A Risk Prediction Model And The Etiology Of Abnormal Uterine Bleeding Caused By Primary Endometrial Disorders

Abnormal uterine bleeding(AUB)is a common gynecological disease,which causes,anemia after acute or chronic blood loss,infertility,hysterectomy,endometrial malignancy,and other adverse outcomes.It interferes with the quality of life,work productivity,and health care cost are seriously affected.In 2011 the International Federation of Gynecology and Obstetrics(FIGO)recommended the“PALM-COEIN”classification system to classify the common causes of AUB.AUB-E caused by primary endometrial disorders is thought to be associated with endometrial dysfunction,although the etiology is not clear.AUB-E that occurs in the context of a structurally normal uterus with regular menstrual cycles without evidence of coagulopathy is likely to have an underlying endometrial cause,primary endometrial disorder.However,a lack of clinically available specific tests.Admittedly,the diagnosis of AUB is challenging for the variability of representation,the complexity of etiology,lack of accuracy,poor timeliness,and dependence on invasive examination.The essential problem that the traditional symptom-based diagnosis and treatment can not give patients accurate diagnosis,treatment,and precaution.Therefore,it is time for medical development to transform the traditional diagnosis and treatment pattern to the individualized and the precision medical model.In this study,based on the current status of AUB-E diagnosis and the new opportunity of precision medicine,we looked at AUB-E patients,relied on the“PALM-COEIN”system,combined with the methods of epidemiology,molecular biology,and data mining with big data to explore the potential risk factors of AUB-E to establish risk prediction model.Also,the proteomics of human endometrium was performed by mass spectrometry to search for the target proteins associated with AUB-E,which provides the etiological reference for clinical practice of AUB-E.Part 1 Establishing a risk prediction model for abnormal uterine bleeding caused by primary endometrial disorders(AUB-E)Objective:Due to the diagnosis of AUB-E depending on exclusion,it is a lack of specific detection means,and etiology is not clear.In this study,a case-control study was carried out to analyze the epidemiological characteristics between AUB-E and control groups,explore the risk factors,and establish a risk prediction model,which might improve the diagnostic accuracy and therapeutic effect of AUB.Methods:In this study,data were collected by a self-designed health questionnaire and summarized the diagnosis process of AUB-E relied on the AUB diagnostic guidelines,both at home and abroad.At the same time,PSM was used to match the controls as the condition of“Age”with a ratio of 1:2.After that,exploring the epidemiological distribution of the two groups was conducted.The Logistic regression model was made with 7 variables with statistical significance,such as education degree,occupation type,tea-drinking habit,sugar diet,history of diagnosis and curettage,history of fallopian tube operation,and BMI,to analyze the relationship between potential risk factors and the incidence of AUB-E,and then the model was evaluated.Results:This study included 84 patients with AUB-E and 170 controls.The multinomial test suggested that the results have a good balance of variables after matching.The median age of all participants was 42(35,47)years and ranged from 22 to 51 years.The results showed that there was a significant difference in the demographic character of the educational level(p=0.000)and the employment(p=0.004)between the two groups.In aspects of lifestyle and medical history,there were significant differences in the distribution of drinking tea(p=0.008),possessing sugar diet(p=0.035),diagnosis and curettage(p=0.000)and the history of fallopian tube disease(p=0.018).Also,BMI(p=0.000)was a significant difference between the two groups.Besides,there were significant differences between the controls and the AUB-E patients with prolonged menstrual cycle(p=0.000),heavy menstrual bleeding(p=0.000)combined with the Hb(p=0.000)and HCT(p=0.000)levels.There were no significant differences in other characteristics.The Logistic regression analysis showed that educational level(x1)(p=0.003),tea-drinking(x3)(p=0.018)and BMI(x7)(p=0.011)were significant for disease prediction.Primary education or below(OR=3.642,95%CI 1.550-8.555)and increasing BMI(OR=1.119,95%CI 1.026-1.221)were risk factors for AUB-E,although tea-drinking habit(OR=0.437,95%CI 0.220-0.866)might be a protective factor.The sensitivity and specificity of the model were 41.0%and 91.9%,the positive predictive value and negative predictive value were 75.56%and 71.84%.The AUC was0.740(p=0.000)with 95%CI 0.671-0.808.The best BMI threshold for AUB-E was≥22.7 kg/m~2.Conclusion:1.A case-control study has collected the information of AUB patients with a self-designed questionnaire.A total of 712 patients were enrolled in this study,including83 patients with AUB-E and 138 controls.2.The PSM method is used to match the object of study.The data after matching has a good balance.3.There were differences in the distribution of educational level,employment,tea-drinking,sugar diet,history of diagnosis and curettage,history of fallopian tube operation,and BMI between the two groups.4.Logistic regression analysis showed that education level,tea-drinking habits,and BMI of independent variables had statistical significance for AUB-E prediction.5.Primary education and below and BMI≥22.7 kg/m~2are risk factors for AUB-E,and tea-drinking is a protective factor.6.AUB-E patients who complained of HMB or prolonged menstruation often combine with latent anemia.Therefore,it should be monitored and immediately corrected.Part 2 Proteomic analysis of endometrium from patients with abnormal uterine bleeding caused by primary endometrial disorders(AUB-E)and SMC1A verificationObjective:Due to the etiology of AUB-E is not clear,the diagnosis of this disease lacks specific identification markers.In this chapter,the human endometrium of AUB-E and normal control groups were analyzed by mass spectrometry to find the biomarkers associated with AUB-E,which provided the basis for the etiology and mechanism of AUB-E.Methods:The endometrial tissues of AUB-E patients(aged 25-40)who visited Women’s Hospital,School of Medicine,Zhejiang University from December 2019 to May 2020 were collected,and the endometrial tissues of age-matched controls with normal menstruation and regular period were also collected.Following the structural causes or pathological changes of endometrium were ruled out,the endometrium was analyzed by label-free proteomics.The quantitative differential proteins with p<0.05 and FC≤0.67(or FC≥1.5)were defined as differentially expressed proteins(DEPs).Principal component analysis(PCA)combined with bioinformatics analysis,followed by volcano map analysis,cluster thermograph analysis and functional annotation for DPEs,and mining for potential target proteins of AUB-E through enrichment analysis,and further verification.Results:There were no significant differences in baseline characteristics between AUB-E and control groups except for the PBAC score(p=0.000).All the samples were in the period of proliferation by HE staining.After label-free quantification,1921 differential proteins(p<0.05)and 291 DEPs were obtained,including 140 up-regulated proteins and 151 down-regulated proteins.11 DEPs from the KEGG enrichment assay were significantly enriched in the Cell cycle,Apoptosis,and Cell adhesion molecule pathway.Western blot was used to identify the potential target protein and only SMC1A was verified down-regulation in AUB-E patients.The expression of SMC1A protein in the endometrial glandular epithelium of AUB-E patients was lower than that in controls.Conclusion:1.There were significant differences in menstrual blood loss(MBL)among the patients from the study sample.2.Proteomic analysis showed that 1921 differential proteins were expressed in proliferative endometrium between AUB-E and control groups,of which 291 proteins were considered to be significantly different,including 140 up-regulated proteins and151 down-regulated proteins.3.The results of PCA analysis showed that there was a good difference between the two groups,and no significant sample selection bias was found.4.Occurrence of AUB-E is regulated by multiple signaling pathways.5.The expression of SMC1A in the human endometrial glandular epithelium of AUB-E patients decreased significantly.Part 3 Effects of down-regulated SMC1A on human endometrial epithelial cells and exploring the mechanismObjective:Based on the proteomic analysis and SMC1A protein validation of human endometrial tissues from AUB-E patients in the previous chapter,this study aimed to investigate the effect of down-regulated SMC1A in human endometrial epithelial cells(hEECs)to explore the relationship between SMC1A protein disorder and AUB-E.Methods:The hEECs were isolated and cultured from fresh endometrial tissue of normal menstrual cycle women followed identified.The expression of SMC1A in hEECs was down-regulated by si RNA technology and we researched the cellular function of cell proliferation,cell migration,cell cycle,apoptosis,and NF-κB pathway P65 expression.Results:The hEECs culture showed that most were short columns or polygons with clumps and positive expression of cytokeratin-7 on the cell surface.Immunofluorescence co-localization showed that SMC1A was expressed in the nucleus of hEECs in vitro.si RNA can effectively knockdown the expression of SMC1A in hEECs,with an efficiency of more than 50%(p=0.012).After transfection of si-SMC1A for 72 h and96 h,hEECs significantly inhibited cell proliferation(p=0.0016 and 0.0068).After 24hours of cell migration,hEECs were significantly decreased in the si-SMC1A group(p<0.0001).There was no change in cell cycle between the two groups:G0/G1 phase%(p=0.99),S phase%(p=0.83),G2/M phase%(p=0.69).Cell apoptosis in the si-SMC1A group was significantly higher than that in the NC group after 72 hours of transfection.However,there was no significant difference in the expression of NF-κB p65 protein between the two groups.Conclusion:1.hEECs were successfully cultured through the digestion and isolation of fresh human endometrial tissue.2.Stable expression of SMC1A in the nucleus of hEECs.3.si-SMC1A can knockdown successfully in hEECs to decrease the expression of SMC1A protein.4.Down-regulation of SMC1A could inhibit the proliferation of hEECs.5.Down-regulation of SMC1A inhibits the migration ability of hEECs.6.Down-regulation of SMC1A had no significant effect on the cell cycle of hEECs.7.Down-regulation of SMC1A promotes apoptosis of hEECs.8.Down-regulated SMC1A protein is not regulated by NF-κB P65.