| Objective: Polycystic ovary syndrome(PCOS)is a common reproductive endocrine disease whose pathogenesis is not fully understood.Hyperandrogenemia(HA)and insulin resistance(IR)are key risk factors which may lead to PCOS reproductive and endocrine and metabolic disorders.At present,symptomatic treatment still dominates the clinical treatment of PCOS,mainly including regulation of menstrual cycle,anti-HA,weight control,IR reduction,improvement of metabolic disorders and lifestyle intervention.Drugs that improve insulin sensitivity such as metformin(MET)have been shown to effectively improve insulin sensitivity,glucose and lipid metabolism disorders,and HA which have been used in PCOS patients with IR.Glucagon-like peptide-1(GLP-1)receptor agonists and thiazolidinediones(TZD)have the advantages of improving body weight and ovulation when compared with MET,a classic drug that has been used as a second-line treatment for PCOS,which has also been used in PCOS patients.As a sex hormone transporter,sex hormone binding globulin(SHBG)is produced by the liver,it binds with high affinity to circulating sex hormones,regulates the concentration of biologically active free testosterone(FT)in the blood and affects its bioavailability,therefore,SHBG can be used to judge the severity of HA and evaluate the effect of treatment.Meanwhile,low serum SHBG levels are considered a marker of metabolic abnormalities and are associated with IR,compensatory hyperinsulinemia,and abnormal glucose and lipid metabolism in PCOS patients.Hepatocyte nuclear factor-4α(HNF-4α)is an important transcriptional regulator of SHBG gene upstream promoter located on chromosome 17p13.1,which can activate SHBG expression in liver.The level of HNF-4α is negatively correlated with the fat content in the liver,and monosaccharide-induced lipogenesis decreases the level of hepatic HNF-4α,which in turn attenuates the expression of hepatic SHBG.At the same time,hyperinsulinemia can inhibit the expression of HNF-4α and reduce the synthesis and production of SHBG in the liver,but the related mechanism is not clear and needs further study.However,due to the small clinical sample size and different quality levels of previous studies,the results of different types of insulin sensitivity improving drugs on body weight,glucose and lipid metabolism,and androgen improvement in PCOS patients are not the same.In addition,most of the previous studies were traditional meta-analyses(TMA),and there was no study comparing the reproductive and endocrine and metabolic effects of different insulin sensitivity improving treatment regimens in PCOS patients at the same time.At present,multiple clinical studies have found that MET and GLP-1receptor agonists have significant effects in improving metabolism in PCOS patients.GLP-1 receptor agonists have obvious advantages in weight loss,but their effect in improving IR is similar to that of MET with no significant difference.In addition,the number of clinical trials on MET and exenatide(EX)in improving HA in overweight PCOS patients is limited,and previous studies only observed androgen changes,without further observation of other hormonal indicators that regulate menstruation.At present,the basic research on SHBG of PCOS patients with drugs including MET to improve insulin sensitivity is relatively blank.Therefore,we designed 1.A network meta-analysis(NMA)to expand the sample size and compare the effects of various types of insulin sensitivity improving drugs and treatment regimens on sex hormones,metabolism and weight-related indicators in overweight women with PCOS;2.A clinical trial to observe the effects of MET monotherapy and MET combined with EX therapy on various sex hormones in overweight PCOS patients and to explore the related risk factors of low serum SHBG levels;3.An animal experiment used MET and EX to intervene in the PCOS model,and to preliminarily explore the effect and possible mechanism of MET and EX on SHBG in liver tissue aiming to provide a more accurate basis for clinicians to treat PCOS and better guide the treatment of clinical overweight and obese patients with PCOS.Methods: Part 1: We searched Pub Med,EMbase,the Cochrane Library,Wan Fang Database,and CNKI databases for randomized controlled trial(RCT)studies of relevant overweight PCOS patients from the date of inception to September 2019.Primary outcomes included sex hormones,glucose metabolism,and weight-related parameters.We used NMA to evaluate the efficacy and safety of different regimens of insulin-sensitivity-modifying drugs,including MET,GLP-1 receptor agonists,and TZD.Part 2: We recruited 60 overweight PCOS subjects from January 2021 to January2022 in the outpatient department of endocrinology,Shengjing Hospital of China Medical University.Randomly assigned to MET or combination(COM)groups to receive 12 weeks of MET monotherapy or MET combined with EX,respectively.All subjects received standard anthropometric measurements,recorded menstrual cycle changes and measured serum sex hormones at baseline,4 weeks of treatment and 12 weeks of treatment,and underwent oral glucose tolerance test(OGTT)at baseline and 12 weeks of treatment to observe the effects of different treatment regimens on sex hormones in overweight PCOS patients and explore the related risk factors of low serum levels of SHBG.Part 3: We applied letrozole dissolved in carboxymethyl cellulose(CMC)gavage combined with high-fat diet intervention for 4 weeks to induce overweight PCOS rat model;the control group was given CMC gavage combined with normal diet intervention for 4 weeks.After modeling,the PCOS rats were randomly divided into three groups:PCOS group,MET group and EX group.The control group continued to be given CMC gavage combined with ordinary diet and given normal saline gavage and subcutaneous injection;PCOS group continued to use letrozole dissolved in CMC gavage combined with high-fat diet and was given normal saline gavage and subcutaneous injection;MET group continued to use letrozole dissolved in CMC combined with high-fat diet and was given intragastrically with MET and subcutaneous injection of normal saline;EX group continued to use letrozole dissolved in CMC gavage combined with high-fat diet and was given normal saline gavage and subcutaneous injection of EX.All interventions lasted 3weeks.Changes in body weight and food intake were recorded,vaginal smears were used to observe changes in estrous cycle,hematoxylin-eosin(HE)staining was used to observe morphological changes of ovary and liver tissue,and OGTT test was performed7 weeks later to observe changes in glucose metabolism.The serum and liver triglyceride(TG)levels and serum sex hormone levels were determined by linked immunosorbent assay(ELISA),and the expression of SHBG,HNF-4α and phosphatidyl inositol 3-kinase(PI3K)/ protein kinase B(AKT)pathway in liver tissue was observed by western blot(WB).Results: Part 1: 1.We included 14 RCTs including 619 overweight PCOS patients and a total of 5 different interventions were included: MET,GLP-1 receptor agonist,TZD,MET+GLP-1 receptor agonist,and MET+TZD.Most adverse reactions were mild and resolved spontaneously within a few weeks of treatment,with only one subject withdrawing from the trial due to severe rash at the injection site following GLP-1receptor agonist use.2.Compared with MET,MET+TZD was associated with increased menstrual recovery(weighted mean difference(WMD)3.68,95% confidence interval(Cr I)[1.65,8.20])and increased SHBG(WMD 4.30,95%Cr I [0.78,7.82]),while MET+GLP-1 receptor agonists were more effective in reducing androstenedione(AND)(WMD-2.53,95%Cr I [-3.96,-1.09])and increased SHBG(WMD 9.22,95% Cr I [5.46,12.98]).3.TZD is less effective than MET in reducing body mass index(BMI)(WMD1.69,95%Cr I [0.72,2.66]).4.MET+GLP-1 receptor agonists and MET+TZD improve total testosterone(TT),free testosterone(FT),fasting glucose(FG),fasting insulin(FINS)and waist circumference(WC)were not significantly different from MET.Part 2: 1.52 subjects(25 in the MET group and 27 in the COM group)completed the 12-week follow-up,3 of whom were lost to follow-up due to unintended pregnancy and 5 of whom were lost to follow-up because they could not be contacted.The adverse reactions of all patients were mild and resolved spontaneously after 2 weeks of treatment.Both MET and EX were safe for there were no patients who dropped out due to drug intolerance in the MET group and COM group and the final compliance was 83.3% in the MET group while 90% in the COM group.Both COM group and MET group could improve menstrual cycle after 12 weeks of treatment(P<0.01);anthropometric parameters,including body weight(P<0.01),BMI(P<0.01)and abdominal circumference(AG)(P<0.01);glucose metabolism,including FG(P<0.05),FINS(P<0.01)and homeostasis model of insulin resistance index(HOMA-IR)(P<0.01).2.The COM group could significantly improve HA after 12 weeks of treatment,including TT(P<0.01),SHBG(P<0.01)and free androgen index(FAI)(P<0.01),while the MET group only improved SHBG(P<0.01)and FAI(P<0.05)after 12 weeks of treatment.Moreover,compared with the MET group,the COM group improved TT [(0.79 ±0.31)ng/m L vs.(0.62 ± 0.24)ng/m L,P=0.032],SHBG [22.40(15.25-34.60)nmol/L vs.27.00(22.60-44.90)nmol/L,P=0.018] and FAI [12.72(6.40-17.71)% vs.7.06(3.90-10.19)%,P=0.004] were more effective.3.After 12 weeks of treatment,the COM group was more effective than the MET group in improving luteinizing hormone(LH)[(9.77 ± 5.81)m IU/m L vs.(6.61 ± 4.72)m IU/m L,P=0.036],progesterone(Prog)[0.54(0.31-1.72)ng/m L vs.1.08(0.52-10.61)ng/m L,P=0.020],however,there was no significant difference in the improvement of estradiol(E2),follicle-stimulating hormone(FSH),LH/FSH and prolactin(PRL)between the two groups.4.After 12 weeks of treatment,in overweight PCOS patients the serum SHBG levels were positively correlated with COM compared with MET intervention(r=0.331,P=0.017),menstrual cycle recovery rate(r=0.285,P=0.041)and the serum E2 levels(r=0.340,P=0.014).5.Logistic regression analysis indicated that COM compared with MET intervention(OR0.170,95%CI [0.032,0.901],P=0.037)and menstrual cycle recovery rate(OR 0.117,95%CI [0.016,0.829],P=0.032)were protective factors of the low serum SHBG levels.Part 3: 1.Compared with the normal control rats,the overweight PCOS rats showed disappearance of estrous cycle,polycystic ovarian changes,and significantly increased body weight,TT,FG,FINS and HOMA-IR after letrozole combined with high-fat diet modeling(P<0.01).2.After 3 weeks of EX and MET treatment,compared with the rats in the PCOS group,the body weight [(170.42±13.01)g vs.(178.33±8.01)g vs.(208.25±11.18)g,P<0.01 ] and food intake [(11.03±0.25)g vs.(12.29±0.14)g vs.(14.07±0.15)g,P<0.01] reduced.3.After 3 weeks of EX and MET treatment,compared with the PCOS group,FINS [(36.88±2.18)m U/L vs.(38.47±2.52)m U/L vs.(47.56±3.65)m U/L,P<0.01],HOMA-IR [(7.50±0.70)vs.(8.59±0.75)vs.(11.63±1.19),P<0.01] and area under the curve of insulin(AUCins)[(4556.00±200.00])m U/L×min vs.(4498.00±202.90)m U/L×min vs.(5565.00±261.80)m U/L×min,P<0.01] decreased,while only FG [(4.58±261.80)0.46)mmol/L vs.(5.50±0.31)mmol/L,P<0.01] in the EX group was lower than the PCOS group.4.After 3 weeks of treatment with EX and MET,compared with the rats in the PCOS group,83.3% of the rats in the EX group and 67.7%of the rats in the MET group returned to regular estrous cycles,and the ovarian morphology of the rats recovered.5.After 3 weeks of EX and MET treatment,compared with the PCOS group,TT [(0.92±0.01)nmol/L vs.(0.95±0.07)nmol/L vs.(1.18±0.04)nmol /L,P<0.01] and FAI [(0.81±0.04)% vs.(0.86±0.02)% vs.(1.31±0.01)%,P<0.01]decreased,while SHBG [(113.34±7.29)nmol/L] vs.(110.52±6.43)nmol/L vs.(89.90± 3.72)nmol/L,P<0.01] increased.6.After 3 weeks of EX and MET treatment,compared with the rats in the PCOS group,the two groups of rats had reduced lipid droplets,improved liver parenchyma regeneration and inflammatory cell infiltration in the liver tissue,accompanied by decreased liver TG [(5.43±0.26)mmol/L vs.(5.59±0.34)mmol/L vs.(6.96 ± 0.33)mmol/L,P<0.01],and up-regulated SHBG and its transcription factor HNF-4α in liver tissue expression,and EX was more efficient than MET.7.After 3 weeks of EX and MET treatment,compared with the PCOS group,both EX and MET activated the PI3K/AKT pathway to improve IR,and EX was more effective than MET.Conclusions: 1.Metformin combined with exenatide therapy may better improve hyperandrogenism in overweight PCOS patients than metformin monotherapy.2.Metformin combined with exenatide therapy may improve reproductive abnormalities in overweight PCOS patients by regulating the hypothalamic-pituitary-ovarian axis better than metformin alone.3.Serum SHBG level in overweight PCOS patients is closely related to menstrual cycle recovery rate and estradiol level.4.Metformin and exenatide may activate liver PI3K/AKT,up-regulate HNF-4α protein expression,reduce fat deposition,and then increase SHBG protein expression,and exenatide is better than metformin. |
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