This randomized, open-label, parallel-group controlled trial was conducted between February 2020 and December 2021 at the gynecological outpatient departments of Sichuan Women’s and Children’s Hospital, Sichuan Provincial Hospital of Traditional Chinese Medicine, the Third Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, and Xinan Women’s and Children’s Hospital. This study aimed to compare AC with clomiphene for the treatment of OPCOS. The ethics review board of the Sichuan Traditional Chinese Medicine Regional Ethical Review Committee approved the study protocol (2019KL-075). The trial was registered with the Chinese Clinical Trial Registry (ChiCTR2000029882). All patients provided written informed consent before participation. The study adhered to the Consolidated Standards of Reporting Trials (33) and Standards for Reporting Interventions in Clinical Trials of Acupuncture guidelines (34) for reporting randomized trials. The trial protocol has been published (35).

From February 2020 to December 2021, participants with persistent OPCOS and healthy subjects were recruited by publishing recruitment advertisements in newspapers or on the information platform of the official website and bulletin boards of various research and recruitment websites by sending out leaflets with recruitment information and conducting free clinics in the community among other activities. Before participation, patients were informed about the study design, the advantages and disadvantages of the treatment, and relevant safety measures that would be used during the trial. All participants provided written informed consent before the start of the study. Physicians with over 3 years of clinical experience performed acupuncture in outpatient clinics. Healthy subjects did not undergo any preliminary treatment and only participated in intestinal microbiome testing. The inclusion and exclusion criteria for the patients and healthy controls are described in Table 1 .

OPCOS patients were randomly assigned (1:1) to either the AC or clomiphene alone. Independent statisticians (not researchers) used SPSS (version 27.0; IBM Corp., Armonk, NY, USA) to generate random sequences. Each random number was printed and placed in a separate lighttight envelope managed by a third party. When the participants met the established inclusion criteria for the study and voluntarily signed the informed consent form, the clinical research coordinator called a third party to obtain the subjects’ random numbers and grouping information. The acupuncturists then arranged the corresponding treatment plans for the participants. Owing to the nature of acupuncture and the control group used in this study, neither the patients nor practitioners could be blinded. At the same time, as the outcome indicators of this study were objective laboratory indicators, such as the LH-to-FSH ratio (LH/FSH), the outcome evaluators were not blinded. However, after the trial period, the statistician responsible for statistical analysis was blinded to the group assignment to prevent bias.

All participants were informed of the benefits of healthy lifestyle habits such as regular exercise and healthy eating. The study group received AC citrate treatment, whereas the control group received only clomiphene citrate. Clomiphene citrate treatment will be administered on the fifth day of menstruation to participants without amenorrhea in the study and control groups. Participants will receive treatment with an initial oral dose of 50 mg for 5 continuous days. The acupuncture treatment will be conducted three times a week from the fifth day of menstruation or withdrawal bleeding until the start of the next menstruation, for up to three menstrual cycles. Acupuncture points will be selected based on the Zang-fu organ system, Yin-Yang theory, and clinical rules for PCOS acupoint selection. Two sets of acupoints will be used; each set will be used on alternate treatments. The first acupoint formula comprises DU-20, DU-24, GB-13, RN-12, ST-25, RN-4, EX-CA-1, KI-12, SP-6, and LR-3. The second acupoint formula comprises BL-23, BL-32, SP-6, and KI-3. Disposable, single-use, sterilized needles (Huatuo, Suzhou Medical Appliance Fact. 215005 Suzhou, China) of sizes 0.25mm × 25mm, 0.25mm × 40mm, and 0.25mm × 50mm will be inserted into the acupoints, and a Deqi sensation obtained by manipulating the needles. Each treatment will last for 30 min; no manipulation will be used once the Deqi sensation is achieved. The acupuncture treatment will be conducted three times a week from the fifth day of menstruation or withdrawal bleeding until the start of the next menstruation. The intervention lasted for three menstrual cycles in both groups. Supplementary Data Sheet 1 presents detailed intervention methods.

The primary outcome was the change in LH/FSH from baseline to after three menstrual cycles. Data were collected before the start of the study (baseline assessment) and after three menstrual cycles. Supplementary Data Sheet 1 presents the reasons for selection.

The secondary outcomes included changes in endometrial thickness, follicular diameter, LH, FSH, testosterone, estradiol, prolactin, progesterone, fasting blood glucose, fasting insulin, c peptide, triglyceride, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and C-reactive protein levels; homeostasis model assessment-IR (HOMA-IR) values; Self-Rating Anxiety Scale (SAS) scores; Self-Rating Depression Scale (SDS) scores; and gut microbiome after three menstrual cycles. Safety results and adverse reactions to acupuncture, such as local subcutaneous bleeding, bruising, hematoma, pain, and fainting, were also documented.

Fecal specimens were collected before the start of the study (baseline assessment) and after three menstrual cycles from patients who met the requirements for collecting intestinal flora in this study. The stool collection methods are detailed in Supplementary Data Sheet 1 . The method and process of metagenomic sequencing, as well as the splitting, assembling, filtering of sequencing results, removal of chimeras, species analysis, functional analysis, etc. are presented in Supplementary Data in detail (S1). Bar plot, and heatmap of taxonomy analysis were used to species composition. The Wilcoxon rank sum test, and linear discriminant analysis effect size were used to analysis the difference for group. We perform Alpha/Beta diversity analysis for each sample/group. The Chao1, Shannon, and Simpson index reflect the alpha diversity of the community. The bray distance of the samples is calculated according to the abundance matrix to measure Beta diversity. We also compared the intestinal flora genes with the KEGG database to obtain each functional gene (KO), and the difference KO between groups was tested using the Wilcoxon rank-sum test. In addition, gene pathway annotations were obtained based on KEGG annotation information, and the Reporter Score37 method was used to analyze pathway differences. It was also compared to other gene databases as a sensitivity analysis.

The sample size was calculated based on the results of a previous study to observe acupuncture’s effects on PCOS and noted an average LH/FSH decrease of 0.5 (36). However, analysis of the effects of clomiphene on PCOS in another study showed an average LH/FSH decrease of 0.53 (37). In our study, PASS software (version 15.0, NCSS, LLC, East Kayville, UT, USA) was used for sample size calculation. We expected AC and clomiphene to reduce LH/FSH by 1 and 0.5, respectively, setting the standard deviation (SD) to 0.7. We assumed 80% power to detect a between-group difference in the LH/FSH of 0.5 at a significance level of 0.05. A total of 37 participants were required in each group. Assuming a 15% dropout rate, we recruited 86 patients. In this study, the intention-to-treat (ITT) analysis population, consisting of all patients randomized to either group, was the primary population for efficacy and safety analyses.

Multiple imputations supplemented the missing data for all outcome indicators to complete the ITT analysis dataset. Specifically, the predicted mean-matching model was used, the number of iterations was set to 100, and five ITT datasets were generated. Sensitivity analysis was also performed using patients with complete outcome data (per protocol [PP]). An independent sample t-test was used to analyze continuous variables for baseline data analysis, with the results presented as mean ± SD. The chi-square test was used to analyze categorical variables, with the results presented as numbers and percentages. For the outcome data after three menstrual cycles and changes relative to the baseline, we used an analysis of covariance with age, body mass index, and baseline outcome data as covariates. We plotted a histogram of the residuals to evaluate the normality of the data.

We used the mean, 95% confidence interval (CI), and statistical significance to analyze the outcome indicators. All data analyses were performed using SPSS version 27. This study considered two-tailed p < 0.05 as indicating statistical significance for the primary outcome indicators. The Bonferroni method was used for the secondary outcome indicators for multiple comparisons. A total of 20 outcome indicators, with average distribution alphas of 0.05 and p < 0.0025, were considered statistically significant. Adverse events (AEs) and serious AEs (SAEs) were also described.

The diversity and relative abundance of the gut microbiota genera and species were analyzed using the Wilcoxon signed-rank sum test and are expressed as medians (interquartile ranges). Linear discriminant analysis effect size (LEfSe) was used to analyze the differences in microbial populations between the AC and control groups, as well as healthy controls, after the treatment while distinguishing the acupuncture-effective samples from the acupuncture-ineffective samples according to the changes in LH/FSH after acupuncture and analyzing the differences in gut flora. Linear discriminant analysis > 2.0 was considered a microorganism with a significant effect. Microbial bioinformatics analysis was performed using R (version 3.1.1, Ross Ihaka and Robert, Auckland, New Zealand). Correlations among clinical variables, bacterial flora, and inflammatory markers were analyzed using linear regression. p values ≤0.05 were considered statistically significant. Further details are provided in Supplementary Data Sheet 1 . Statistical analyses were performed using SPSS version 27.

A total of 173 patients were screened, of whom 87 were excluded. Finally, 86 patients were randomly assigned to the AC group (mean age: 26.95 [SD: 3.8]) and the clomiphene alone group (mean age: 27.28 [SD: 2.74]). Five (15.0%) patients dropped out during the study, including one (2.3%) in the AC group and four (9.3%) in the clomiphene group ( Figure 1 ). The baseline characteristics of the patients are presented in Table 2 . There were no significant differences between the AC and clomiphene groups regarding any demographic features and no statistically significant differences in health-related indicators such as hormone levels, blood lipid levels, C-reactive protein levels, and SAS and SDS scores. The PP analysis included 81 patients with OPCOS (42 in the AC and 39 in the clomiphene group), and the differences in baseline features between the AC and clomiphene groups were similar to those observed in the ITT analysis ( Supplementary Table S1 ).

The ITT analysis of the first multiple interpolation set revealed that the adjusted mean decrease in LH/FSH was -0.8 (95%CI: -1.14 to -0.46) in the AC group and -0.22 (95%CI: -0.47 to 0.01) in the clomiphene group ( Supplementary Table S1 ). The adjusted between-group difference in the five summarized ITT sets was 0.53 (95%CI: 0.24 to 0.82; p < 0.001; Table 3 ). The adjusted mean LH/FSH decreased by -0.77 (95%CI: -1.12 to -0.43) in the AC group and -0.19 (95%CI: -0.46 to 0.07) in the clomiphene group. The adjusted between-group difference was 0.52 (95%CI: 0.22 to 0.82; p = 0.001) according to the PP analysis ( Supplementary Table S2 ).

The ITT analysis of the first multiple interpolation set showed that the follicular diameter increased by 9.25 (95%CI: 7.97 to 10.53) in the AC group and 6.53 (95%CI: 5.25 to 7.82) in the clomiphene group ( Supplementary Table S1 ). The adjusted between-group difference in the five summarized ITT sets was -2.82 (95%CI: -4.59 to -1.06; p = 0.002). The LH decreased by -5.5 (mIU/mL) (95%CI: -7.37 to -3.63) in the AC group and -1.04 (mIU/mL) (95%CI: -2.54 to 0.44) in the clomiphene group ( Supplementary Table S1 ). The adjusted between-group difference in the five summarized ITT sets was 4.09 (95%CI: 2.55 to 5.63; p < 0.001). The AC group showed a decrease of -24.52 (ng/dL) (95%CI: -33.03 to -16.01), and the clomiphene group showed an increase of 2.31 (ng/dL) (95%CI: -6.3 to 10.93) in testosterone levels. The AC group showed a decrease of -2.77 (mIU/L) (95%CI: -5.25 to -0.29), and the clomiphene group showed an increase of 2.21 (mIU/L) (95%CI: 0 to 4.43) in fasting insulin levels. The AC group showed a decrease of -0.82 (95%CI: -1.4 to -0.24), and the clomiphene group showed an increase of 0.52 (95%CI: 0.03 to 1.02) in HOMA-IR values. There were no statistically significant differences in the other outcomes ( Table 3 ; Supplementary Tables S1 - S3 ).

In total, 59 subjects were included in the metagenomic analysis, with 21 patients in the AC group, 19 in the clomiphene group, and 19 in the healthy control group. The basic information is presented in Supplementary Table S4 . There were significant differences in the structure, diversity, and function of the intestinal flora between patients with OPCOS and healthy controls. After AC treatment, the abundance of Erysipelatoclostridium and Proteus decreased at the genus level (p < 0.05). In contrast, the abundance of Agathobacter faecis increased (p < 0.05), and Erysipelatoclostridium spiroforme, Streptococcus lutetiensis, and Lactococcus lactis decreased (p < 0.05) at the species level. However, there were no statistically significant differences in terms of diversity. An LEfSe revealed that A. faecis was significantly enriched after acupuncture. The functional analysis revealed statistically significant differences in 54 cases of Kyoto Encyclopedia of Genes and Genomes ontology (KO) before and after acupuncture (p < 0.05). The top 10 KOs were mainly related to proteins and transporters involved in genetic material metabolism, carbohydrate transport and metabolism, and oxidative stress. See Supplementary Data Sheets 1 and 2 for the detailed gut microbiota analysis results.

During the study, there were 55 acupoint bleeding events, which were treated by applying pressure with sterile cotton swabs. There were also 23 local acupoint bruises, seven cases of local pain after acupuncture, and one local hematoma at the acupoint, none of which required further treatment. These AEs are common phenomena in clinical acupuncture, and the treatment methods are also based on the routine clinical treatment methods provided in the textbook “Acupuncture and Moxibustion”. No SAEs occurred during the study period.