| ObjectivesIn this study,a randomized controlled trial was conducted to evaluate the effectiveness of acupuncture combined with CC on infertility for overweight/obesity patients with PCOS.Besides,the changes of gut microbiota between overweight/obese patients with PCOS and health participants were analyzed based on metagenomic sequencing method to explore the role of gut microbiota in the pathogenesis of overweight/obese PCOS.At the meanwhile,the changes of gut microbiota after acupuncture treatment were analyzed based on metagenomic sequencing method to further clarify the microbiological mechanism of acupuncture.Methods1.Clinical TrialThis is a randomized,controlled trial.A total of 81 obese women with PCOS were recruited and randomly assigned to a study group and a control group in a 1:1 ratio,with 40subjects in study group and 41 in control group.The study group received acupuncture and clomiphene citrate treatment,while the control group only received clomiphene citrate.Acupuncture intervention was 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cycles.During the treatment,when a participant was during menstruation or pregnancy,the acupuncture intervention was withdrawn.CC was taken from the 5th day of withdrawal bleeding or menstrual bleeding,50mg once daily for 5 days,with the maximum dose not exceeding 150mg daily.The primary outcome was clinical pregnancy rate.The secondary outcomes comprised hormone biomarkers,including LH,FSH,LH/FSH,T and E2,metabolic biomarkers,including BMI,FBG,FINS,HOMA-IR,TG,CHOL,HDL-C and LDL-C,and adverse effects rate.The outcomes were measured at baseline and post-intervention.Data was analyzed using IBM SPSS statistics 22.0.2.Gut microbiota analysisAccording to the inclusion and exclusion criteria,15 participants in study group and 14participants in control group were enrolled,and 10 healthy participants were included in this part of study.Feces were collected from healthy participants and obese PCOS patients before the treatment to analyze the characteristics of species distribution and functional genes of gut microbiota in obese women with PCOS based on metagenomics sequencing analysis.Besides,feces were collected from participants in study group and control group before and after the treatment to explore the difference of species distribution and functional genes of gut microbiota between the two groups based on metagenomics sequencing analysis.Results1.Clinical TrialThe baseline of two groups were balanced and comparable(P>0.05).The clinical pregnancy rate of study group was 36.1%,while 13.5%in control group(P<0.05)。Serum LH and FSH level in study group were decreased after treatment compared to baseline(P<0.05),while serum LH/FSH,FSH and E2 level did not have a significant difference compared to baseline(P>0.05).Serum T level in control group was decreased after treatment compared to baseline(P<0.05),while serum LH,FSH,LH/FSH and E2level did not have a significant difference compared to baseline(P>0.05).The changes of serum LH,FSH,LH/FSH,and T level in study group did not have a significant difference compared to control group(P>0.05),while the change of serum E2 level had a significant difference compared to control group(P<0.05).BMI,FBG,FINS,HOMA-IR,TC,CHOL and LDL-C level in study group were decreased after treatment compared to baseline(P<0.05),and HDL-C level was increased after treatment compared to baseline(P<0.05).BMI and LDL-C level were decreased and HDL-C level was increased after treatment in control group compared to baseline(P<0.05),while the level of FBG,FINS,HOMA-IR,TC and CHOL had no difference after treatment compared to baseline(P>0.05).When compared to the control group,the changes of BMI,FBG,FINS,HOMA-IR,TC,CHOL and LDL-C level had a significant difference in study group(P<0.05).The two groups had no significant statistical difference in the incidence of AEs(P>0.05).2.Gut microbiota analysis(1)The difference of gut microbiota between PCOS group and health groupThe two groups had no significant statistical difference in theαdiversity(Shannon index P=0.913>0.05,Simpson index P=0.887>0.05).βdiversity based on Bray-Curtis indicated that there were significant differences in the composition of gut microbiota between the two groups(Phylum level:F=0.04,R~2=1.06,P=0.03;Genus level:F=0.27,R~2=1.31,P=0.03;Species level:F=0.34,R~2=1.27,P=0.03).The dominant gut microbiota at phylum level in the two groups were Bacteroidota and Firmicutes,Bacteroides and Prevotella at genus level,Bacteroides dorei、Prevotella corpori、Bacteroides plebelus etc.at species level.Wilcoxon Rank-sum test showed that Bacteroides、Barnesiella、Neobitarella and Parabacteroides at phylum level,Bacteroides barnesiae、Bacteroides acidifaciens、Bacteroides dorei、Bacteroides massiliensis、Bacteroides sartorii、Barnesiella intestinihominis、Neobitarella massiliensis、Odoribacter massiliensis、Parabacteroides distasonis、Prevotella corpori at species level were enriched in PCOS group,while Akkermansia at phylum level,and Faecalibacterium prausnitzii、Anaerostipes hadrus and Phascolarctobacterium succinatutens at species level were enrich less in PCOS group.LEf Se analysis showed that the biomarker in PCOS group included Bacteroides dorei,Bacteroides massiliensis,Bacteroides sartorii,Bacteroides sp002493165,Parabacteroides distasonis,Barnesiella intestinihominis,and Neobitarella massiliensis;the biomarker in health group comprised Phascolarctobacterium,Akkermansia muciniphila and Faecalibacterium prausnitzii.Spearman’s rank correlation analysis showed that Barnesiella and Barnesiella intestinihominis was negatively correlated with CHOL,Akkermansia was negatively correlated with FBG and BMI,Bacteroides sartorii was negatively correlated with E2,Faecalibacterium prausnitzii was positively with LH.Function analysis suggested that there were 271 KOs and 42 pathways were different between health group and PCOS group,Carbohydrate metabolism and Global and overview maps pathways were enriched in PCOS group,and Metabolism,Cellular Processes,Genetic information processing,Environmental Information Processing and Human Diseases pathways were enriched in health group.(2)Gut microbiota difference analysis in the study groupIn the study group,compared to the baseline,there was no significant difference in theαdiversity(Shannon index P=0.855>0.05,Simpson index P=0.881>0.05)after treatment.Wilcoxon Rank-sum test suggested that there were significant differences in genus level and species level after treatment compared to baseline.After treatment,at the genus level,the relatively abundance of Erysipelatoclostridium and Proteus were decreased(P<0.05);at the species level,the relatively abundance of Agathobacter faecis and Lactococcus lactis were increased(P<0.05),while the relatively abundance of Erysipelatoclostridium spiroforme and Streptococcus lutetiensis were decreased(P<0.05).Besides,after treatment,the relatively abundance of F.prausnitzii,A.muciniphila and P.succinatutens had an increasing trend compared to baseline,the relatively abundance of A.hadrus,Bacteroides and P.distasonis had a decreasing trend compared to baseline,but no statistical difference was found before and after treatment(P>0.05).LEf Se analysis showed that the biomarker after treatment is Agathobacter faecis.Function analysis showed that there were 54 KOs and 41 pathways were different between health group and PCOS group,KEGG pathway enrich analysis suggested that Carbohydrate metabolism,ABC transporters,Phosphotransferase system,and pathways related to oxidative stress were enriched after treatment.(3)Gut microbiota difference analysis in the control groupIn the control group,compared to the baseline,there was no significant difference in theαdiversity(Shannon index P=0.0.810>0.05,Simpson index P=0.700>0.05)after treatment.F.prausnitzii、A.muciniphila had an increasing trend compared to baseline,P.distasonis had a decreasing trend compared to baseline,but no statistical difference was found before and after treatment(P>0.05).Wilcoxon Rank-sum test showed there was no difference gut microbiota between baseline and after treatment,so as the LEf Se analysis and function analysis.Conclusions1.Acupuncture combined with CC is safe and effective in the treatment of infertility patients with overweight/obese PCOS,and the efficacy is better than that of CC alone.2.The dysbiosis of gut microbiota is closely related to the development of overweight/obese PCOS.Gut microbiota may have a significant role in the pathological process of overweight/obese PCOS.3.Acupuncture can treat overweight/obese PCOS by regulating the dysbiosis of gut microbiota. |
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