Acupuncture of Polycystic Ovary Syndrome: Delving into Bile Acid Metabolism

Acupuncture of Polycystic Ovary Syndrome: Delving into Bile Acid Metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Acupuncture of Polycystic Ovary Syndrome: Delving into Bile Acid Metabolism Haolin Zhang, Yang Chen, Dai Heng, Jingzhi Luo, Haining Wang, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6600841/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Background: polycystic ovary syndrome (PCOS), a common endocrine disorder, is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Given bile acids' role in metabolic regulation and acupuncture's therapeutic potential for endocrine-metabolic disorders, this study examines acupuncture’s effects on bile acid metabolism and insulin sensitivity in PCOS patients and rat models. Methods: 33 PCOS patients and 28 age/BMI-matched controls were enrolled in a clinical trial (NCT04193371). 16 PCOS patients received 4-month acupuncture followed by 4-month observation. Serum bile acids, insulin sensitivity, hormones, and inflammatory markers were analyzed. A dihydrotestosterone (DHT)-induced PCOS rat model was treated with electroacupuncture (EA), with outcomes assessed via estrous cycles and ovarian histology. The mechanisms of EA are dissected through RNA sequencing. Results: acupuncture improved BMI, insulin resistance, and hyperandrogenism in PCOS patients, with sustained post-treatment effects. Specific bile acids (e.g., TCA, LCA) correlated with metabolic improvements. In PCOS rats, EA restored estrous cyclicity, reduced ovarian cysts, and downregulated hepatic FXR expression. RNA-seq revealed significant alterations in bile acid metabolism-related genes and pathways. Conclusions: acupuncture therapy offers potential therapeutic benefits to patients with PCOS by modulating bile acid metabolism, with a particular focus on specific types of bile acids. Polycystic ovary syndrome acupuncture bile acids insulin resistance FXR pathway Figures Figure 1 Figure 2 Figure 3 Background Polycystic Ovary Syndrome (PCOS), the most prevalent endocrine-metabolic disorder in women of reproductive age, is characterized by menstrual irregularities, hyperandrogenism, and anovulation. Its incidence, particularly among adolescents, has risen significantly in recent decades( 1 ). Current medical interventions for PCOS primarily focus on symptom management. Acupuncture, however, has garnered global research interest due to its unique mechanisms and favorable safety profile. Both as a standalone and adjunctive therapy, acupuncture demonstrates potential in ameliorating endocrine-metabolic dysregulation in PCOS( 2 ). Previous studies, including our own, have established its therapeutic efficacy across disease stages, particularly in mitigating insulin resistance( 3 ). Nevertheless, the underlying molecular mechanisms remain incompletely understood. Emerging evidence highlights the gut microbiota-bile acid axis as a critical mediator in PCOS pathogenesis and metabolic comorbidities( 4 ). Dysbiosis-driven alterations in bile acid composition may disrupt key signaling pathways, such as the Farnesoid X receptor (FXR)-FGF15/19 axis, thereby exacerbating metabolic dysfunction, including insulin resistance( 5 ). Notably, PCOS patients exhibit distinct gut microbiota profiles and bile acid imbalances, which correlate with compromised immunity, intestinal barrier dysfunction, and hyperandrogenism( 6 ). These findings position bile acids as promising diagnostic biomarkers for PCOS. Pharmacological insights further support this link: metformin partially alleviates symptoms by inhibiting intestinal FXR signaling and enhancing glucose metabolism( 7 ). Additionally, elevated circulating conjugated primary bile acids have been directly associated with hyperandrogenism in PCOS( 8 ). Our prior work revealed that acupuncture restores gut microbial diversity in PCOS patients, specifically enriching beneficial microbes Akkermansia and Prevotella , which inversely correlate with insulin resistance severity( 9 ). Collectively, these findings suggest that acupuncture’s therapeutic effects on PCOS, particularly its insulin-sensitizing actions, may be mediated through modulation of the gut microbiota-bile acid-FXR axis. Methods Participants To delve deeper of the relationship between bile acids and acupuncture treatment, we profiled the serum bile acids under acupuncture treatment. 33 participants diagnosed with PCOS, and 28 age/BMI matched healthy controls were recruited from the registered clinical trial (NCT04193371). Ethical approval was obtained from the Regional Ethical Review Board of Peking University Third Hospital (Beijing, China; approval number, PKU3-IRB-M2019021) between 2019 and 2022, informed consent was obtained from all patients, The inclusion criteria for the study were age from 20 to 40 and BMI ranging from 25 to 40. Participants would be excluded if they: ( 1 ) diagnosis of any other endocrine disorder (e.g., androgen-secreting tumors, thyroid dysfunction or diabetes etc.); ( 2 ) receive pharmacological or acupuncture treatment within the last 3 months; ( 3 ) pregnancy or breastfeeding in the last 6 months. Follow up and assessment of outcomes All participants adhered to regular lifestyle management for 4 months. While 16 out of 33 participants were randomly selected to undergo 4 months of acupuncture treatment, in addition, participants were followed up 4 months after the completion of the treatment period (Fig. 1 a). Glucose tolerance and insulin sensitivity were assessed by using the oral glucose tolerance test (OGTT). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as: [fasting insulin (µU mL-1) × fasting glucose (mmol L-1)] / 22.5, while the HOMA-β (islet β-cell function) was calculated as [20 × fasting insulin (µU mL-1) / (fasting plasma glucose (mmol L-1) − 3.5) ].Hyperinsulinemia was estimated by calculating the area under the curve (AUC) using the trapezoid method( 4 ). Samples were analyzed across three time points (pre-treatment, post 4-month treatment, and post 4-month follow-up), sexual hormones include estrogen (E2), progestin (P), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T), and androstenedione (A2) were measured with the Immulite 2000 immunoassay system (Siemens Healthcare Diagnostics, Siemens, Germany). Sex hormone-binding globulin (SHBG) was measured using the Beckman DxI800 immunoassay system (Beckman Coulter, Brea, CA, USA). The free androgen index (FAI) was calculated using the following formula: [T.TES (nmol L-1) × 100/SHBG (nmol L-1)]. Serum anti-Müllerian hormone (AMH) concentrations, fibroblast growth factor (FGF)-19, Ghrelin, Interleukin (IL)-6, beta (β)-Endorphin and adrenocorticotropic-hormone (ACTH) levels were measured by an ultrasensitive two-site enzyme-linked immunosorbent (ELISA) assay (Ansh Laboratories LLC; Webster, TX, USA). Plasma glycated hemoglobin (HbA1c) and Gamma amino butyric acid (GABA) were quantified using UPLC-MS/MS method( 4 ). Tumor Necrosis Factor alpha (TNF-a) was determined by immune-assay (Luminex 100™, Austin, TX, US), glutamate (Glu) and dopamine (DA) plasma concentrations were measured using high-performance liquid chromatography(Phenomenex, Torrance, CA USA)( 3 ). Serum bile acid metabolome was quantified using an Eksigent Ultra Liquid Chromatography 100 system coupled to an AB 5600 TripleTOF system (AB SCIEX) (Fig. 1 b). Animal experiment Animals This study was conducted with the approval of the Animal Care and Use Committee at the Peking University Health Science Center. Female Sprague–Dawley Wistar rats were randomly divided into three groups (Control, PCOS, and PCOS + EA, n = 6/group) and housed under controlled conditions (21ºC–22ºC, 55–65% humidity, 12–12 h light–dark cycle) with free access to normal chow and water. The study employed 4-week-old rats obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Establishment of the PCOS-like rat model Slow-release silicon tube with dihydrotestosterone (DHT, 7.5mg, daily release amount of 83ug, inner diameter and outer diameter of 0.078” (ID) and 0.125” (O.D), 3 cm length) were implanted subcutaneously in the dorsal neck of 21 days of age rats anesthetized by intraperitoneal injection, 0.5 cm from the paraspinal column. Suture after ensuring the location of the embedded silicon tube did not affect the normal feeding and activities of the rats. Closely observed the wound healing and weighed weekly. Vaginal smears The stage of the estrous cycle was determined by microscopic analysis of the predominant cell type in vaginal smears. Seven consecutive days after one month of modeling, the last four consecutive days of the electroacupuncture intervention, and seven consecutive days prior to sampling, vaginal wall cells were taken on a saline moistened cotton swab and applied flat on slides. Cells were fixed and then stained using Shorr's stain and observed under a microscope to determine the estrous cycle. the proestrus stage was typified by predominantly nucleated cells, the estrus stage exhibited cornified squamous epithelial cells, the metestrus stage was marked by a combination of cornified cells and leukocytes, and the diestrus stage showed a predominance of leukocytes. Oral glucose tolerance test 6 weeks after the electroacupuncture intervention, the rats were fasted for 8h and fasting blood glucose was measured (0min), 2g/kg of 50% Glucose was administered by gastric gavage, and blood glucose was measured at each time point for 30, 60, and 120min thereafter. Low-frequency EA Rats in the PCOS + EA group were treated with low-frequency EA at 9 weeks of age. The treatments were given from 08:00 to 10:00 on Monday, Wednesday and Friday for 6 weeks (for a total of 17 treatments). The rats were fixed using a fixator, single-use sterile acupuncture needles were inserted bilaterally at traditional acupuncture point locations CV12(Zhongwan), CV4(Guanyuan), ST25(Tianshu), ST29 (Guilai), CV3(Zhongji), ST36(Zusanli) and SP6 (Sanyinjiao). On CV12, ST25, ST29 and CV3, the needles were attached to an electrical stimulator (HANS-LH202, Huayang Co., Ltd, China) for 20min at 2 Hz, 2 mA and 10min at 2Hz, 3mA. Measurement of hormones At 16 weeks of age, all rats were fasted for eight hours and sacrificed. Blood was collected via puncture of the retro-orbital venous plexus and transferred to EDTA-containing tubes. Serum was obtained by centrifugation (3,000 rpm, 20 min) and stored at − 80°C for use. Fasting insulin (FINS) levels was measured using radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China). The levels of testosterone (T), SHBG were determined with 125-I labeled radioimmunoassay kits (Beijing North Institute of Biological Technology, Beijing, China). The kits contained standard samples for quality control and were used in accordance with manufacturer instructions. RNA isolation and quantitative PCR Total RNA from liver tissue was extracted using the MolPure® Cell/Tissue Total RNA kit and reverse transcribed to cDNA using the PrimeScript RT kit. qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, order Q711-02) on a Line-Gene 9600 instrument. qPCR primers were as follows: (Sequence (5'->3'), (Sequence (5'->3')). qPCR primers were as follows: (Sequence (5'->3')) FXR: forward primer: CTCCTCTCGTCCTATTATTCC reverse primer: TCAGCCAACATTCCCATC β-Actin: forward primer: GCCACCAGTTCGCCATGGAT reverse primer: CCCACGATGGGAGGGGAAGA PCR amplification conditions were as follows: initial denaturation at 95 ℃ for 30 s, 40 cycles of amplification, denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s, and extension at 60 ℃ for 30 s. Melt curve analysis was performed after the reaction to determine the homogeneity of amplicons. RNA-seq analysis Total RNA was extracted from the livers of rats in the control, the PCOS and the PCOS-EA group. RNA-seq library preparation was performed by Biomarker Technologies. Sequencing libraries were constructed, and paired-end mRNA-seq data with read lengths of 150 bp were generated, with two or three biological replicates for each cell line. The raw reads were first aligned to the mouse reference genome (mm10) using the STAR software( 10 ) with default parameters. The expression matrix was generated by FeatureCounts software( 10 ). DEGs were calculated using the R package DEseq( 11 ). Expression levels were represented as Fragments Per Kilobase of exon model per Million mapped fragments(FPKM). DEGs were identified based on the following criteria: absolute fold change > 1.5 and the False Discovery Rate (FDR) < 0.05. To compare the functions of the DEGs across the five cell types, we used the Metascape website( https://metascape.org/gp/index.html#/main/step1 ). Statistical analysis All statistical analyses were conducted using SPSS software version 20.0 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were represented as mean, standard deviation, and range. Pearson correlations examined associations between the bile acids and clinical indicators. Independent t-tests determined intergroup differences. Group comparisons underwent (One-way ANOVA) with significance denoted as p < 0.05. Results Serum bile acid profiling of Patients with PCOS under Acupuncture treatment 33 PCOS patients and 28 age/BMI-matched controls were enrolled in a clinical trial (NCT04193371). 16 PCOS patients received 4-month acupuncture followed by 4-month observation (Fig. 1 a). Acupuncture treatment significantly reduced body mass index (BMI) and waist-hip ratio (%) in patients with PCOS. Key insulin resistance indices, including hemoglobin A1c (HbA1c), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and Area Under the Curve (AUC) for glucose tolerance, demonstrated marked improvement alongside hyperandrogenism markers (androstenedione [A2], sex hormone-binding globulin [SHBG], and free androgen index [FAI]). These therapeutic effects persisted for 4 months post-treatment (Table 1 ). Table 1 Characteristics and clinical indices of 59 participants Characteristics Non-PCOS PCOS A&L A&L SA&L SA&L Non-PCOS VS PCOS A&L 4months VS PCOS A&L 8months VS PCOS SA&L 4 months VS PCOS SA&L 8 months VS PCOS 4 months 8 months 4 months 8 months Age (years) 31.07 ± 5.36 28.45 ± 5.06 28.69 ± 5.24 29.23 ± 5.21 28.20 ± 5.03 29.45 ± 4.76 NS NS NS NS NS BMI (kg/m 2 ) 28.65 ± 2.53 30.18 ± 3.48 27.57 ± 2.95 27.01 ± 2.86 29.80 ± 3.84 28.79 ± 3.94 NS * ** NS NS Waist-hip ratio (%) 87.68 ± 5.49 87.30 ± 5.67 85.55 ± 5.98 85.11 ± 5.84 87.14 ± 6.08 87.00 ± 6.56 NS NS NS NS NS Body fat rate (%) 40.11 ± 3.69 42.17 ± 5.23 38.81 ± 4.94 36.30 ± 6.75 40.02 ± 6.28 39.24 ± 6.76 NS * ** NS NS Lean weight 44.21 ± 3.66 44.30 ± 2.95 43.16 ± 2.51 43.87 ± 3.99 44.09 ± 3.28 44.51 ± 3.24 NS NS NS NS NS HbA1c 5.50 ± 0.55 5.63 ± 0.38 5.23 ± 0.28 5.13 ± 0.40 5.48 ± 0.31 5.58 ± 0.35 NS *** *** NS NS Fasting Glucose (mmol/L) 5.38 ± 0.76 5.11 ± 0.70 5.09 ± 0.40 5.30 ± 0.92 5.29 ± 0.56 5.35 ± 0.77 NS NS NS NS NS Fasting insulin (mU/L) 19.06 ± 15.22 19.45 ± 8.14 12.59 ± 4.94 14.30 ± 6.95 15.81 ± 6.7 13.98 ± 8.72 NS ** * NS * HOMA-IR 4.95 ± 5.98 4.48 ± 2.11 2.89 ± 1.27 3.44 ± 1.84 3.72 ± 1.58 3.28 ± 2.08 NS ** NS NS NS HOMA-B 202.35 ± 108.34 394.76 ± 721.38 159.97± 50.87 174.44± 79.84 190.19± 92.59 172.92 ± 103.24 NS NS NS NS NS AUC 120 glucose 988.93 ± 227.97 992.27 ± 144.40 886.78 ± 180.29 889.50 ± 113.50 938.80 ± 195.01 885.68 ± 106.37 NS * * NS * AUC 120 insulin 11631.54 ± 7110.74 14242.35 ± 6696.57 10071.38 ± 4518.92 9842.00 ± 5320.59 10303.00 ± 5548.73 11265.00 ± 6883.02 NS * * NS NS PRL ng/ml 11.73 ± 5.93 12.32 ± 8.39 12.70 ± 8.14 11.62 ± 6.08 14.37 ± 10.19 13.51 ± 11.74 NS NS NS NS NS LH mIU/ml 4.50 ± 2.99 7.40 ± 4.66 5.82 ± 3.24 5.46 ± 3.10 5.78 ± 2.67 8.69 ± 4.42 ** NS NS NS NS FSH mIU/ml 6.79 ± 2.81 5.51 ± 1.49 6.31 ± 1.27 5.92 ± 1.01 5.44 ± 1.24 6.07 ± 1.99 * NS NS NS NS E2 pmol/L 178.20± 50.77 200.19 ± 106.80 152.69± 38.86 142.00± 55.71 166.01± 56.38 161.54± 43.82 NS NS NS NS NS P nmol/L 1.74 ± 2.95 1.04 ± 0.53 0.98 ± 0.40 0.92 ± 0.34 0.99 ± 0.36 0.90 ± 0.30 NS NS NS NS NS A 2 nmol/L 7.27 ± 3.28 10.13 ± 4.58 8.96 ± 4.84 8.36 ± 4.52 10.43 ± 4.50 10.46 ± 3.27 ** NS NS NS NS T nmol/L 1.55 ± 0.50 2.29 ± 0.82 1.99 ± 0.91 2.17 ± 0.95 2.19 ± 0.74 2.13 ± 0.71 *** NS NS NS NS FAI 6.29 ± 4.03 12.76 ± 6.46 7.46 ± 4.32 8.78 ± 5.21 12.60 ± 6.75 9.97 ± 4.23 **** ** NS NS NS SHBG nmol/L 31.70 ± 14.64 21.86 ± 12.91 32.36 ± 17.22 29.16 ± 10.35 21.51 ± 10.97 24.13 ± 7.70 ** * NS NS NS AMH ng/ml 3.83 ± 3.59 7.62 ± 4.40 4.92 ± 3.88 6.12 ± 4.48 7.62 ± 5.78 10.37 ± 5.65 *** * NS NS NS P values comparing different groups have been computed using Student’s t-test. * P < 0.05, ** P < 0.01 Serum bile acid (BA) profiling revealed pronounced reductions in taurocholic acid (TCA), lithocholic acid (LCA), taurodeoxycholic acid (TDCA), and tauroursodeoxycholic acid (TUDCA) levels in PCOS participants (Fig. 1 b, c). A decreased cholic acid (CA) to chenodeoxycholic acid (CDCA) ratio suggested a shift from primary to secondary BA synthesis pathways, while altered glycolithocholic acid (GLCA)-to-CDCA ratios indicated dysregulated secondary BA metabolism. Logistic regression confirmed these BA profiles as discriminators between PCOS patients and healthy controls (Fig. 1 d). Notably, LCA—a microbiota-derived BA that enhances insulin sensitivity via TGR5-mediated glucagon-like peptide-1 (GLP-1) secretion—showed a negative correlation with HOMA-IR (Fig. 1 e). This aligns with established mechanisms linking BA signaling to insulin resistance in PCOS( 12 , 13 ). Furthermore, TDCA and taurochenodeoxycholic acid (TCDCA) correlated with prolactin (PRL), while glycocholic acid (GCA) levels associated with adrenocorticotropic hormone (ACTH), implicating BAs in neuroendocrine regulation. Compared to sham controls, 4-month acupuncture followed by 4-month observation significantly reversed serum levels of glycoursodeoxycholic acid (GUDCA), TUDCA, TCA, and LCA (Fig. 1 f). This is consistent with prior evidence: TUDCA improves glucose tolerance and reduces adiposity, while metformin elevates TUDCA in insulin-resistant models( 14 ). Similarly, GUDCA enhances insulin sensitivity in diabetic cohorts, corroborating acupuncture’s efficacy in metabolic modulation( 15 ). Downstream molecular targets were quantified via enzyme-linked immunosorbent assay (ELISA), while serum γ-aminobutyric acid (GABA) and dopamine (DA) levels were measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Acupuncture treatment significantly reduced serum fibroblast growth factor 19 (FGF-19) levels, suggesting suppression of intestinal farnesoid X receptor (FXR) signaling—a pathway implicated in hepatic metabolic regulation and recognized as a therapeutic target for metabolic disorders( 15 ). This aligns with prior evidence that acupuncture modulates FXR activity (Fig. 1 g). Notably, the acupuncture-treated group exhibited marked alterations in neuroendocrine and immune markers compared to sham controls (Fig. 1 g). β-endorphin, DA, and GABA levels increased significantly, whereas glutamate (Glu) decreased. Given that β-endorphin, DA, and GABA inhibit gonadotropin-releasing hormone (GnRH) secretion, while Glu stimulates it, these shifts suggest that acupuncture may regulate GnRH-mediated ovulatory dysfunction in PCOS( 3 ). Concurrent reductions in interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) further indicate acupuncture’s anti-inflammatory effects. Additionally, elevated ghrelin (GHRL) levels post-acupuncture correlated with improved insulin sensitivity, consistent with studies linking GHRL to insulin resistance mitigation in PCOS( 4 ). These findings collectively highlight acupuncture’s multi-target regulatory effects on metabolic, neuroendocrine, and inflammatory pathways in PCOS. Electroacupuncture Ameliorates PCOS Rat Model and Modulate Hepatic FXR Signaling To validate clinical findings, we established a PCOS rat model via subcutaneous dihydrotestosterone (DHT) slow-release silicone tube implantation (Fig. 2 a) and treated the rat model with electroacupuncture. The model group exhibited a significant increase in body weight compared to controls, which was attenuated post-acupuncture (Fig. 2 b). Reproductive assessments revealed irregular estrous cycles (Fig. 2 c) and ovarian histopathology characterized by immature and atretic follicles in PCOS rats, consistent with sporadic ovulation (Fig. 2 d). Hormonal profiling showed elevated LH/FSH ratios and markedly reduced progesterone and sex hormone-binding globulin (SHBG) levels in PCOS rats (Fig. 2 e). SHBG reduction, indicative of sex hormone receptor downregulation or insensitivity—a diagnostic hallmark of PCOS—was reversed by electroacupuncture. To investigate the mechanisms, we analyzed hepatic FXR expression via qPCR. PCOS rats displayed significant FXR upregulation compared to controls, which was reversed after electroacupuncture (Fig. 2 f). These results confirm dysregulated bile acid metabolism in PCOS pathogenesis and demonstrate that acupuncture alleviates reproductive-endocrine dysfunction, partly through FXR pathway modulation. Liver transcriptome analysis in a PCOS rat model treated with Electroacupuncture revealed the Modulation of Bile Acid Metabolism RNA sequencing (RNA-seq) was performed to compare hepatic transcriptomes between control rats, untreated PCOS rats and PCOS rats treated with electroacupuncture (PCOS-EA). Differential gene expression (DEGs) analysis revealed significant upregulation of multiple genes including Cyp2b1 , Fkbp5 , Cdkn1a , Osmr , Igmbp2 in PCOS versus controls (Fig. 3 a). KEGG enrichment analysis of DEGs highlighted dysregulation in pathways critical to hormone synthesis and bile acid metabolism, including steroid hormone biosynthesis, cytochrome P450-mediated xenobiotic metabolism, bile secretion, primary bile acid biosynthesis, and PI3K-Akt signaling (Fig. 3 c). These findings align with prior evidence linking bile acid biosynthesis inhibition to PCOS pathogenesis( 15 ), suggesting impaired fatty acid metabolism and bile acid homeostasis in PCOS. Electroacupuncture reversed the expression of multiple DEGs in PCOS rats (Fig. 3 b). KEGG pathway analysis demonstrated restored activity in linoleic acid metabolism, xenobiotic metabolism, steroid hormone biosynthesis, and PI3K-Akt signaling (Fig. 3 d). Gene Ontology (GO) enrichment identified acupuncture-induced modulation of bile acid-associated biological processes (Fig. 3 e-f). Collectively, these results indicate that electroacupuncture ameliorates bile acid dysmetabolism in PCOS. Discussion Bile acids play a pivotal role in the pathogenesis of polycystic ovary syndrome (PCOS)( 16 ). We discovered that acupuncture may confer therapeutic benefits by modulating bile acid metabolism. The study demonstrated that specific bile acids, notably taurocholic acid (TCA) and lithocholic acid (LCA), are significantly altered in PCOS patients, with acupuncture showing a regulatory impact on these bile acid profiles. These changes were associated with enhanced insulin sensitivity and reduced hyperandrogenism, with sustained benefits observed up to four months post-treatment. These effects may be related to gut microbiota-driven bile acid pathways( 15 , 16 ). These clinical observations were corroborated in a PCOS rat model, where electroacupuncture restored estrous cyclicity, ovarian morphology, and reproductive hormone balance. Hepatic transcriptomic analysis further revealed electroacupuncture-induced reverse of genes involved in steroid hormone synthesis and bile acid metabolism. The modulation of the farnesoid X receptor (FXR) pathway by acupuncture, as suggested by our results, which is a known therapeutic target in PCOS( 15 ). Collectively, these data implicate acupuncture in the modulation of the gut microbiota-bile acid-insulin resistance axis, offering a systemic therapeutic approach for PCOS. Our study advances understanding of acupuncture’s systemic regulatory effects on endocrine and metabolic dysfunction in PCOS, underscoring its potential as a multifaceted therapeutic strategy. Subsequent research should seek to elucidate the interplay between bile acid metabolism, gut microbiota, and PCOS clinical manifestations, as well as the precise mechanisms underlying acupuncture's influence on these dynamics. This study also has limitations. Due to the implementation of stricter inclusion and exclusion criteria in this study, the resulting sample size was relatively limited, the findings should be validated in larger, multicenter trials with extended follow-up durations to confirm the long-term effects of acupuncture on PCOS. Finally, future research should aim to develop an optimized, accessible acupuncture protocol that can be readily integrated into clinical practice. Conclusions In summary, bile acids appear to be pivotal in the etiology of PCOS. Acupuncture may offer therapeutic benefits by modulating bile acid metabolism, especially targeting specific bile acids. The potential mechanisms might revolve around the influence of acupuncture on gut microbiota-driven bile acid metabolic pathways. The bile acids pathway we have identified in this study hold potential as therapeutic targets for acupuncture in PCOS women, aiming to prevent or slow down the progression of PCOS and provide a potential solution for these patients. Declarations Authors' contributions H.Z., Y.C. and D.H. contributed equally to this work. R.L., D.L., and J.Q. supervised the study; H.Z., Y.C., and D.H. contributed to the design and development of the study protocol; R.L., D.L., and J.Q. conceptualized and coordinated the study; H.W. , H.Z., and W.Q. were responsible for patient recruitment and data collection; L.S. and W.W. were responsible for the generation of random numbers;, R.W.,Y.Y., X.L., and provided scientific advice regarding the development of the intervention; D.H., Z.H., Y.Y., C.Z.and S.Y. acquired the patient, animal data and administered the treatments; C.S.,L.S., and S.Y. provided guidance on the statistical methodology and prepared the data analysis; H.Z., Y.C., D.H. W.M. and J.L. contributed to the writing of the manuscript, and all authors have read and approved the final manuscript. Ethics approval and consent to participate Ethical approval for this study was obtained from the Regional Ethical Review Board of Peking University Third Hospital (Beijing, China). Approval number of the clinical trial: PKU3-IRB-M2019021. All participants provided written informed consent；Approval number of the animal experiments: A2022045. Efforts were made to minimize animal suffering, including anesthesia administration and humane euthanasia. All participants provided written informed consent. Availability of data and materials Data supporting the findings of this study are not publicly available due to privacy or ethical restrictions. Anonymized data may become available to third parties after request to the email address [email protected] Competing interests The authors declare that they have no competing interests. Funding The research was supported by the National Natural Science Foundation of China (Grant No. 82174151), National Natural Science Foundation of Beijing (Grant No. 7242260), Special Grant for Capital’s Funds for Health Improvement and Research (Grant No. 2022-2-4098, 2022-2-4095), Extensive Collection & Harmony Integration ” Projects of Peking University Third Hospital(Grant No.2024-01-17), National Natural Science Foundation of the Peoples’ Republic of China (Grant No. 82288102), Peking University Third Hospital “Key Young Talents” Training Program (Grant No. BYSYFY2021032), National Key R&D Program of China (Grant No. 2022YFC3500400) and the Proof of Concept Program of Zhongguancun Science City and Peking University Third Hospital (Grant No. HDCXZHKC2023208). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknolwdgements We thank the Department of Biochemistry and Biophysics, Center for Precision Medicine Multi-Omics Research of Peking University Health Science Center for data processing and analysis. We thank all the women whose participation made this study possible. None of those participants were compensated for their contribution . References Azziz R. Polycystic Ovary Syndrome. Obstet Gynecol. 2018;132(2):321-36. Lim CED, Ng RWC, Cheng NCL, Zhang GS, Chen H. Acupuncture for polycystic ovarian syndrome. Cochrane Database Syst Rev. 2019;7(7):CD007689. Zhang H, Wang W, Zhao J, Jiao P, Zeng L, Zhang H, et al. Relationship between body composition, insulin resistance, and hormonal profiles in women with polycystic ovary syndrome. Front Endocrinol (Lausanne). 2022;13:1085656. Yang Q, Vijayakumar A, Kahn BB. Metabolites as regulators of insulin sensitivity and metabolism. Nat Rev Mol Cell Biol. 2018;19(10):654-72. Tong TT, Bai LB, Yau LF, Li JY, Huang H, Jiang ZH. Pharmacological effects of bile acids on polycystic ovary syndrome via the regulation of chemerin. Chin Med. 2025;20(1):45. Yue F, Xing L, Wu S, Wei L, Zhou Z, Shi Y, et al. Constant light exposure alters gut microbiota and short-/medium-chain fatty acids and aggravates PCOS-like traits in HFD-fed rats. Obesity (Silver Spring). 2022;30(3):694-706. Sun L, Xie C, Wang G, Wu Y, Wu Q, Wang X, et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nat Med. 2018;24(12):1919-29. Zhang B, Shen S, Gu T, Hong T, Liu J, Sun J, et al. Increased circulating conjugated primary bile acids are associated with hyperandrogenism in women with polycystic ovary syndrome. J Steroid Biochem Mol Biol. 2019;189:171-5. Qi X, Yun C, Sun L, Xia J, Wu Q, Wang Y, et al. Gut microbiota-bile acid-interleukin-22 axis orchestrates polycystic ovary syndrome. Nat Med. 2019;25(8):1225-33. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30. Dobin A, Gingeras TR. Mapping RNA-seq Reads with STAR. Curr Protoc Bioinformatics. 2015;51:11 4 1- 4 9. Higgins V, Asgari S, Hamilton JK, Wolska A, Remaley AT, Hartmann B, et al. Postprandial Dyslipidemia, Hyperinsulinemia, and Impaired Gut Peptides/Bile Acids in Adolescents with Obesity. J Clin Endocrinol Metab. 2020;105(4):1228-41. An J, Wang L, Song S, Tian L, Liu Q, Mei M, et al. Electroacupuncture reduces blood glucose by regulating intestinal flora in type 2 diabetic mice. J Diabetes. 2022;14(10):695-710. Zhang Y, Cheng Y, Liu J, Zuo J, Yan L, Thring RW, et al. Tauroursodeoxycholic acid functions as a critical effector mediating insulin sensitization of metformin in obese mice. Redox Biol. 2022;57:102481. Yun C, Yan S, Liao B, Ding Y, Qi X, Zhao M, et al. The microbial metabolite agmatine acts as an FXR agonist to promote polycystic ovary syndrome in female mice. Nat Metab. 2024;6(5):947-62. Duan L, An X, Zhang Y, Jin D, Zhao S, Zhou R, et al. Gut microbiota as the critical correlation of polycystic ovary syndrome and type 2 diabetes mellitus. Biomed Pharmacother. 2021;142:112094. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Revision requested 30 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 25 Jun, 2025 Editor assigned by journal 08 May, 2025 Submission checks completed at journal 08 May, 2025 First submitted to journal 06 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6600841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":477711872,"identity":"d667408f-802b-4a37-9596-606034c69326","order_by":0,"name":"Haolin Zhang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haolin","middleName":"","lastName":"Zhang","suffix":""},{"id":477711873,"identity":"5000bd75-4582-433c-92fa-c82f314f5727","order_by":1,"name":"Yang Chen","email":"","orcid":"","institution":"Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Chen","suffix":""},{"id":477711874,"identity":"5baa7cb5-7734-4fa6-82a3-d519d6981861","order_by":2,"name":"Dai Heng","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dai","middleName":"","lastName":"Heng","suffix":""},{"id":477711875,"identity":"b3742ec7-af38-4a93-a93e-31f5abd3cbf6","order_by":3,"name":"Jingzhi Luo","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingzhi","middleName":"","lastName":"Luo","suffix":""},{"id":477711876,"identity":"cdd1f56f-6cf3-446b-889d-bba710f88eea","order_by":4,"name":"Haining Wang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haining","middleName":"","lastName":"Wang","suffix":""},{"id":477711877,"identity":"512e8fc0-6b2e-40ba-9b6c-7c7e16e9abe0","order_by":5,"name":"Wei Wang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":477711878,"identity":"0ed280a7-1be3-4e25-89e2-6722e4420aff","order_by":6,"name":"Zejun Huo","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zejun","middleName":"","lastName":"Huo","suffix":""},{"id":477711879,"identity":"eef9419b-36a9-4ecd-83b8-e054c061d695","order_by":7,"name":"Rui Wei","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wei","suffix":""},{"id":477711880,"identity":"ada21c4e-9357-446b-abdd-a2b7a15ced0b","order_by":8,"name":"Yang Ye","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Ye","suffix":""},{"id":477711881,"identity":"91bfb3bf-a731-4ee2-909f-949ea953a7e5","order_by":9,"name":"Xiaoyu Long","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Long","suffix":""},{"id":477711882,"identity":"0086fae3-32ac-491e-81b1-1629500ed6a2","order_by":10,"name":"Chunmei Zhang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Zhang","suffix":""},{"id":477711883,"identity":"16f9c6f1-6841-4392-87ab-b45823e6acb9","order_by":11,"name":"Wen Ma","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Ma","suffix":""},{"id":477711884,"identity":"05fa48ca-78fc-461b-8dac-f77949a73e36","order_by":12,"name":"Li Shi","email":"","orcid":"","institution":"Peking University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Shi","suffix":""},{"id":477711885,"identity":"a9772791-d85a-482f-85b2-1e09c158f66e","order_by":13,"name":"Yang Yang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":477711886,"identity":"45da9271-7b35-4fb4-bef2-6201711802d9","order_by":14,"name":"Chunhua Shan","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunhua","middleName":"","lastName":"Shan","suffix":""},{"id":477711887,"identity":"52638030-4a07-40ad-9bc9-f8c1585477cd","order_by":15,"name":"Shuhan Yang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuhan","middleName":"","lastName":"Yang","suffix":""},{"id":477711888,"identity":"89a201a9-0761-4ae3-8fb8-cd5a38edc38b","order_by":16,"name":"Rong Li","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Li","suffix":""},{"id":477711889,"identity":"bf5ee084-53a0-4d87-b997-fbe9f50f368c","order_by":17,"name":"Dong Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDACdsYGCSAlZ8BwAMRlJkILM0SLMSlaGBhAWhI3wLmEtTA33vi4ozZ9O+PpNAmGCuvEBvazBwg5rNly5pnjuTsbzm6TYDiTntjAk5dASEubNG/bsdwNB4BaGNsOJzZI8BgQpSXdAKzlH/FaahIgWhqI0wL0S9sBQ6BfNlskHEs3buPJIaCFvf3hjY9tdfLmEmc33vhQYy3bz34Gvxb7A2DqMDB2gKwEIJMNvxVwUMfAwN9ApNpRMApGwSgYcQAAxglE2ULxzS4AAAAASUVORK5CYII=","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":true,"prefix":"","firstName":"Dong","middleName":"","lastName":"Li","suffix":""},{"id":477711890,"identity":"7324ed8a-83f6-4d5e-93b3-fd72a615ea01","order_by":18,"name":"Jie Qiao","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Qiao","suffix":""}],"badges":[],"createdAt":"2025-05-06 08:38:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6600841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6600841/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-025-01297-6","type":"published","date":"2026-01-09T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85744305,"identity":"709ce139-b641-4193-992d-08ec7b1cc0c1","added_by":"auto","created_at":"2025-07-01 09:16:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum bile acid profiling in Patients. (\u003c/strong\u003ea) Study cohort overview. The study included 4 months of treatment with acupuncture or with sham acupuncture, accompanied by lifestyle management, and 4 months of follow-up with no intervene. 33 PCOS Participants were randomly assigned into the active acupuncture or the sham acupuncture. All participants received lifestyle management. Serum samples for bile acid analysis were taken before treatment, after 4 months treatment and after 4 month follow up. (b) Diagram depicting the bile acid synthesis and cholesterol clearance pathway.\u003cstrong\u003e \u003c/strong\u003e(c) Levels of indicated serum bile acids and ratio between bile acids in PCOS and healthy controls. P values were determined by two-tailed t-test and data are presented as medians with SEM (*, p\u0026lt;0.05; **, p\u0026lt;0.01). (d) Receiver operating characteristic (ROC) curves of differentially expressed bile acids, highlighting a panel of 6 bile acid features using the criteria of adjust \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 between participants with PCOS and the controls. (e) Scatterplot illustrating correlations between selected bile acid concentrations and clinical indicators, with Pearson correlation coefficients.\u003cstrong\u003e \u003c/strong\u003e(f) Display of specific serum bile acid concentrations in paired untreated PCOS participants versus those who underwent 4 months of acupuncture treatment and a 4-month follow-up. The change rate, calculated by the formula |Treated - untreated|/untreated, is depicted as a box plot (*, p\u0026lt;0.05).\u003cstrong\u003e \u003c/strong\u003e(g) Serum concentrations of FGF-19, GHRL, beta-endorphin, IL-6, TNF- α, GABA, Glu and DA across three time points (pre-treatment, post 4-month treatment, and post 4-month follow-up) determined by ELISA(*, p\u0026lt;0.05; ***, p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6600841/v1/bce299f7821b2fe8c7e2a603.png"},{"id":85744306,"identity":"e40c349f-e91f-4eca-a5d2-d728c5b33d20","added_by":"auto","created_at":"2025-07-01 09:16:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1075964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotype of DHT-induced PCOS rats after electroacupuncture treatment. (\u003c/strong\u003ea) Schematic representation of electroacupuncture treatment and the experimental procedure in DHT-induced PCOS rats.(b) The weighing of the body weight of rats. n=6. (c) The estrous cycle of each rat was detected for 7 consecutive days. (d) HE staining microscopic examination of ovarian sections. (e) The concentrations of serum hormones, including FSH, LH, progesterone, and SHBG, were measured using ELISA (****, p\u0026lt;0.0001). n=6. f. The expression level of FGF19 was detected by qRT-PCR (*, p\u0026lt;0.05; **, p\u0026lt;0.01). n=3.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6600841/v1/d7e1174b552fb25cdb15527f.png"},{"id":85745901,"identity":"dc709d20-8008-4c87-b690-61ffe3e2e519","added_by":"auto","created_at":"2025-07-01 09:24:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1584642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-Seq Analysis of DHT-Induced PCOS Rat Livers. \u003c/strong\u003e(a) Differential gene expression analysis between Control and PCOS. Red indicated upregulated genes (fold change \u0026gt; 1.5, FDR \u0026lt; 0.05); Blue indicates downregulated genes (fold change \u0026gt; 1.5 and FDR \u0026lt; 0.05). (b) Differential gene expression analysis between PCOS-EA and PCOS. Red indicated upregulated genes (fold change \u0026gt; 1.5, FDR \u0026lt; 0.05); Blue indicates downregulated genes (fold change \u0026gt; 1.5 and FDR \u0026lt; 0.05). (c) Enrichment of Signaling pathways related to upregulated and downregulated genes in Control and PCOS, as analyzed through Metascape. (d) Enrichment of Signaling pathways related to upregulated and downregulated genes in PCOS-EA and PCOS, as analyzed through Metascape. (e) Enrichment of biological process related to bile-acid of DEGs in Control and PCOS, as analyzed through Metascape. (f) Enrichment of biological process related to bile-acid of DEGs in PCOS-EA and PCOS, as analyzed through Metascape.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6600841/v1/e56a1c45100b5dc1e923cfd2.png"},{"id":100069500,"identity":"64fa1237-ecd7-4399-9761-00f68e8c0684","added_by":"auto","created_at":"2026-01-12 16:14:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5143774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6600841/v1/138eac8f-0df4-49a0-8714-63bf769e75fc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acupuncture of Polycystic Ovary Syndrome: Delving into Bile Acid Metabolism","fulltext":[{"header":"Background","content":"\u003cp\u003ePolycystic Ovary Syndrome (PCOS), the most prevalent endocrine-metabolic disorder in women of reproductive age, is characterized by menstrual irregularities, hyperandrogenism, and anovulation. Its incidence, particularly among adolescents, has risen significantly in recent decades(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Current medical interventions for PCOS primarily focus on symptom management. Acupuncture, however, has garnered global research interest due to its unique mechanisms and favorable safety profile. Both as a standalone and adjunctive therapy, acupuncture demonstrates potential in ameliorating endocrine-metabolic dysregulation in PCOS(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Previous studies, including our own, have established its therapeutic efficacy across disease stages, particularly in mitigating insulin resistance(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Nevertheless, the underlying molecular mechanisms remain incompletely understood.\u003c/p\u003e \u003cp\u003eEmerging evidence highlights the gut microbiota-bile acid axis as a critical mediator in PCOS pathogenesis and metabolic comorbidities(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Dysbiosis-driven alterations in bile acid composition may disrupt key signaling pathways, such as the Farnesoid X receptor (FXR)-FGF15/19 axis, thereby exacerbating metabolic dysfunction, including insulin resistance(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Notably, PCOS patients exhibit distinct gut microbiota profiles and bile acid imbalances, which correlate with compromised immunity, intestinal barrier dysfunction, and hyperandrogenism(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These findings position bile acids as promising diagnostic biomarkers for PCOS. Pharmacological insights further support this link: metformin partially alleviates symptoms by inhibiting intestinal FXR signaling and enhancing glucose metabolism(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, elevated circulating conjugated primary bile acids have been directly associated with hyperandrogenism in PCOS(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Our prior work revealed that acupuncture restores gut microbial diversity in PCOS patients, specifically enriching beneficial microbes \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e, which inversely correlate with insulin resistance severity(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Collectively, these findings suggest that acupuncture\u0026rsquo;s therapeutic effects on PCOS, particularly its insulin-sensitizing actions, may be mediated through modulation of the gut microbiota-bile acid-FXR axis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eTo delve deeper of the relationship between bile acids and acupuncture treatment, we profiled the serum bile acids under acupuncture treatment. 33 participants diagnosed with PCOS, and 28 age/BMI matched healthy controls were recruited from the registered clinical trial (NCT04193371). Ethical approval was obtained from the Regional Ethical Review Board of Peking University Third Hospital (Beijing, China; approval number, PKU3-IRB-M2019021) between 2019 and 2022, informed consent was obtained from all patients, The inclusion criteria for the study were age from 20 to 40 and BMI ranging from 25 to 40. Participants would be excluded if they: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) diagnosis of any other endocrine disorder (e.g., androgen-secreting tumors, thyroid dysfunction or diabetes etc.); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) receive pharmacological or acupuncture treatment within the last 3 months; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) pregnancy or breastfeeding in the last 6 months.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFollow up and assessment of outcomes\u003c/h3\u003e\n\u003cp\u003eAll participants adhered to regular lifestyle management for 4 months. While 16 out of 33 participants were randomly selected to undergo 4 months of acupuncture treatment, in addition, participants were followed up 4 months after the completion of the treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Glucose tolerance and insulin sensitivity were assessed by using the oral glucose tolerance test (OGTT). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as: [fasting insulin (\u0026micro;U mL-1) \u0026times; fasting glucose (mmol L-1)] / 22.5, while the HOMA-β (islet β-cell function) was calculated as [20 \u0026times; fasting insulin (\u0026micro;U mL-1) / (fasting plasma glucose (mmol L-1)\u0026thinsp;\u0026minus;\u0026thinsp;3.5) ].Hyperinsulinemia was estimated by calculating the area under the curve (AUC) using the trapezoid method(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Samples were analyzed across three time points (pre-treatment, post 4-month treatment, and post 4-month follow-up), sexual hormones include estrogen (E2), progestin (P), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T), and androstenedione (A2) were measured with the Immulite 2000 immunoassay system (Siemens Healthcare Diagnostics, Siemens, Germany). Sex hormone-binding globulin (SHBG) was measured using the Beckman DxI800 immunoassay system (Beckman Coulter, Brea, CA, USA). The free androgen index (FAI) was calculated using the following formula: [T.TES (nmol L-1) \u0026times; 100/SHBG (nmol L-1)]. Serum anti-M\u0026uuml;llerian hormone (AMH) concentrations, fibroblast growth factor (FGF)-19, Ghrelin, Interleukin (IL)-6, beta (β)-Endorphin and adrenocorticotropic-hormone (ACTH) levels were measured by an ultrasensitive two-site enzyme-linked immunosorbent (ELISA) assay (Ansh Laboratories LLC; Webster, TX, USA). Plasma glycated hemoglobin (HbA1c) and Gamma amino butyric acid (GABA) were quantified using UPLC-MS/MS method(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Tumor Necrosis Factor alpha (TNF-a) was determined by immune-assay (Luminex 100\u0026trade;, Austin, TX, US), glutamate (Glu) and dopamine (DA) plasma concentrations were measured using high-performance liquid chromatography(Phenomenex, Torrance, CA USA)(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Serum bile acid metabolome was quantified using an Eksigent Ultra Liquid Chromatography 100 system coupled to an AB 5600 TripleTOF system (AB SCIEX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n\u003ch3\u003eAnimal experiment\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e This study was conducted with the approval of the Animal Care and Use Committee at the Peking University Health Science Center. Female Sprague\u0026ndash;Dawley Wistar rats were randomly divided into three groups (Control, PCOS, and PCOS\u0026thinsp;+\u0026thinsp;EA, n\u0026thinsp;=\u0026thinsp;6/group) and housed under controlled conditions (21\u0026ordm;C\u0026ndash;22\u0026ordm;C, 55\u0026ndash;65% humidity, 12\u0026ndash;12 h light\u0026ndash;dark cycle) with free access to normal chow and water. The study employed 4-week-old rats obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEstablishment of the PCOS-like rat model\u003c/h3\u003e\n\u003cp\u003eSlow-release silicon tube with dihydrotestosterone (DHT, 7.5mg, daily release amount of 83ug, inner diameter and outer diameter of 0.078\u0026rdquo; (ID) and 0.125\u0026rdquo; (O.D), 3 cm length) were implanted subcutaneously in the dorsal neck of 21 days of age rats anesthetized by intraperitoneal injection, 0.5 cm from the paraspinal column. Suture after ensuring the location of the embedded silicon tube did not affect the normal feeding and activities of the rats. Closely observed the wound healing and weighed weekly.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVaginal smears\u003c/h2\u003e \u003cp\u003eThe stage of the estrous cycle was determined by microscopic analysis of the predominant cell type in vaginal smears. Seven consecutive days after one month of modeling, the last four consecutive days of the electroacupuncture intervention, and seven consecutive days prior to sampling, vaginal wall cells were taken on a saline moistened cotton swab and applied flat on slides. Cells were fixed and then stained using Shorr's stain and observed under a microscope to determine the estrous cycle. the proestrus stage was typified by predominantly nucleated cells, the estrus stage exhibited cornified squamous epithelial cells, the metestrus stage was marked by a combination of cornified cells and leukocytes, and the diestrus stage showed a predominance of leukocytes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOral glucose tolerance test\u003c/h3\u003e\n\u003cp\u003e6 weeks after the electroacupuncture intervention, the rats were fasted for 8h and fasting blood glucose was measured (0min), 2g/kg of 50% Glucose was administered by gastric gavage, and blood glucose was measured at each time point for 30, 60, and 120min thereafter.\u003c/p\u003e\n\u003ch3\u003eLow-frequency EA\u003c/h3\u003e\n\u003cp\u003eRats in the PCOS\u0026thinsp;+\u0026thinsp;EA group were treated with low-frequency EA at 9 weeks of age. The treatments were given from 08:00 to 10:00 on Monday, Wednesday and Friday for 6 weeks (for a total of 17 treatments). The rats were fixed using a fixator, single-use sterile acupuncture needles were inserted bilaterally at traditional acupuncture point locations CV12(Zhongwan), CV4(Guanyuan), ST25(Tianshu), ST29 (Guilai), CV3(Zhongji), ST36(Zusanli) and SP6 (Sanyinjiao). On CV12, ST25, ST29 and CV3, the needles were attached to an electrical stimulator (HANS-LH202, Huayang Co., Ltd, China) for 20min at 2 Hz, 2 mA and 10min at 2Hz, 3mA.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of hormones\u003c/h2\u003e \u003cp\u003eAt 16 weeks of age, all rats were fasted for eight hours and sacrificed. Blood was collected via puncture of the retro-orbital venous plexus and transferred to EDTA-containing tubes. Serum was obtained by centrifugation (3,000 rpm, 20 min) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for use. Fasting insulin (FINS) levels was measured using radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China). The levels of testosterone (T), SHBG were determined with 125-I labeled radioimmunoassay kits (Beijing North Institute of Biological Technology, Beijing, China). The kits contained standard samples for quality control and were used in accordance with manufacturer instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA from liver tissue was extracted using the MolPure\u0026reg; Cell/Tissue Total RNA kit and reverse transcribed to cDNA using the PrimeScript RT kit. qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, order Q711-02) on a Line-Gene 9600 instrument. qPCR primers were as follows: (Sequence (5'-\u0026gt;3'), (Sequence (5'-\u0026gt;3')). qPCR primers were as follows: (Sequence (5'-\u0026gt;3'))\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFXR:\u003c/h2\u003e \u003cp\u003eforward primer: CTCCTCTCGTCCTATTATTCC\u003c/p\u003e \u003cp\u003ereverse primer: TCAGCCAACATTCCCATC\u003c/p\u003e \u003cp\u003eβ-Actin:\u003c/p\u003e \u003cp\u003eforward primer: GCCACCAGTTCGCCATGGAT\u003c/p\u003e \u003cp\u003ereverse primer: CCCACGATGGGAGGGGAAGA\u003c/p\u003e \u003cp\u003ePCR amplification conditions were as follows: initial denaturation at 95 ℃ for 30 s, 40 cycles of amplification, denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s, and extension at 60 ℃ for 30 s. Melt curve analysis was performed after the reaction to determine the homogeneity of amplicons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the livers of rats in the control, the PCOS and the PCOS-EA group. RNA-seq library preparation was performed by Biomarker Technologies. Sequencing libraries were constructed, and paired-end mRNA-seq data with read lengths of 150 bp were generated, with two or three biological replicates for each cell line. The raw reads were first aligned to the mouse reference genome (mm10) using the STAR software(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) with default parameters. The expression matrix was generated by FeatureCounts software(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). DEGs were calculated using the R package DEseq(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Expression levels were represented as Fragments Per Kilobase of exon model per Million mapped fragments(FPKM). DEGs were identified based on the following criteria: absolute fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and the False Discovery Rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To compare the functions of the DEGs across the five cell types, we used the Metascape website(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html#/main/step1\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html#/main/step1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using SPSS software version 20.0 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were represented as mean, standard deviation, and range. Pearson correlations examined associations between the bile acids and clinical indicators. Independent t-tests determined intergroup differences. Group comparisons underwent (One-way ANOVA) with significance denoted as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSerum bile acid profiling of Patients with PCOS under Acupuncture treatment\u003c/h2\u003e \u003cp\u003e33 PCOS patients and 28 age/BMI-matched controls were enrolled in a clinical trial (NCT04193371). 16 PCOS patients received 4-month acupuncture followed by 4-month observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Acupuncture treatment significantly reduced body mass index (BMI) and waist-hip ratio (%) in patients with PCOS. Key insulin resistance indices, including hemoglobin A1c (HbA1c), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and Area Under the Curve (AUC) for glucose tolerance, demonstrated marked improvement alongside hyperandrogenism markers (androstenedione [A2], sex hormone-binding globulin [SHBG], and free androgen index [FAI]). These therapeutic effects persisted for 4 months post-treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics and clinical indices of 59 participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNon-PCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u0026amp;L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u0026amp;L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSA\u0026amp;L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSA\u0026amp;L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNon-PCOS VS PCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eA\u0026amp;L 4months VS PCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eA\u0026amp;L 8months VS PCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSA\u0026amp;L 4 months VS PCOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSA\u0026amp;L 8 months VS PCOS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 months\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.07\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.45\u0026thinsp;\u0026plusmn;\u0026thinsp;5.06\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.69\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.23\u0026thinsp;\u0026plusmn;\u0026thinsp;5.21\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.03\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.76\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBMI (kg/m\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e28.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e30.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e27.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e27.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e29.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e28.79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWaist-hip ratio (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e87.68\u0026thinsp;\u0026plusmn;\u0026thinsp;5.49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e87.30\u0026thinsp;\u0026plusmn;\u0026thinsp;5.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e85.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e85.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e87.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e87.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.56\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBody fat rate (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e40.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e42.17\u0026thinsp;\u0026plusmn;\u0026thinsp;5.23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e38.81\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e36.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e40.02\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e39.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLean weight\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e44.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e44.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e43.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e43.87\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e44.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e44.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHbA1c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e5.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e5.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e***\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e***\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFasting Glucose (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e5.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFasting insulin (mU/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e19.06\u0026thinsp;\u0026plusmn;\u0026thinsp;15.22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e19.45\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12.59\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e14.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e15.81\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e13.98\u0026thinsp;\u0026plusmn;\u0026thinsp;8.72\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHOMA-IR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHOMA-B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e202.35\u0026thinsp;\u0026plusmn;\u0026thinsp;108.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e394.76\u0026thinsp;\u0026plusmn;\u0026thinsp;721.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e159.97\u0026plusmn; 50.87\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e174.44\u0026plusmn; 79.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e190.19\u0026plusmn; 92.59\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e172.92\u0026thinsp;\u0026plusmn;\u0026thinsp;103.24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAUC 120 glucose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e988.93\u0026thinsp;\u0026plusmn;\u0026thinsp;227.97\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e992.27\u0026thinsp;\u0026plusmn;\u0026thinsp;144.40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e886.78\u0026thinsp;\u0026plusmn;\u0026thinsp;180.29\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e889.50\u0026thinsp;\u0026plusmn;\u0026thinsp;113.50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e938.80\u0026thinsp;\u0026plusmn;\u0026thinsp;195.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e885.68\u0026thinsp;\u0026plusmn;\u0026thinsp;106.37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAUC 120 insulin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e11631.54\u0026thinsp;\u0026plusmn;\u0026thinsp;7110.74\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14242.35\u0026thinsp;\u0026plusmn;\u0026thinsp;6696.57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e10071.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4518.92\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e9842.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5320.59\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10303.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5548.73\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e11265.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6883.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePRL ng/ml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;5.93\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e12.32\u0026thinsp;\u0026plusmn;\u0026thinsp;8.39\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12.70\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e11.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e14.37\u0026thinsp;\u0026plusmn;\u0026thinsp;10.19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e13.51\u0026thinsp;\u0026plusmn;\u0026thinsp;11.74\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLH mIU/ml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e5.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e5.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFSH mIU/ml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE2 pmol/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e178.20\u0026plusmn; 50.77\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e200.19\u0026thinsp;\u0026plusmn;\u0026thinsp;106.80\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e152.69\u0026plusmn; 38.86\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e142.00\u0026plusmn; 55.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e166.01\u0026plusmn; 56.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e161.54\u0026plusmn; 43.82\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP nmol/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003enmol/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e10.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8.96\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e8.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.52\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e10.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT nmol/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e***\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFAI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e12.76\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;4.32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e8.78\u0026thinsp;\u0026plusmn;\u0026thinsp;5.21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e12.60\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e****\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSHBG nmol/L\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31.70\u0026thinsp;\u0026plusmn;\u0026thinsp;14.64\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e21.86\u0026thinsp;\u0026plusmn;\u0026thinsp;12.91\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e32.36\u0026thinsp;\u0026plusmn;\u0026thinsp;17.22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e29.16\u0026thinsp;\u0026plusmn;\u0026thinsp;10.35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e21.51\u0026thinsp;\u0026plusmn;\u0026thinsp;10.97\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e24.13\u0026thinsp;\u0026plusmn;\u0026thinsp;7.70\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAMH ng/ml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e7.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e6.12\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e10.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.65\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e***\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eP values comparing different groups have been computed using Student\u0026rsquo;s t-test. * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003cp\u003eSerum bile acid (BA) profiling revealed pronounced reductions in taurocholic acid (TCA), lithocholic acid (LCA), taurodeoxycholic acid (TDCA), and tauroursodeoxycholic acid (TUDCA) levels in PCOS participants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). A decreased cholic acid (CA) to chenodeoxycholic acid (CDCA) ratio suggested a shift from primary to secondary BA synthesis pathways, while altered glycolithocholic acid (GLCA)-to-CDCA ratios indicated dysregulated secondary BA metabolism. Logistic regression confirmed these BA profiles as discriminators between PCOS patients and healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Notably, LCA\u0026mdash;a microbiota-derived BA that enhances insulin sensitivity via TGR5-mediated glucagon-like peptide-1 (GLP-1) secretion\u0026mdash;showed a negative correlation with HOMA-IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). This aligns with established mechanisms linking BA signaling to insulin resistance in PCOS(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, TDCA and taurochenodeoxycholic acid (TCDCA) correlated with prolactin (PRL), while glycocholic acid (GCA) levels associated with adrenocorticotropic hormone (ACTH), implicating BAs in neuroendocrine regulation. Compared to sham controls, 4-month acupuncture followed by 4-month observation significantly reversed serum levels of glycoursodeoxycholic acid (GUDCA), TUDCA, TCA, and LCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This is consistent with prior evidence: TUDCA improves glucose tolerance and reduces adiposity, while metformin elevates TUDCA in insulin-resistant models(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Similarly, GUDCA enhances insulin sensitivity in diabetic cohorts, corroborating acupuncture\u0026rsquo;s efficacy in metabolic modulation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDownstream molecular targets were quantified via enzyme-linked immunosorbent assay (ELISA), while serum γ-aminobutyric acid (GABA) and dopamine (DA) levels were measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Acupuncture treatment significantly reduced serum fibroblast growth factor 19 (FGF-19) levels, suggesting suppression of intestinal farnesoid X receptor (FXR) signaling\u0026mdash;a pathway implicated in hepatic metabolic regulation and recognized as a therapeutic target for metabolic disorders(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This aligns with prior evidence that acupuncture modulates FXR activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eNotably, the acupuncture-treated group exhibited marked alterations in neuroendocrine and immune markers compared to sham controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). β-endorphin, DA, and GABA levels increased significantly, whereas glutamate (Glu) decreased. Given that β-endorphin, DA, and GABA inhibit gonadotropin-releasing hormone (GnRH) secretion, while Glu stimulates it, these shifts suggest that acupuncture may regulate GnRH-mediated ovulatory dysfunction in PCOS(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Concurrent reductions in interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) further indicate acupuncture\u0026rsquo;s anti-inflammatory effects.\u003c/p\u003e \u003cp\u003eAdditionally, elevated ghrelin (GHRL) levels post-acupuncture correlated with improved insulin sensitivity, consistent with studies linking GHRL to insulin resistance mitigation in PCOS(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These findings collectively highlight acupuncture\u0026rsquo;s multi-target regulatory effects on metabolic, neuroendocrine, and inflammatory pathways in PCOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eElectroacupuncture Ameliorates PCOS Rat Model and Modulate Hepatic FXR Signaling\u003c/h2\u003e \u003cp\u003eTo validate clinical findings, we established a PCOS rat model via subcutaneous dihydrotestosterone (DHT) slow-release silicone tube implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and treated the rat model with electroacupuncture. The model group exhibited a significant increase in body weight compared to controls, which was attenuated post-acupuncture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eReproductive assessments revealed irregular estrous cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and ovarian histopathology characterized by immature and atretic follicles in PCOS rats, consistent with sporadic ovulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Hormonal profiling showed elevated LH/FSH ratios and markedly reduced progesterone and sex hormone-binding globulin (SHBG) levels in PCOS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). SHBG reduction, indicative of sex hormone receptor downregulation or insensitivity\u0026mdash;a diagnostic hallmark of PCOS\u0026mdash;was reversed by electroacupuncture.\u003c/p\u003e \u003cp\u003eTo investigate the mechanisms, we analyzed hepatic FXR expression via qPCR. PCOS rats displayed significant FXR upregulation compared to controls, which was reversed after electroacupuncture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). These results confirm dysregulated bile acid metabolism in PCOS pathogenesis and demonstrate that acupuncture alleviates reproductive-endocrine dysfunction, partly through FXR pathway modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLiver transcriptome analysis in a PCOS rat model treated with Electroacupuncture revealed the Modulation of Bile Acid Metabolism\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA sequencing (RNA-seq) was performed to compare hepatic transcriptomes between control rats, untreated PCOS rats and PCOS rats treated with electroacupuncture (PCOS-EA). Differential gene expression (DEGs) analysis revealed significant upregulation of multiple genes including \u003cem\u003eCyp2b1\u003c/em\u003e, \u003cem\u003eFkbp5\u003c/em\u003e, \u003cem\u003eCdkn1a\u003c/em\u003e, \u003cem\u003eOsmr\u003c/em\u003e, \u003cem\u003eIgmbp2\u003c/em\u003e in PCOS versus controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). KEGG enrichment analysis of DEGs highlighted dysregulation in pathways critical to hormone synthesis and bile acid metabolism, including steroid hormone biosynthesis, cytochrome P450-mediated xenobiotic metabolism, bile secretion, primary bile acid biosynthesis, and PI3K-Akt signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings align with prior evidence linking bile acid biosynthesis inhibition to PCOS pathogenesis(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), suggesting impaired fatty acid metabolism and bile acid homeostasis in PCOS.\u003c/p\u003e \u003cp\u003eElectroacupuncture reversed the expression of multiple DEGs in PCOS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). KEGG pathway analysis demonstrated restored activity in linoleic acid metabolism, xenobiotic metabolism, steroid hormone biosynthesis, and PI3K-Akt signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Gene Ontology (GO) enrichment identified acupuncture-induced modulation of bile acid-associated biological processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f). Collectively, these results indicate that electroacupuncture ameliorates bile acid dysmetabolism in PCOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBile acids play a pivotal role in the pathogenesis of polycystic ovary syndrome (PCOS)(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). We discovered that acupuncture may confer therapeutic benefits by modulating bile acid metabolism. The study demonstrated that specific bile acids, notably taurocholic acid (TCA) and lithocholic acid (LCA), are significantly altered in PCOS patients, with acupuncture showing a regulatory impact on these bile acid profiles. These changes were associated with enhanced insulin sensitivity and reduced hyperandrogenism, with sustained benefits observed up to four months post-treatment. These effects may be related to gut microbiota-driven bile acid pathways(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). These clinical observations were corroborated in a PCOS rat model, where electroacupuncture restored estrous cyclicity, ovarian morphology, and reproductive hormone balance. Hepatic transcriptomic analysis further revealed electroacupuncture-induced reverse of genes involved in steroid hormone synthesis and bile acid metabolism. The modulation of the farnesoid X receptor (FXR) pathway by acupuncture, as suggested by our results, which is a known therapeutic target in PCOS(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Collectively, these data implicate acupuncture in the modulation of the gut microbiota-bile acid-insulin resistance axis, offering a systemic therapeutic approach for PCOS. Our study advances understanding of acupuncture\u0026rsquo;s systemic regulatory effects on endocrine and metabolic dysfunction in PCOS, underscoring its potential as a multifaceted therapeutic strategy. Subsequent research should seek to elucidate the interplay between bile acid metabolism, gut microbiota, and PCOS clinical manifestations, as well as the precise mechanisms underlying acupuncture's influence on these dynamics.\u003c/p\u003e \u003cp\u003eThis study also has limitations. Due to the implementation of stricter inclusion and exclusion criteria in this study, the resulting sample size was relatively limited, the findings should be validated in larger, multicenter trials with extended follow-up durations to confirm the long-term effects of acupuncture on PCOS. Finally, future research should aim to develop an optimized, accessible acupuncture protocol that can be readily integrated into clinical practice.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, bile acids appear to be pivotal in the etiology of PCOS. Acupuncture may offer therapeutic benefits by modulating bile acid metabolism, especially targeting specific bile acids. The potential mechanisms might revolve around the influence of acupuncture on gut microbiota-driven bile acid metabolic pathways. The bile acids pathway we have identified in this study hold potential as therapeutic targets for acupuncture in PCOS women, aiming to prevent or slow down the progression of PCOS and provide a potential solution for these patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Z., Y.C. and D.H. contributed equally to this work. R.L., D.L., and J.Q. supervised the study; H.Z., Y.C., and D.H. contributed to the design and development of the study protocol; R.L., D.L., and J.Q. conceptualized and coordinated the study; H.W. , H.Z., and W.Q. were responsible for patient recruitment and data collection; L.S. and W.W. were responsible for the generation of random numbers;, R.W.,Y.Y., X.L., and provided scientific advice regarding the development of the intervention; D.H., Z.H., Y.Y., C.Z.and S.Y. acquired the patient, animal data and administered the treatments; C.S.,L.S., and S.Y. provided guidance on the statistical methodology and prepared the data analysis; H.Z., Y.C., D.H. W.M. and J.L. contributed to the writing of the manuscript, and all authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Regional Ethical Review Board of Peking University Third Hospital (Beijing, China). Approval number\u0026nbsp;of the clinical trial: PKU3-IRB-M2019021. All participants provided written informed consent；Approval number\u0026nbsp;of the animal experiments: A2022045. Efforts were made to minimize animal suffering, including anesthesia administration and humane euthanasia. All participants provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are not publicly available due to privacy or ethical restrictions. Anonymized data may become available to third parties after request to the email address [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the National Natural Science Foundation of China (Grant No. 82174151), National Natural Science Foundation of Beijing (Grant No. 7242260), Special Grant for Capital\u0026rsquo;s Funds for Health Improvement and Research (Grant No. 2022-2-4098, 2022-2-4095), Extensive Collection \u0026amp; Harmony Integration \u0026rdquo; Projects of Peking University Third Hospital(Grant No.2024-01-17), National Natural Science Foundation of the Peoples\u0026rsquo; Republic of China (Grant No. 82288102), Peking University Third Hospital \u0026ldquo;Key Young Talents\u0026rdquo; Training Program (Grant No. BYSYFY2021032), National Key R\u0026amp;D Program of China (Grant No. 2022YFC3500400) and the Proof of Concept Program of Zhongguancun Science City and Peking University Third Hospital (Grant No. HDCXZHKC2023208). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknolwdgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Department of Biochemistry and Biophysics, Center for Precision Medicine Multi-Omics Research of Peking University Health Science Center for data processing and analysis. We thank all the women whose participation made this study possible. None of those participants were compensated for their contribution\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAzziz R. Polycystic Ovary Syndrome. Obstet Gynecol. 2018;132(2):321-36.\u003c/li\u003e\n \u003cli\u003eLim CED, Ng RWC, Cheng NCL, Zhang GS, Chen H. Acupuncture for polycystic ovarian syndrome. Cochrane Database Syst Rev. 2019;7(7):CD007689.\u003c/li\u003e\n \u003cli\u003eZhang H, Wang W, Zhao J, Jiao P, Zeng L, Zhang H, et al. Relationship between body composition, insulin resistance, and hormonal profiles in women with polycystic ovary syndrome. Front Endocrinol (Lausanne). 2022;13:1085656.\u003c/li\u003e\n \u003cli\u003eYang Q, Vijayakumar A, Kahn BB. Metabolites as regulators of insulin sensitivity and metabolism. Nat Rev Mol Cell Biol. 2018;19(10):654-72.\u003c/li\u003e\n \u003cli\u003eTong TT, Bai LB, Yau LF, Li JY, Huang H, Jiang ZH. Pharmacological effects of bile acids on polycystic ovary syndrome via the regulation of chemerin. Chin Med. 2025;20(1):45.\u003c/li\u003e\n \u003cli\u003eYue F, Xing L, Wu S, Wei L, Zhou Z, Shi Y, et al. Constant light exposure alters gut microbiota and short-/medium-chain fatty acids and aggravates PCOS-like traits in HFD-fed rats. Obesity (Silver Spring). 2022;30(3):694-706.\u003c/li\u003e\n \u003cli\u003eSun L, Xie C, Wang G, Wu Y, Wu Q, Wang X, et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nat Med. 2018;24(12):1919-29.\u003c/li\u003e\n \u003cli\u003eZhang B, Shen S, Gu T, Hong T, Liu J, Sun J, et al. Increased circulating conjugated primary bile acids are associated with hyperandrogenism in women with polycystic ovary syndrome. J Steroid Biochem Mol Biol. 2019;189:171-5.\u003c/li\u003e\n \u003cli\u003eQi X, Yun C, Sun L, Xia J, Wu Q, Wang Y, et al. Gut microbiota-bile acid-interleukin-22 axis orchestrates polycystic ovary syndrome. Nat Med. 2019;25(8):1225-33.\u003c/li\u003e\n \u003cli\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30.\u003c/li\u003e\n \u003cli\u003eDobin A, Gingeras TR. Mapping RNA-seq Reads with STAR. Curr Protoc Bioinformatics. 2015;51:11 4 1- 4 9.\u003c/li\u003e\n \u003cli\u003eHiggins V, Asgari S, Hamilton JK, Wolska A, Remaley AT, Hartmann B, et al. Postprandial Dyslipidemia, Hyperinsulinemia, and Impaired Gut Peptides/Bile Acids in Adolescents with Obesity. J Clin Endocrinol Metab. 2020;105(4):1228-41.\u003c/li\u003e\n \u003cli\u003eAn J, Wang L, Song S, Tian L, Liu Q, Mei M, et al. Electroacupuncture reduces blood glucose by regulating intestinal flora in type 2 diabetic mice. J Diabetes. 2022;14(10):695-710.\u003c/li\u003e\n \u003cli\u003eZhang Y, Cheng Y, Liu J, Zuo J, Yan L, Thring RW, et al. Tauroursodeoxycholic acid functions as a critical effector mediating insulin sensitization of metformin in obese mice. Redox Biol. 2022;57:102481.\u003c/li\u003e\n \u003cli\u003eYun C, Yan S, Liao B, Ding Y, Qi X, Zhao M, et al. The microbial metabolite agmatine acts as an FXR agonist to promote polycystic ovary syndrome in female mice. Nat Metab. 2024;6(5):947-62.\u003c/li\u003e\n \u003cli\u003eDuan L, An X, Zhang Y, Jin D, Zhao S, Zhou R, et al. Gut microbiota as the critical correlation of polycystic ovary syndrome and type 2 diabetes mellitus. Biomed Pharmacother. 2021;142:112094.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polycystic ovary syndrome, acupuncture, bile acids, insulin resistance, FXR pathway","lastPublishedDoi":"10.21203/rs.3.rs-6600841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6600841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003epolycystic ovary syndrome (PCOS), a common endocrine disorder, is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Given bile acids' role in metabolic regulation and acupuncture's therapeutic potential for endocrine-metabolic disorders, this study examines acupuncture’s effects on bile acid metabolism and insulin sensitivity in PCOS patients and rat models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e33 PCOS patients and 28 age/BMI-matched controls were enrolled in a clinical trial (NCT04193371). 16 PCOS patients received 4-month acupuncture followed by 4-month observation. Serum bile acids, insulin sensitivity, hormones, and inflammatory markers were analyzed. A dihydrotestosterone (DHT)-induced PCOS rat model was treated with electroacupuncture (EA), with outcomes assessed via estrous cycles and ovarian histology. The mechanisms of EA are dissected through RNA sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eacupuncture improved BMI, insulin resistance, and hyperandrogenism in PCOS patients, with sustained post-treatment effects. Specific bile acids (e.g., TCA, LCA) correlated with metabolic improvements. In PCOS rats, EA restored estrous cyclicity, reduced ovarian cysts, and downregulated hepatic FXR expression. RNA-seq revealed significant alterations in bile acid metabolism-related genes and pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eacupuncture therapy offers potential therapeutic benefits to patients with PCOS by modulating bile acid metabolism, with a particular focus on specific types of bile acids.\u003c/p\u003e","manuscriptTitle":"Acupuncture of Polycystic Ovary Syndrome: Delving into Bile Acid Metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 09:16:21","doi":"10.21203/rs.3.rs-6600841/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-30T06:20:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-17T15:24:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129541183970293730989685920678954301636","date":"2025-08-08T23:54:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T16:21:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83853764508692756625462741362343088502","date":"2025-07-07T15:52:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-25T15:34:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-08T07:12:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-08T07:05:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-05-06T08:36:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bced6514-bb53-4bda-b50b-83198f91dda5","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:07:37+00:00","versionOfRecord":{"articleIdentity":"rs-6600841","link":"https://doi.org/10.1186/s13020-025-01297-6","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2026-01-09 15:57:54","publishedOnDateReadable":"January 9th, 2026"},"versionCreatedAt":"2025-07-01 09:16:21","video":"","vorDoi":"10.1186/s13020-025-01297-6","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01297-6","workflowStages":[]},"version":"v1","identity":"rs-6600841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6600841","identity":"rs-6600841","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}