Proteomics Of Endometriosis

Endometriosis(EMs) is a common disease of the female reproductive system in which endometrial tissue is found in an extrauterine location.The disease is frequently associated with infertility,dysmenorrhoea,dyspareunia,chronic pelvic pain and menorrhagia.The prevalence of the disease is estimated to range from 3 to 10%in women of reproductive age.Several theories have been proposed to explain the pathogenesis of this disease,but the mechanism of endometroiosis is not yet clear.Many literatures have shown that the expression of several biomarkers,such as CA-125,CA19-9 and EMAb in the serum of women with endometriosis,are higher than the controls.These biomarkers have already been used as auxiliary methods in diagnosing endometriosis, but the sensitivity and specificity is very low.This study investigated the different expression of proteins in serum and eutopic endometrium in women with and without endometriosis using high-throughput proteome techniques.Due to the attributes of surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) in providing rapid proteomic profiling of proteins/peptides from different biological specimens,the technology had been applied extensively in cancer research.The aim of this study was to screen biomarkers of endometriosis in serum,and also elucidate the molecular mechanisms of the formation of EMs,based on above findings,clinical diagnostic patterns of EMs were established.Our research also identified differential proteins in eutopic endometrium with and without endometriosis using two-dimensional electrophoresis (2-DE) and MALDI-MS/MS.The differential proteins identified may play important roles in the presence and advance of endometriosis.Part One Screening of Biomarkers and Establishment of Diagnostic Pattern for Endometriosis Using SELDI-TOF-MSObjectives:To investigate and screen differential proteins in serum of patients with and without endometriosis(EMs) and establish the serum molecular diagnostic patterns.Methods:Serum samples were collected from 79 with and 72 without endometriosis.A special serum protein or peptide spectra was determined by SELDI-TOF-MS measurement after treating the sample onto WCX2 protein chip for each case.The serum protein profiles were analysis by Biomarker Wizard Software, and the diagnostic pattern of endometriosis and blind validity were determined by Biomarker Patterns Software(BPS).Results:(1) According to serum protein fingerprints of 79 patients and 72 control women,a total of 231 protein peaks were identified at the M/Z value ranging from 2000Da to 30000Da,16 significant differential proteins were found between the two groups(P＜0.01),11 proteins were upregulated in serum of endometriosis with the M/Z value of 3963.50,4172.09,4195.31,4304.10,4493.25,8121.06,8180.75, 16947.30,17199.80,17823.80 and 18354.20,5 proteins were downregulated with the M/Z value of 2014.83,2032.87,5637.32,6103.08 and 10255.60 respectively.Serum protein profiles obtained from these samples were analyzed by BPS to generate endometriosis decision tree which combined with 3 candidate proteins with the M/Z value of 4172.09,5637.32 and 1963.80,with a sensitivity of 80.0%and specificity of 80.7%,accuracy of 79.3%.Blind validity generated sensitivity of 73.4%,specificity of 72.2%,positive predictive value of 74.4%and negative predictive value of 71.2%. (2) 147 protein peaks were identified between two groups of secretory phase,6 significant differential proteins were found,among which 2 proteins upregulated in serum of endometriosis with the M/Z value of 8170.70 and 18332.80,4 proteins downregulated with the M/Z value of 5631.90,5894.80,6096.70 and 10224.10 respectively.The decision tree combined with 5 candidate proteins with a sensitivity of 96.7%and specificity of 100%.Blind assessment got sensitivity of 100%, specificity of 34.5%,positive predictive value of 65.5%and negative predictive value of 100%.(3) 196 protein peaks were identified in proliferative phase,and 3 proteins have significant difference with 2 upregulated and 1 downregulated.The decision tree combined with 6 candidate proteins with a sensitivity of 94.3%,specificity of 97.1% and accuracy of 95.7%.Blind assessment got sensitivity of 90.7%,specificity of 93.0%,positive predictive value of 92.9%and negative predictive value of 90.9%. Conclusion:SELDI-TOF MS was an ideal technological platform for proteomic research because of its high throughout and stability.Phase of menstruation impact the results of differential proteins we detected.As a result,the decision tree established in proliferative phase was of the highest sensitivity and specificity among the 3 trees and important clinical value.Part Two Study of differential Proteomics in Eutopic Endometrium of EndometriosisObjectives:To analyze and screen differential proteins and their expression of eutopic endometrium from women with and without endometriosis.Methods:Extracted the total protein form eutopic endometrium,then separated using two-dimensional electrophoresis(2-DE) and stained by Coomassie blue.Image analysis and statistical quantification of relative protein abundances was performed using Image Master 2D Platinum software.Interesting differential spots were manually excised from gels and identified via MALDI-TOF-MS/MS.Database searching was performed using the IPI system,and then verified the results of 2-DE via Western bolt.Results:After separated,stained and scanned,we obtained 2-D differential profiles of protein from eutopic endometrium of women with and without endometriosis.The ImageMaster software detected 1278±88(n=3),1073±97(n=3) protein spots in 2-DE gels from endometriosis and control group respectively in proliferative phase.Using the former as reference gels,756 spots were matched in two gels among which 89 ones showed twice or more than twice differences:45 spots were upregulated in group with endometriosis and 44 downregulated.30 spots were picked out for identification via MALDI-TOF-MS/MS,after excluding the reduction 20 proteins were identified successfully,including cytoskeletal proteins,calcium binding protein,proteins participating lipid metabolism and so on which upregulated in the eutopic endometrium from endometriosis,and enzymes participating ATP metabolism,peroxiredoxin which downregulated in the eutopic endometrium from endometriosis.In secretory phase,1052±109(n=3) and 1044±78(n=3) protein spots were detected in the control and endometriosis group respectively,and 64 proteins were of statistical significance.30 spots were picked out for identification and 20 proteins were identified successfully,among which enzymes participating ATP metabolism and cytoskeletal proteins upregulated in the eutopic endometrium from women with endometriosis,and peroxiredoxin,transcription associated proteins such as hnRNP K,H₁,C downregulated.Conclusion:2-DE coupled with MALDI-TOF-MS was a high throughput,high efficiency proteomics platform.The expression of many proteins have changed in the eutopic endometrium,and some of these proteins may be the key ones in the development of endometriosis.