| Objective1.To analyze the clinical characteristics and laboratory indications of repro ductive age women in PCOS with IR,and PCOS with Non-IR.To screen for serum differential proteins between them based on high-throughput proteins chi p.Serum proteins closely related to PCOS with Non-IR were finally identified.2.To further validate screening results of proteins chip and ELISA results from PCOS animal model,a PCOS-IR/Non-IR rat model was established and evaluated.From the levels of clinical and basic research,only to provide a bas is forthe future exploration of pathogenesis of PCOS with Non-IR.Methods1.In this study,patients who were first diagnosed with polycystic ovary syndrome of reproductive age at Infertility clinic of Re productive medicine of the Second Hospital of Lanzhou University from October 2017 to December 2018 were selected as experimental group,and patients who came to department and had regular menstruation and normal fertility were selected as control group.Finally,a total of157 PCOS and 41 Non-PCOS patients were obtained,and all participants were left with residual serum samples after routine examination and stored at-80°C.According to HOMA-IR(≥2.69),patients with PCOS were divided into IR and Non-IR group.The final experimental group was 41 cases of Control,102 cases of IR and 55 cases of Non-IR group,the clinical characteristics and laboratory indications of three groups were compared and analyzed.Using high-throughput proteins chip technology(including 640 cytokines)to compare expression of proteins in mixed serum of parts of samples between Control 1,Control 2(as a technical repeat),IR and Non-IR group,SD value was calculated by the technical repeat,and the difference≥3 times of SD value as standard,and the data were analyzed and proteins which were significantly differentially expressed in IR and Non-IR group serum were selected.Subsequent expansion of clinical sample size verification study was performed by ELISA kit.Graphpad prism 6.0 and R 3.5.1 software was used to data visualization,Cluster3.0Software was used to cluster analysis of proteins chip,statistics results were presented as mean±standard deviation,SPSS22.0 software was used to analyze data,p<0.05indicates that the data was statistically significant.2.Induction of PCOS rat model by continuous subcutaneous injection of DEHA(dehydroepiandrosterone)(6mg/100g)for 20 days in the neck,evaluation of model by body weight,vaginal smear,HE staining of ovay,fasting blood glucose(FBG),fasting insulin(FIN),and sex hormone determination.According to HOMA-IR(≥2.8),model rat were divided into IR and Non-IR group.And to further validate proteins screened by human serum protein chip that might be closely related to PCOS Non-IR from the levels of PCOS animals by ELISA.Graphpad prism6.0 software was used to draw,statistics results were presented as mean±standard deviation,SPSS22.0 software was used to analyze data,p<0.05 indicates that the data was statistically significant.Results1.Analysis of clinical characteristics and laboratory indications of Control,PCOS with IR and PCOS with Non-IR groupAmong of 157 patients with PCOS,41 patients in Control group with an average age of 26.29±3.38 years.According to HOMA-IR(≥2.69),157 patients with PCOS were divided into 102 patients with IR,the an average age was 24.92±3.48 years,and55 patients with Non-IR,the average age was 24.93±3.22 years,there was no significant difference in age.The menstrual features of PCOS patients with IR or Non-IR were mainly characterized by a disorder or prolonged menstrual cycle.Most of PCOS patients with IR or Non-IR were in polycystic changes in bilateral ovaries.LH,LH/FSH and T levels in IR and Non-IR group were significantly increased which were statistically significant(p<0.05),while there were no significant differences between IR and Non-IR group.Compared with Control group and Non-IR group,FBG,FIN and HOMA-IR were significantly increased in IR group(p<0.05).FBG in Non-IR group was significantly higher than Control group(p<0.05),there were no significant differences in FIN and HOMA-IR between Non-IR group and Control group.Comparing BMI between three groups,obesity patients accounted for 34.3%in PCOS IR group,and accounted for 5.5%in PCOS Non-IR group,while BMI of Control group were basically within the normal range(18.5~22.9 kg/m~2).2.Screening and verification of serum differential proteins in PCOS with Non-IR based on proteins chip2.1 Screening for differential proteins in Non-IR groupReproducibility analysis,PCA analysis,k-means cluster analysis,t-test analysis,and functional analysis of differential proteins were performed on protein chip results.Finally,comparing with Control and IR groups,we screened 10 proteins that were significantly differentially expressed in Non-IR group which have important clinical diagnostic significance,including Mer,Calcitonin,CA125,Procalcitonin,CA19-9,IL-1 F8,Pepsinogen II,CEA,AFP and CA15-3.Functional analysis revealed that CA125,CA19-9,CA15-3,CEA and AFP were all serum tumor markers closely associated with tumorigenesis.Calculating concentration of these five serum tumor markers in three groups according to the standard curve.The results showed that CA125,CA19-9,CEA and AFP were significantly increased in Non-IR group when compared with Control group and IR group(p<0.05).CA15-3 was significantly increased both in IR group and Non-IR group(p<0.05),but there was no significant difference between IR group and Non-IR group.2.2 Expanding sample size for validation based on ELISAScreening for serum samples based on clinical information of patients,41 cases in Control group,102 cases in IR group,and 55 cases in Non-IR group were finally used.Compared with Control group and IR group,CA125,CA19-9,CEA and AFP were significantly increased in Non-IR group(p<0.05),but there were no significant differences between Control and IR group.CA15-3 in IR and Non-IR group was significantly increased(p<0.05),while there was no significant difference between IR group and Non-IR group.3.Evaluation of PCOS rat model and verification results by ELISA3.1 Evaluation of the PCOS rat modelOn the 11th day of administration,body weight of IR group and Non-IR group was significantly higher than that of Control group(p<0.05),while there was no significant difference between IR group and Non-IR group.On the day of 14th,17th and 20th of administration,IR group and Non-IR group showed a significant increase in body weight(p<0.05)compared with Control group,and IR group showed higher than Non-IR group(p<0.05).The vaginal cytology and changes of estrous cycle were evaluated.The results showed that the proportion of complete estrous cycle both in IR group and Non-IR group decreased significantly,18.2%in IR group,and 16.7%in Non-IR group,while each rat in Control group had normal estrous cycles.Ovarian wet weight of IR group and Non-IR group were significantly increased(p<0.05),there was no significant difference between IR and Non-IR group.By HE of ovary,statistical analysis showed that the proportion of primary follicles(PF)formed in IR group and Non-IR group was significantly increased(p<0.05),while the proportion of anterior follicles(AF)and corpus luteum(LH)formed in IR group and Non-IR group was significantly lower(p<0.05).There were no significant differences in PF,AF and LH between IR group and Non-IR group.Compared with Control group and Non-IR group,FBG,FIN and HOMA-IR were significantly increased in IR group(p<0.05).Non-IR group was significantly higher than Control group in FBG(p<0.05),and there were no significant differences between Non-IR group and Control group in FIN and HOMA-IR.IR group and Non-IR group were significantly increased in E2 and T levels(p<0.05),and there were no significant differences between IR group and Non-IR group.There were no significant differences in P,PRL,LH,FSH and LH/FSH among three groups.3.2 Verification results of serum tumor markers in control,IR and Non-IR ratsCompared with Control,AFP levels in IR group and Non-IR group were significantly increased(p<0.05),and Non-IR group was higher than IR group(p<0.05).Moreover,there were no significant differences in CA125,CA19-9,CA15-3,and CEA levels among three groups.Conclusions1.Reproductive age women with PCOS are often with prolonged menstrual cycle,and usually accompanied by insulin resistance and hyperandrogenism.PCOS with insulin resistance accounted for a higher proportion of obese patients,most of patients showed bilateral polycystic ovary.2.A significant up-regulation of multiple serum tumor biomarkers in PCOS with Non-IR,and combined with a validation study of animal levels,It is suggested that women with PCOS Non-IR have an increased risk of endometrial cancer.3.This study provides meaningful evidence for clinical content of expanded PCOS Non-IR patients and the importance of strengthening follow-up work. |
PDF Full Text Request