of the gut microbiota changed signicantly after the BZYQ treatment. In particular, at the phylum level, the abundance of Spirochaetae was signicantly higher after treatment than that before treatment. At the genus level, the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, and Fusicatenibacter were signicantly higher after treatment than those before treatment, but the abundance of Megamonas was signicantly lower. A total of 106 differential metabolites and 14 KEGG enrichment pathways were quantied. The disorder in the gut microbiota and fecal metabolites of obese patients with PCOS and SPSD were closely related to hyperandrogenemia and insulin resistance. The level of tetracosanoic acid was negatively correlated with serum DHEA level (p<0.05), while the palmitoleic acid level was negatively correlated with serum T level (p<0.05). Conclusions: BZYQ could ameliorate the serum androgen level and had an impact on

tolerance was tested in the outpatient laboratory before and after the BZYQ treatment. Fecal samples were detected by 16S rRNA high-throughput sequencing and nontargeted metabolomic methods to determine the structure of the gut microbiota and metabolites, respectively.
Results: BZYQ could signi cantly alleviate the serum DHEAS (p<0.001) and T level (p<0.001) in obese patients with PCOS and SPSD. The structure of the gut microbiota changed signi cantly after the BZYQ treatment. In particular, at the phylum level, the abundance of Spirochaetae was signi cantly higher after treatment than that before treatment. At the genus level, the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, and Fusicatenibacter were signi cantly higher after treatment than those before treatment, but the abundance of Megamonas was signi cantly lower. A total of 106 differential metabolites and 14 KEGG enrichment pathways were quanti ed. The disorder in the gut microbiota and fecal metabolites of obese patients with PCOS and SPSD were closely related to hyperandrogenemia and insulin resistance. The level of tetracosanoic acid was negatively correlated with serum DHEA level (p<0.05), while the palmitoleic acid level was negatively correlated with serum T level (p<0.05).
Conclusions: BZYQ could ameliorate the serum androgen level and had an impact on the gut microbiota and metabolites in obese patients with PCOS and SPSD. Background Polycystic ovarian syndrome (PCOS) is a chronic disease characterized by reproductive endocrine and metabolic dysfunction. Patients with PCOS often manifest different degrees of menstrual abnormalities, infertility, amount of hair, acne, and obesity. The incidence rate of PCOS can reach 5-15% [1], and this dysfunction occurs in puberty and childbearing age. The cause of this disease remains unclear [2].
Epidemiological data show that approximately 53.5-85.5% of patients with PCOS are overweight or obese, with central obesity as the most typical case [3]. Obesity is recognized as the most dangerous factor for insulin resistance. For obese patients with PCOS, weight loss is still one of the important treatments [4].
In recent years, research on traditional Chinese medicine (TCM) and intestinal microecology has become a hot topic. Some single-herb or TCM compounds can help maintain the balance of intestinal microecology [4]. Oral herbal decoction and other dosage forms are the most important means of using TCM for the clinical treatment of diseases. This natural therapy plays a role in local parts or even the whole body through the digestive tract, which is the most important region for organism to parasitize microorganisms. Previous studies have shown that the gut microbiota is closely related to PCOS [6 − 8].
Disordered gut microbiota could increase intestinal permeability by affecting intestinal metabolites, such as lipopolysaccharide, entering the systemic circulation, and causing antigen-antibody reaction in the body. Abnormal intestinal environment could activate the immune system and chronic in ammation and increase serum insulin level and androgen levels in the ovary, interfering with normal follicular development [9,10].
Buzhong Yiqi prescription (BZYQ) was used to treat infertile obese women with phlegm-dampness syndrome caused by spleen de ciency (SPSD) as recorded in the Fu Qing Zhu Nv Ke, an ancient book of TCM in the Qing Dynasty. Previous studies have shown that BZYQ can improve symptoms of diarrhea and indirectly restore host homeostasis by recovering gut microbiota, such as Lactobacillus, Bi dobacterium, Enterococcus, and Bacillus subtilis [11,12]. Although BZYQ has long been applied to treat obese patients with PCOS and SPSD, the detailed underlying mechanism is still unclear.
Metabolomics is always used conveniently to study metabolism at the molecular level [13]. Fecal metabolites are the cometabolism products of the gut microbiota and host, and these molecules could re ect not only the status of the gut microbiota but also the relationship between commensal bacteria and the host. Metabolomics of PCOS is mainly focused on the metabolism of carbohydrate, lipid, amino acid, and hormones [14]. This thrust indicates that metabolic abnormality is not limited to the ovary but is a systemic metabolic disorder in PCOS, which increases the long-term risk of multiple diseases in patients [15]. In addition, the metabolic disorder of patients with PCOS is signi cantly affected by their phenotypes [16]. Although this disorder is greatly disturbing, few reports are available on the combined study of gut microbiota and fecal metabolomics of obese patients with PCOS and SPSD, especially those treated with BZYQ.
In this study, 16 s rRNA sequencing and nontargeted metabolomics were used to analyze the fecal samples of obese patients with PCOS and SPSD and reveal the impact of BZYQ on the gut microbiota and intestinal metabolites.

Subjects
In this study, 50 obese patients with PCOS and SPSD (BMI ≥ 28 kg/m 2 and 16-35 years old) were recruited from the Traditional Chinese Medicine Gynecology Clinic of Changhai Hospital in Shanghai from June 2016 to November 2017. During the study period, a total of 35 cases did not complete the study, and 15 cases completed the study by providing detailed clinical data and gut microbiota sequencing data. To explore the intestinal metabolites, six patients with the best treatment effect were further selected to complete the analysis of the fecal nontargeted metabolomics (Fig. 1). The obesity criteria were based on the consensus on the prevention of Chinese adult obesity [2]. The diagnostic criteria of PCOS were based on those revised in the 2003 Rotterdam Conference. SPSD was assessed according to the criteria set by Li et al. [17]. Patients with the following characteristics were excluded: had used oral contraceptives, antiandrogens, insulin sensitizers in the past 3 months prior to the experiment; pregnant; with other known hyperandrogenemia and ovulation disorders, such as 21 hydroxylase de ciency, congenital adrenal hyperplasia, Cushing's syndrome, androgen secreting tumors, thyroid diseases, and hyperprolactinemia; with mental diseases or organic diseases; had used corticosteroids or sex steroids; with a history of drug and alcohol abuse in the past 2 years prior to the experiment; and had used antibiotics, probiotics, or prebiotics in the past 3 months prior to the experiment. This study was approved by the Chinese Ethics Committee of Registering Clinical Trials (No. ChiCTRCTEC2016050), and each subject voluntarily signed the informed consent form before the trial. Fecal samples were collected 3-5 days after menstruation, and the patients received guidance for carbohydrate-based diet (300 g/day) 3 days before sampling. Sterile plastic spoon and sterile test tube were used to collect ~ 10 g of fresh fecal samples from each participant. The samples were transported to the laboratory in an ice box within 2 h from sampling and stored at − 80 °C. Then, 16S rRNA gene sequencing was performed to analyze the structure of the gut microbiota, and nontarget metabolomics was applied to analyze the fecal metabolites.

Detection Of Gut Microbiota
Fecal samples from 15 patients were collected before and after the BZYQ treatment. Sample DNA extraction, PCR ampli cation, Illumina MiSeq sequencing, and post-processing of data were performed as described in previous studies [9]. The OTU abundance data were used to analyze the intestinal microbial diversity and changes in the abundance before and after BZYQ treatment, and the community composition of each sample at different classi cation levels was obtained. Mothur software (v.1.30.1, https://mothur.org/) was used to analyze the alpha diversity before and after BZYQ treatment. R language was used to draw a bar map of the community structure at the phylum and genus levels. Beta diversity was analyzed via principal co-ordinate analysis (PCoA). Wilcox rank sum test was used to analyze the differences in the species between the two groups at the phylum and genus levels. Linear discriminant analysis (LDA) was conducted to estimate the in uence of the abundance of species on the differences in the gut microbiota after BZYQ treatment.

Nontarget Metabolomic Analysis
The nontarget metabolomic experimental steps were based on a validated method as previously described [18]. The data were analyzed on the free online platform of Majorbio Cloud Platform (https://www.majorbio.com). Principal component analysis (PCA) and orthogonal partial least squaresdiscriminant analysis (OPLS-DA) were conducted to distinguish the overall differences in the metabolic pro les and nd the different metabolites before and after BZYQ treatment (n = 6). The metabolites with variable importance for the projection (VIP) greater than 1 and p values less than 0.05 were considered as differential variables. The expression mode of the metabolites in each sample was displayed in the cluster heat map, and the p and VIP values of the metabolites were displayed in a VIP bar chart. The metabolic pathway annotation was carried out through the KEGG database (https://www.kegg.jp/kegg/pathway.html) to obtain the pathways participated by the differential metabolites. Pathway enrichment was analyzed on Python (scipy.stats), and the most relevant biological pathway was selected using Fisher's precise test.
Statistical analysis SPSS software (version 21.0) was used for statistical analysis. Paired t test was used to analyze the quantitative demographic and clinical data with normal distribution, and the data were expressed as mean with standard deviation. Wilcoxon rank sum test was used to analyze the quantitative sequencing data with nonnormal distribution, and the p values were checked multiple times using Benjamini and Hochberg false discovery rate. A double-tailed p < 0.05 indicated statistically signi cant difference.

Changes In The Gut Microbiota Structure
In this study, 16S rRNA high-throughput sequencing was used to detect the structures of the gut microbiota before and after BZYQ treatment (n = 15). The results showed no signi cant difference in the alpha diversity (Sobs, Chao, Shannon, and Simpson) and beta diversity (PCA and PCoA) before and after BZYQ treatment. At the phylum level, BZYQ treatment increased the abundance of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and the ratio of Firmicutes/Bacteroidetes (2.0 vs. 1.91) but decreased the abundances of Fusobacteria and Verrucomicrobia. However, no signi cant differences were found on these phyla. Although the abundance of Spirochaetae increased signi cantly after BZYQ treatment (p = 0.0006), the sample was limited and short of clinical signi cance (Fig. 3a).
The top 20 species were selected in accordance with abundance for comparison due to the large number of species at the genus level. BZYQ treatment increased the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, unclassi ed_f__Lachnospiraceae, and Fusicatenibacter (p < 0.05) but decreased the abundance of Megamonas (p < 0.05, Fig. 3b). Prior to the BZYQ treatment, 26 unique genera were found in the patients compared with those after BZYQ treatment, and Mitsuokella was the most abundant, accounting for 64.11% (Fig. 3c). After BZYQ treatment, 13 unique genera were found, of which Edwardsiella was the most abundant at 28.05% (Fig. 3d). Furthermore, the LDA scores showed that Dialister, Holdemania, Megamonas, Ruminiclostridium_9, and vadinBC27_wastewater_sludge_group were the characteristic species before the BZYQ treatment. After the BZYQ treatment, 19 genera, such as Fusicatenibacter, Blautia, and Dorea, were the characteristic species (Fig. 3e).
Comparison Of Fecal Metabolites Before And After Bzyq Treatment and after BZYQ treatment (n = 6) were signi cantly grouped. In this study, 962 different ion peaks were found, and 106 different metabolites (VIP > 1, p < 0.05) were quanti ed (Fig. 4d). KEGG pathway analysis demonstrated the involvement of 10 different metabolites, such as taurocholic acid, palmitic acid, and stearic acid (Fig. 5a, Table 1). The 10 differential metabolites participated in 26 KEGG pathways, of which the lipid metabolism pathway contained the most differential metabolites (Fig. 5b). The differential metabolites were signi cantly enriched in 14 KEGG pathways, including the biosynthesis of unsaturated fatty acids (p < 0.001), fatty acid biosynthesis (p < 0.001), cutin, suberine, and wax biosynthesis (p < 0.001) and biosynthesis of plant secondary metabolites (p < 0.001, Fig. 5c). VIP, variable importance for the projection; FC, fold change (before/after); AUC, area under the curve under receiver operating characteristic curve (ROC); CI, con dence interval.
Correlation analysis among the gut microbiota, fecal metabolites, and serum sex hormones after BZYQ treatment A signi cant positive correlation was observed between the abundance of Paraprevotella and serum LH level in the patients after BZYQ treatment (p < 0.01). The serum DHEAS and T levels were negatively correlated with the abundances of Lachnospiraceae_NC2004_group and Faecalibacterium (p < 0.05) but positively correlated with the abundance of [Ruminococcus]_gnavus_group (p < 0.05). HOMA-IR was positively correlated with the abundance of Blautia (p < 0.01, Fig. 6a).
The correlation between the 10 fecal metabolites involved in the KEGG pathways and the top 50 genera was analyzed in accordance with their abundance. These fecal metabolites, except for Sphingosine_1_phosphate, had signi cant correlations with speci c genera (Fig. 6b). In particular, the abundance of Bacteroides was signi cantly correlated with the palmitoleic acid level (p < 0.001), and abundance of the [Eubacterium]_ventriosum_group was positively correlated with the tetracosanoic acid level (p < 0.001, Fig. 6b). The results showed that combined with changes in the fecal metabolites and bacterial abundance after BZYQ treatment, BZYQ had an effect on the abundance of the gut microbiota and fecal metabolite level, which showed signi cant correlation (Fig. 6c).
In addition, the tetracosanoic acid level was negatively correlated with the serum DHEAS level (p < 0.05). Palmitoleic acid level was negatively correlated with the serum T level (p < 0.05). The levels of eicosenoic acid, palmitic acid, and erucic acid were all positively correlated with HOMA-IR (p < 0.05, Fig. 6d).

Discussion
PCOS is an endocrine metabolic disorder with multiple causes and polymorphic clinical symptoms.
Chinese medicine can obviously improve the clinical symptoms of obese patients with PCOS, with small side effects, without drug dependence, and with other advantages [19]. BZYQ is an effective prescription for the treatment of obese patients with PCOS and SPSD [20], but its mechanism on the intestinal environment has not been reported yet. In this study, the impact of BZYQ on the gut microbiota and fecal metabolites of obese patients with PCOS and SPSD were discussed through the study of intestinal microecology and nontargeted metabolomics.
Gut microbiota is a general term for sojourn microorganisms in the human intestine, which has physiological functions, such as participating in the body's nutritional metabolism, antagonizing pathogenic microorganisms, immunity, and maintaining the balance of the internal environment [21]. In TCM theory, the gut microbiota is closely related to the physiological function of the "spleen" [22]. When the gut microbiota is disturbed, gastrointestinal discomfort, decline in digestive and absorption function, and other clinical manifestations will be manifested, similar to the spleen de ciency syndrome in TCM [23]. Obese patients with PCOS often have spleen de ciency symptoms, including obesity, fatigue, loss of appetite, and thin stool. In recent years, increasing studies have con rmed that spleen-invigorating TCM compounds help regulate the gut microbiota and maintain the balance in intestinal microecology [24]. BZYQ is one of the spleen-invigorating TCM prescriptions and have a remarkable impact on recovering the gut microbiota of the host. The regulation of gut microbiota may be one of the mechanisms of the treatment for the spleen de ciency syndrome.
The results of the 16S rRNA high-throughput sequencing demonstrated that the composition structure of the gut microbiota in obese patients with PCOS and SPSD at the phylum and genus levels had changed signi cantly. At the phylum level, the abundance of Spirochaetae increased signi cantly after BZYQ treatment, but its abundance in the sample was too small, and the clinical signi cance was small. At the genus level, the bacteria in the top 20 abundances were compared. After BZYQ treatment, the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, unclassi ed_f__Lachnospiraceae, and Fusicatenibacter increased signi cantly, whereas the abundance of Megamonas decreased signi cantly.
[Eubacterium] _rectale_group is a bacterium that produces butyrate (an anti-in ammatory compound) and plays a key role in ghting in ammation [25]. Cattaneo et al. [26] found that the serum levels of proin ammatory cytokines IL-1β, NLRP3, and CXCL2 in the elderly with cognitive impairment cerebral amyloidosis were negatively correlated with the abundance of [Eubacterium]_rectale_group. The increased abundance of [Eubacterium] _rectale_group in patients with in ammatory bowel disease indicates the enhancement of the anti-TNF-α [27].
PCOS is a chronic in ammatory disease, and chronic nonspeci c in ammatory factors affect follicle development, resulting in infertility and adverse pregnancy outcomes by in uencing ovarian function, androgen synthesis in vivo, and insulin resistance [28,29]. The present study suggested that BZYQ may improve chronic in ammation in obese patients with PCOS and SPSD by improving the abundance of [Eubacterium]_rectale_group]. Megamonas could produce short-chain fatty acids (SCFA) [30], which are converted from indigestible carbohydrates by the gut microbiota [31]. Meanwhile, den Besten et al. [32] found that SCFA could activate peroxisome proliferator-activated receptor-γ in the liver and muscle, thereby regulating uptake of glucose and oxidation of fatty acid. In addition, the gut microbiota could in uence the insulin sensitivity by SCFA-mediating in ammatory responses [33]. In the present study, the abundance of Megamonas in the patients decreased after BZYQ treatment, which was bene cial for reducing intestinal permeability and maintaining intestinal homeostasis. The Lefse multi-level differential analysis of species showed that the characteristic genera were Dialister, Holdemania, Megamonas, Ruminiclostridium_9, and vadinBC27_wastewater_sludge_group 5 species before treatment, but the characteristic genera after treatment were Fusicatenibacter, Blautia, and Dorea. Studies have con rmed that lipopolysaccharides produced by Gram-negative bacteria are key molecules involved in the early development of in ammation and metabolic diseases, and these bacteria have an endotoxin effect. Gram-negative bacteria could stimulate the production of many in ammatory factors and produce chronic systemic in ammation by binding to the CD14-toll receptor 4 complex on the surface of innate immune cells [34]. These bacteria could also promote insulin resistance via the phosphorylation of insulin receptor substrate 1 through signaling pathways, such as nuclear factor κB. Thus, Dialister was speculated to be associated with chronic in ammation and insulin resistance in obese patients with PCOS and SPSD.
BZYQ also exhibited implications on the fecal metabolites of obese patients with PCOS and SPSD. The contents of taurocholic acid and xanthine were upregulated after BZYQ treatment, while eight differential metabolites, such as palmitic acid, stearic acid, and sphingosine, were downregulated according to the nontargeted metabolomic studies. Taurocholic acid is a primary bile acid that binds amide to the amino group of the cholic acid carboxyl group and taurine. Previous studies [35] have proven that taurocholic acid has a signi cant inhibitory effect on acute and chronic in ammations, and its mechanism of action is related to its inhibition of macrophage in ltration and the production of pro-in ammatory adipokines.
Palmitic acid is a long-chain saturated fatty acid, which is an important component of blood lipids. Some studies [36] have found that free palmitic acid can induce stress of the endoplasmic reticulum and then induce β cell apoptosis and inhibit insulin synthesis and secretion when glucose concentration is too high. Moreover, in the macrophages, palmitic acid induces the in ammatory responses by increasing FABP4/aP2 protein expression [37]. BZYQ may improve the disorder of glucose metabolism and chronic in ammation of obese patients with PCOS and SPSD by reducing palmitic acid abundance. Sphingosine and its metabolic enzymes are key mediators in the human body. Sphingosine kinase and its lipid product, namely, sphingosine 1-phosphate, are involved in signal transduction and diseases, especially in chronic in ammatory diseases and autoimmunity. These molecules play an important role in the occurrence and development of the disease. In the present study, the abundance of sphingosine in obese patients with PCOS and SPSD was reduced after BZYQ treatment, and this event may help reduce the in ammatory response in the body.
In addition, the differential metabolites were signi cantly enriched in 14 KEGG pathways, such as the biosynthesis of unsaturated fatty acids, fatty acids, cutin, and wax. Most enrichment pathways were lipid metabolism enrichment pathways. The pathogenesis of abnormal lipid metabolism was related to ApoA1, the related regulators of lipid metabolism, adiponectin, leptin, and endogenin [38]. Obirikorang et al. [39] found that a decrease in total blood adiponectin levels can induce IR, obesity, and type 2 diabetes. The gut microbiota is affected by sex hormones and also has an impact on the serum the sex hormone levels [40]. Through the correlation analysis of the gut microbiota-fecal metabolites-serum sex hormones and HOMA-IR values, the abundances of the different bacterial groups after BZYQ treatment were adjusted in different directions, and most of the metabolites involved in KEGG showed an upward trend. Among these bacteria, the abundance of Paraprevotella in obese patients with PCOS and SPSD was positively correlated with serum LH levels, and the correlation was signi cant. The abundances of Lachnospiraceae_NC2004_group and Faecalibacterium were negatively correlated with the serum DHEAS and T levels, whereas the abundance of [Ruminococcus]_gnavus_group was positively correlated with the aforementioned parameters. The abundance of Blautia was positively correlated with HOMA-IR values.
Published studies have proven that Blautia contributes to maintaining glucose stability and its dysregulation impairs the intracellular insulin signaling [41]. The abundance of [Eubacterium]_ventriosum_group was positively correlated with tetracosanoic_acid, but this acid was negatively correlated with serum DHEAS levels. The abundance of Bacteroides was positively correlated with serum T levels but negatively correlated with serum levels. A linear relationship existed among the gut microbiota, fecal metabolite, and hyperandrogenemia. BZYQ may have improved the disorder of hyperandrogen by regulating the abundance of Bacteroides, [Eubacterium]_ventriosum_group, and their differential fecal metabolites.
Although the number of subjects was relatively small, the authors complied strictly by controlling the inclusion and exclusion criteria and excluded the most factors with potential impact on the gut microbiota. In addition, the authors conducted diet guidance and trained sampling method for subjects before sampling. Hence, heterogeneity was greatly reduced in the group, so the authors are con dent that the results are greatly meaningful. In this study, fecal metabolomics combined with gut microbiota was used to explore the relationship between the intestinal environment and clinical parameters in obese patients with PCOS and SPSD who were treated with BZYQ.

Conclusions
BZYQ could ameliorate the serum DHEAS and T level and had an impact on the gut microbiota and metabolites in obese patients with PCOS and SPSD. Relationships existed among the gut microbiota, fecal metabolites, and hyperandrogenism. BZYQ could ameliorate a part of the endocrine disorders in these patients, and this process may be achieved by regulating the abundances of Bacteroides, [Eubacterium] _ventriosum_group, and Blautia and the level of important fecal metabolites, such as palmitoleic acid, tetracosanoic acid, and eicosenoic acid. ChiCTRCTEC2016050). All participants voluntarily signed the informed consent before participating.

Consent for publication
Consents for publication were obtained from all participants.

Availability of data and materials
The raw clinical data of patients is not available due to hospital privacy regulations. The sequencing and LC-MS data are availability on the free online platform of Majorbio Cloud Platform (www.majorbio.com) with the account provided by corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.