E. coli Nissle 1917 ameliorates mitochondrial injury of granulosa cells in polycystic ovary syndrome through promoting gut immune factor IL-22 via gut microbiota and microbial metabolism

The experiments were approved by the Ethics Committee of Hunan Provincial Maternal and Child Health Hospital (2022037). A total of 30 female C57BL/6 mice (21 days old, 8~10 g) were purchased from Lake Jingda Laboratory Animal Center, Hunan. The mice were raised at a temperature of 18 to 29°C, a relative humidity of 40 to 70, and 12/12h of light. Mice were free to eat and drink. The standard feed and drinking water for mice were autoclaved. Mice were fed adaptively for one week. Mice were divided into Control group, DHEA-induced PCOS group and E. coli Nissle 1917 (EcN) group, 10 mice/group. Mice in DHEA-induced PCOS group and EcN group were subcutaneously injected with DHEA (6 mg/100 g body weight, HY-14650, MCE, USA) once a day for 3 weeks to construct the PCOS model. DHEA was dissolved with sesame oil (0.09 mL) and ethanol (95%, 0.01 mL) at room temperature (24). In making mold period, the estrus cycle (proestrus, estrus, metestrus, and diestrus) was examined by vaginal smears for 14 days. When the estrous cycle was disturbed, the PCOS mouse model was successfully established (21, 24). After modeling, mice in DHEA-induced PCOS group and EcN groups were treated by gavage with 0.9% NaCl and EcN (10⁹ CFU/0.2 mL, dissolved in 0.9% NaCl, HG-VDH1186, HonorGene) for 4 weeks (25). During the experiment, mice in Control group were injected with sesame oil (0.09 mL) and ethanol (95%, 0.01 mL) for 3 weeks, subsequently by gavage with 0.9% NaCl for 4 weeks as control. All the drugs intervention were performed at 8 am per day. The experimental flow chart was showed in the Supplementary Figure 1. At the end of the experiment, mice were euthanized with 150 mg/kg pentobarbital sodium (I.P). Peripheral blood, ovary, granulosa cells, and fecal samples (Terminal colorectal contents, 10 samples/group) were collected for subsequent detection. Granulosa cell (GC) was released and collected by killing mice 44 h after intraperitoneal injection of 5 IU PMSG and then puncturing antral follicles with a 26.5-g needle (26). Fecal samples (Terminal colorectal contents, 10 samples/group) were collected from the terminal colorectal contents and stored in a 1.5 mL sterile enzyme free tube at -80°C.

Due to the sexual maturation of mice at 6-7 weeks of age, we conducted estrus cycle testing for 14 consecutive days after a week of modeling intervention. The estrus cycle (proestrus, estrus, metestrus, and diestrus) were examined by vaginal smears (21, 24). Briefly, the mice were kept in the left hand to be tested, and the right hand was used to absorb a small amount of normal saline preheated at 37°C with a sterilized dropper. The dropper head was put into the vaginal opening of the mouse, gently and repeatedly squeeze the dropper rubber head 3~5 times to moisten the mouse’s vagina. Then, 1 drop of normal saline was put in the dropper onto a clean slide. The droplet was laid flat on the slide with the dropper head, and then placed in a ventilated dry place at room temperature to dry thoroughly. The sample area was covered with 95% alcohol and fixed for 5~8 min. The samples were stained with hematoxylin dyeing solution for 20~30s. The samples were differentiated by drops of 1% hydrochloric acid ethanol solution, and immediately stained with eosin solution for 1 min after 5s. Then the sections were washed with running water to remove excess staining solution and observed under a microscope (BA210T, Motic). Subsequently, estrus status was determined by the characteristics of the vulva and the epithelial cells of the vaginal exfoliation at different stages of the estrus cycle (Supplementary Figures 2 and 3). The criteria are as follows: 1) A large number of nuclear epithelial cells appear to be in proestrus; 2) Squamous epithelial cells accounted for most cell types during estrus; 3) Nuclear epithelial cells, squamous epithelial cells and white blood cells can be seen in metestrus. 4) Almost all cells in diestrus are white blood cells.

A total of 10 female C57BL/6 mice (21 days old) were purchased from Lake Jingda Laboratory Animal Center, Hunan. All mice were randomly divided into two groups: DHEA-induced PCOS -FMT group and E. coli Nissle 1917-FMT group, 5 mice/group (Supplementary Figure 1). Fecal samples (Terminal colorectal contents) were collected from donor mice in the above groups (DHEA-induced PCOS group and E. coli Nissle 1917 group). Fecal samples (Terminal colorectal contents, 200 mg) were resuspended in 1mL of sterile saline for subsequent FMT experiments (27). The solution was mixed vigorously for 10 s, then centrifuged at 800×g for 3 min to collect the supernatant and used as graft material. To prevent changes in bacterial composition, fresh graft material was prepared on the day of transplantation within 10 min before tube feeding. Before the FMT, mice were given orally an antibiotic cocktail containing ampicillin (0.1 mg/mL), streptomycin (0.5 mg/mL) and colistin (0.1 mg/mL) to ablate gut microbiota for 4 days, normal drinking water for 3 days (27). Mice in DHEA-induced PCOS-FMT group and E. coli Nissle 1917-FMT group were given 0.1 mL fecal supernatant of mice in DHEA-induced PCOS group and E. coli Nissle 1917 group by gavage every day for 7 days (16, 27). All the drugs intervention were performed at 8 am per day. At the end of the experiment, mice were euthanized with 150 mg/kg pentobarbital sodium (I.P). Peripheral blood, ovary and granulosa cell specimens were collected for subsequent detection.

A total of 25 female C57BL/6 mice (21 days old) were purchased from Lake Jingda Laboratory Animal Center, Hunan. All mice were randomly divided into 5 groups: Control group, DHEA-induced PCOS group, α-IL-22 group, E. coli Nissle 1917-FMT group, α-IL-22 +E. coli Nissle 1917-FMT group, 5 mice/group (Supplementary Figure 1). Except for Control group, the other groups established the PCOS mouse model according to the above methods. After 21 days of modeling in the α-IL-22 group, mice were given a single intraperitoneal (I.P) dose of 400 µg α-IL-22 (IL22JOP, Thermo Fisher, USA) and gavage of 0.1mL sterile saline (28). After 21 days of modeling, mice in the E. coli Nissle 1917-FMT group were given 0.1mL fecal supernatant of mice in the E. coli Nissle 1917 group daily for 7 days. Mice in the α-IL-22+E. coli Nissle 1917-FMT group were given α-IL-22 and fecal supernatant interventions for 7 days. All the mice were treated with 400 µg of mouse IgG2aκ (11-4724-42, Thermo Fisher, USA) and 0.1 mL of sterile saline intragastric for 7 days as control. All the drugs intervention were performed at 8 am per day. At the end of the experiment, mice were euthanized with 150 mg/kg pentobarbital sodium (I.P). Peripheral blood, ovary and granulosa cell specimens were collected for subsequent detection.

All subjects were recruited from Hunan Provincial Maternal and Child Health Hospital and were aged 22 to 38 years. All subjects were divided according to their diagnosis into women presenting with male azoospermia or fallopian tube obstruction (Control, n=15) and newly diagnosed patients with PCOS (n=15) (Table 1). Other causes of hyperandrogenemia or ovulation dysfunction (Cushing’s syndrome, 21-hydroxylase deficiency, thyroid disease, androgen-secreting neoplasms, congenital adrenal hyperplasia, and hyperprolactinemia) were excluded in subjects with PCOS. According to the 2003 Rotterdam criteria (16), at least two of the following are required for diagnosis: (1) hypo-ovulation and/or anovulation; (2) Clinical and/or biochemical signs of hyperandrogenism; (3) Polycystic ovary. The control group was from subjects with normal menstrual cycle, ovarian morphology, and hormone levels. All PCOS patients and controls in the above group were in the first IVF cycle, and fasting peripheral blood was collected at 8~10 am for detection after standardized intervention.

The staff of the sample bank will standardize and collect the follicular fluid produced in the process of assisted reproductive technology treatment of the included patients. At the first puncture of each ovary from which the egg was retrieved, the follicular fluid samples were collected from a single large follicle and centrifuged to remove cells for subsequent analysis. The tops of cell precipitates were collected from concomitant follicular fluid samples of PCOS patients, which were aspirated and washed in PBS. Then, the cell precipitates were resuspended in PBS and cultured in Ficoll (LTS1077, TBD Science) to layer the solution and separate from red blood cells through centrifugation. The cell layer at the Ficoll/PBS interface were aspirated and washed with PBS to remove residual Ficoll. The final granulosa cells were precipitated in PMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (5000 U/mL) at 37°C in a moist atmosphere of 5% CO₂ and used for subsequent assays.

The blood and follicular fluid samples were placed at room temperature for 2 h and centrifuged at 1000g for 15 min at 2-8°C to obtain serum and follicular fluid supernatant. Then, they were stored at -80°C for detection. Serum and follicular fluid supernatants were used to measure the progesterone (P, CSB-E05104m, CUSABIO), Luteinizing Hormone (LH, CEA441Mu, CEA441Hu, cloud-clone corp), Testosterone (T, CEA458Ge, cloud-clone corp), Follicle Stimulating Hormone (FSH, CEA830Mu, SEA830Hu, cloud-clone corp), IL-22 (KE10041, KE00008, Proteintech), insulin (INS, CSB-E05069h, CUSABIO), Dehydroepiandrosterone Sulfate (DHEA-S, ab108669, abcam), Androstenedione (ASD, CEA456Ge, cloud-clone corp), Estradiol (E2, CEA461Ge, cloud-clone corp), sex hormone binding globulin (SHBG, SEA396Hu, cloud-clone corp), IL-6 (KE00118, Proteintech), IL-1β (DLB50, Bio-Techne China Co., Ltd) with a microplate reader (MB-530, HEALES).

The levels of triglyceride (TG, A110-1-1, NJJCBI), total cholesterol (T-CHO, A111-1-1, NJJCBI), high-density lipoprotein cholesterol (HDL-C, A112-1-1, NJJCBI), low-density lipoprotein cholesterol (LDL-C, A113-1-1, NJJCBI) and glucose (Glu, F006-1-1, NJJCBI) in serum and follicular fluid supernatant were detected by GPO-PAP, microplate, and peroxidase methods, respectively.

Ovarian tissue was fixed, embedded, and sectioned. Sections were deparaffinized to water by xylene and graded ethanol. Sections were stained with hematoxylin and eosin sequentially. Sections were dehydrated with graded alcohol (95-100%) and observed by a microscope (BA210T, Motic).

Granulosa cells were collected and total RNA was extracted by TRIzol reagent (Thermo, USA). The cDNA was synthesized using a Kit (HiFiScript cDNA Synthesis Kit, CWBio, China). The primer sequence was H-COX4 (146 bp): F-AGGCCCAAGGAGAGAAGCTA, R-AGGCCCATCAACCTTACACG. H-GAPDH (100 bp): F-TCCTGCCCTTTGAGTTTGATGATGCT, R-ACTATGCCACCCCAGGAATGCTT. M-COX4 (149 bp): F-TCCCCACTTACGCTGATCG, R-GATGCGGTACAACTGAACTTTCT and M-GAPDH (122 bp), F-GCGACTTCAACAGCAACTCCC, R-CACCCTGTTGCTGTAGCCGTA. The relative mRNA expression levels of COX4 were analyzed by UltraSYBR Mixture (CW2601, CWBio, China) and 2^−ΔΔCT method.

The cells were washed with ice-pre-cooled PBS and added with 200 uL RIPA lysate (AWB0136, Abiowell). The total protein was extracted after ultrasonic crushing for 1.5 min. Protein concentrations were determined using the BCA method. Protein samples (200 μg) were separated by SDS-PAGE (12%, AWT0047, Abiowell). Then, proteins were transferred to a polyvinylidene difluoride membrane, which was activated with methanol and blocked with skim milk powder (5%, AWB0004, Abiowell). Membrane was incubated with antibodies, which were included anti-progesterone receptor A (PR-A, ab32085, 1:5000, Abcam, UK), anti-LC3II/I (#43566, 1:1000, CST, USA), anti-Beclin1 (11306-1-AP, 1:1000, Proteintech, USA), anti-p62 (18420-1AP, 1:4000, Proteintech, USA), anti-CytC (10993-1-AP, 1:4000, Proteintech, USA) and anti-β-actin (66009-1-Ig, 1:5000, Proteintech, USA). Then, it was incubated with secondary anti-IgG (SA00001-1/2, 1:6000/5000, Proteintech, USA) at 37°C for 90 min. ECL Plus hypersensitive luminescence solution (AWB0005, Abiowell) was used for visualization, and software (ChemiScope6100, CLINX) was used for imaging analysis.

According to the manufacturer’s instructions, the levels of MMP (AWC0128a, Abiowell) was detected by JC-1 method. In brief, cells were re-suspended in 0.5 mL cell culture medium and mixed with 0.5 mL JC-1 staining working medium. The cells were incubated in an incubator at 37°C for 20 min. Cell precipitates were collected by centrifugation. Cells were resuspended and washed twice with JC-1 staining buffer (1×). The cells were suspended with JC-1 staining buffer (1×) and analyzed by flow cytometry (A00-1-1102, Beckman). The MMP (%) = UR (red fluorescence)/LR (green fluorescence).

Cells were fixed in 2.5% glutaraldehyde and 1% osmic acid (18456, TED PELLA INC) for 6-12 h and 1-2h, respectively. The cells were dehydrated using gradient ethanol (30 ~100%) and propylene oxide (M25514↗, MERYER). Subsequently, the cells were immersed in propylene oxide: epoxy resin (1:1) and pure epoxy resin for 1-2 h and 2-3 h for embedding, and oven baked for 60h. The embedded block was taken out and repaired, then ultrathin sections were cut and copper mesh was retrieved. The sections were stained for electron (lead and uranium). Finally, sections were observed with a transmission electron microscope (7700, Hitachi) and images were recorded with a digital camera (ER-B, AMT).

DNA was extracted by the kit (Tiangen, cat. #DP328-02) from fecal samples. PCR amplification and library construction were conducted by phusion enzyme (K1031, APExBIO) and the V3-V4 region primers (357F 5’- ACTCCTACGGRAGGCAGCAG-3’ and 806R 5’-GGACTACHVGGGTWTCTCATAT-3’) of the 16S rRNA gene. Illumina NovaseQ6000 PE250 was applied for mixed sequence to collect Raw Data. Qiime 2 (2020.2) was adopted to improve the data quality control, and calculate the Alpha diversity index, PCA analysis and relative abundance of microbiota. Each ASV/OTU sequence was annotated by referring to the SilvA-132-99 database, and the corresponding species information and abundance distribution were obtained. R software (Venn Diagram package) and Jvenn web page (http://www.bioinformatics.com.cn/static/others/jvenn/example.html↗) were used to analyze common and unique ASVs in groups. LDA Effect Size analysis (LefSe, https://github.com/SegataLab/lefse↗) was applied for evaluate the differential microbiota.

LC-MS/MS analysis was performed by the UHPLC system (1290, Agilent Technologies). The MS raw data files were converted for analysis through the R package XCMS (version 3.2). MetaboAnalyst platform (https://www.metaboanalyst.ca/↗) was used for bioinformatics analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.kegg.jp/↗) pathway database was used for metabolite function prediction.

Statistical analysis of the data in this study was performed using Graphpad Prism8.0 statistical software. The measurement data are expressed as the mean ± standard deviation (SD). First, the normality and homogeneity of variance are tested. The test conformed to the normal distribution and the variance was uniform. The unpaired t test was used between groups. The one-way ANOVA or the analysis of variance of repeated measurement data is used for the comparison among groups. Tukey’s performed post hoc tests. P < 0.05 indicated that the difference was statistically significant.

From d14~d49, the body weight of mice in DHEA-induced PCOS group was higher than that in Control group, which was decreased after EcN intervention (Figure 1A). The estrus cycle detection showed that the Control group and E. coli Nissle 1917 group had normal cycles, while the DHEA-induced PCOS group had disrupted cycles (Figure 1B). The representative picture of the vaginal smears in Control group and DHEA-induced PCOS group (14 days) was shown in Supplementary Figures 2 and 3. There was no significant change of glucose level in mice of Control, DHEA-induced PCOS and EcN groups (Figure 1C). The INS level was rose in PCOS mice, which was down-regulated by EcN (Figure 1C). HE observation of ovarian morphology showed that the ovaries of the Control group contained follicles at different stages of development and several corpus luteum (Figure 1D). The characteristic polycystic ovary morphology was found in the ovaries of mice in the DHEA-induced PCOS group, with less corpus luteum, thinner granulosa cell layer, increased number of antral follicles and fewer primordial follicles (Figure 1D). EcN intervention resulted in relief of DHEA-induced PCOS symptoms (Figure 1D). The serum FSH and P levels were decreased and the serum Testo and LH levels were increased in PCOS mice (Figures 1E–H). EcN restored serum FSH and P levels and decreased Testo and LH levels in PCOS mice (Figures 1E–H). In addition, serum IL-22 levels were reduced in PCOS mice and were restored by EcN intervention (Figure 1I). The LC3II/I, Beclin 1 and Cytochrome C expression increased, while the expression of p62 and PR-A decreased in ovarian tissues of PCOS mice (Figures 1J, K). EcN inhibited the expression of LC3II/I, Beclin 1 and Cytochrome C and promoted the expression of p62 and PR-A in ovarian tissues of PCOS mice (Figures 1J, K). MMP level of PCOS mice was significantly decreased, which was reversed by EcN intervention (Figure 1L). The expression of COX4 in granulosa cells of PCOS mice was significantly decreased, and EcN intervention promoted the expression of COX4 in granulosa cells (Figure 1M). TEM showed that compared with Control group, there were many damaged mitochondria with the fracture, reduction or disappearance of ridge structure in the DHEA-induced PCOS group, which were engulfed by autophagosomes, indicating that autophagy was activated (Figure 1N). EcN intervention reduced the damaged mitochondria and autophagosomes in granulosa cells to inhibit the autophagy (Figure 1N). EcN improved mitochondrial damage and promoted IL-22 expression in granulosa cells of PCOS mice.

The number of gut microbiota in PCOS mice was increased (Figure 2A). EcN treatment decreased the number of gut microbiota in PCOS mice (Figure 2A). The Alpha index of PCOS mice was significantly increased (Figure 2B). EcN treatment reduced the alpha index of PCOS mice, but there was no significant difference (Figure 2B). PcoA analysis showed that the samples of DHEA-induced PCOS group were far away from the Control group, while the samples of E. coli Nissle 1917 group were close to the Control group and there was crossover (Figure 2C). The microbiota at phylum level was mainly composed of Firmicutes, Bacteroidota, Verrucomicrobiota, Proteobacteria, Campilobacterota, Desulfobacterota, Patescibacteria, Actinobacteriota and Cyanobacteria (Figure 2D). The microbiota at genus level was mainly composed of Muribaculaceae, Lactobacillus, Dubosiella, Akkermansia, Clostridia_UCG-014, Bacteroides, Lachnospiraceae_NK4A136_group, Parabacteroides, Alloprevotella, Helicobacter, Desulfovibrio, Parasutterella, Candidatus_Saccharimonas, Ileibacterium, Escherichia-Shigella, Alistipes and Allobaculum (Figure 2E). The differences between groups showed that Dubosiella, RF39 and Ileibacterium were significantly enriched in DHEA-induced PCOS group, while Adlercreutzia, Allobaculum, Escherichia-Shigella and Ileibacterium were significantly enriched in E. coli Nissle 1917 group (Figure 2F). These results demonstrated that EcN improved the microbial diversity and composition of PCOS mice.

PCA analysis showed that QC samples were clustered together, which proved that the instrument was stable (Figure 3A). The Heatmap showed the variation in abundance of differential metabolites across samples (Figure 3B). KEGG prediction showed that metabolites were enriched in the amino sugar and nucleotide sugar metabolism pathway (Figure 3C). All these proved that EcN has the most significant influence on amino sugar and nucleotide sugar metabolism pathway in PCOS mice.

RF39 was positively correlated with Luteinizing hormone, Testosterone, N−Acetylglucosamin and N−Acetylmannosamin, negatively correlated with IL-22 and Progesterone (Figure 4). Ileibacterium was positively associated with IL-22 and Progesterone, negatively associated with Luteinizing hormone, Testosterone, N−Acetylglucosamin, L−Fucose and N−Acetylmannosamin (Figure 4). Dubosiella was positively correlated with Testosterone (Figure 4). Allobaculum was positively correlated with N−Acetylglucosamin and N−Acetylmannosamin (Figure 4). Adlercreutzia was positively associated with IL-22 and Progesterone, negatively associated with Luteinizing hormone and Testosterone (Figure 4). IL-22 was positively associated with Progesterone, negatively associated with Luteinizing hormone, Testosterone, N−Acetylglucosamin, L−Fucose and N−Acetylmannosamin (Figure 4). These results proved that IL-22 was positively associated with Ileibacterium, Adlercreutzia and Progesterone, negatively associated with RF39, Luteinizing hormone, Testosterone, N−Acetylglucosamin, L−Fucose and N−Acetylmannosamin.

There was no significant change of body weight between DHEA-induced PCOS-FMT group and E. coli Nissle 1917-FMT group (Figure 5A). HE observation showed that the characteristic polycystic ovary morphology was found in the DHEA-induced PCOS-FMT group, with less corpus luteum, thinner granulosa cell layer, increased number of antral follicles and fewer primordial follicles (Figure 5B). E. coli Nissle 1917-FMT intervention alleviated PCOS symptoms (Figure 5B). Compared with the DHEA-induced PCOS-FMT group, the glucose level has no change, while the INS level was clearly reduced in the E. coli Nissle 1917-FMT group (Figure 5C). Compared with the DHEA-induced PCOS-FMT group, the levels of FSH, P and IL-22 were increased, and the LH and Testo levels were decreased in the E. coli Nissle 1917-FMT group (Figures 5E–H). E. coli Nissle 1917-FMT intervention increased the expression of p62 and PR-A, and decreased the expression of LC3II/I, Beclin 1 and cytochrome C in ovarian tissues of PCOS mice (Figures 5I, J). In addition, the expression of COX4 and MMP level in granulosa cells were increased after E. coli Nissle 1917-FMT intervention (Figures 5K, L). Electron microscopy showed that the damaged mitochondria and autophagosomes in the ovarian tissues of mice was reduced after E. coli Nissle 1917-FMT intervention (Figure 5M). These results demonstrated that E. coli Nissle 1917-FMT ameliorated mitochondrial damage in granulosa cells of PCOS mice.

Body weight in PCOS mice was higher than that in Control group, which was up-regulated by the αIL-22 intervention, down-regulated by the E. coli Nissle 1917-FMT and αIL-22+E. coli Nissle 1917-FMT intervention (Figure 6A). E. coli Nissle 1917-FMT inhibited the action of αIL-22 in PCOS mice (Figure 6A). HE observation showed that ovaries in the Control group contained follicles at different stages of development and several corpus luteum (Figure 6B). The characteristic polycystic ovary morphology was found in the ovaries of mice in the DHEA-induced PCOS group, with less corpus luteum, thinner granulosa cell layer, increased number of antral follicles and fewer primordial follicles (Figure 6B). αIL-22 exacerbated PCOS symptoms (Figure 6B). E. coli Nissle 1917-FMT intervention alleviated PCOS symptoms and partially reversed the effect of αIL-22 on PCOS mice (Figure 6B). There was no significant change of glucose level in different groups (Figure 6C). αIL-22 promoted the INS level in PCOS mice, which was down-regulated by E. coli Nissle 1917-FMT intervention (Figure 6C). Compared with the DHEA-induced PCOS group, the serum FSH, P and IL-22 levels were decreased, and the LH and T levels were increased in the αIL-22 group (Figures 6D–H). Compared with αIL-22 group, serum FSH, P and IL-22 levels were significantly increased, and the LH and T levels were significantly decreased in αIL-22 + E. coli Nissle 1917-FMT group (Figures 6D–H). αIL-22 increased the expression of LC3II/I, Beclin 1 and cytochrome C, while decreased the expression of p62 and PR-A in ovarian tissues of PCOS mice (Figures 6I, J). Compared with the αIL-22 group, the expression of LC3II/I, Beclin 1 and cytochrome C in ovarian tissues of mice were decreased, and the expression of p62 and PR-A was increased in αIL-22 + E. coli Nissle 1917-FMT group (Figures 6I, J). αIL-22 decreased MMP level and COX4 expression, and increased the damaged mitochondria and autophagosomes in ovarian granulosa cells of PCOS mice, which was reversed by E. coli Nissle 1917-FMT intervention (Figures 6K–M). These results demonstrated that IL-22 mediated mitochondrial damage in granulosa cells of PCOS mice improved by E. coli Nissle 1917-FMT.

Further analysis at the clinical level showed that there was no significant changes of age and BMI between Control and PCOS groups (Table 1). Serum FSH level in PCOS patients was lower than Control group (Figure 7A). The levels of E2, T, ASD, DHEA-S, LH, and SHBG were increased in PCOS patients (Figure 7A). The PR-A protein expression was down-regulated in PCOS patients (Figure 7B). In addition, patients with PCOS had the decrease of HDL-C level, and the increase of INS, TG, T-CHO, and LDL-C levels, while no significant changes in blood glucose level (Figure 7C). The serum levels of IL-6 and IL-1β increased, and IL-22 decreased in PCOS patients (Figure 7D). These results demonstrated that PCOS patients were associated with the decrease of IL-22 level, PR-A level, and mitochondrial damage in granulosa cells, which was the therapeutic target of E. coli Nissle 1917.

The data presented in the study are deposited in the NCBI BioProject repository, accession number PRJNA930556.