Differentiated Expression Of Placental Protein And Serum Biomarkers In Preeclampsia

Background and Object:Preeclampsia(PE)is a disease specific to pregnancy.Its global incidence is about 2%-8%.In developing countries,10%-15% of maternal deaths are related to this disease.Currently,in China,PE is still the main cause of maternal morbidity,fetal morbidity and mortality.PE can present with a variety of serious symptoms.The main clinical features of PE are increased blood pressure and proteinuria after 20 weeks of pregnancy,and it may also present with one of the following adverse manifestations: thrombocytopenia,renal dysfunction,liver damage,pulmonary edema,neurological symptoms or visual impairment.Most PE patients present obvious disease progression from mild to severe.But some cases show atypical disease process,which can rapidly develop in a very short time,and even directly present serious complications,endangering the life of the mother and the child.In other cases,the blood pressure is not significantly increased,so the diagnosis is often difficult to establish.To-date,PE is not curable with medication.Termination of pregnancy and delivery of fetus is the only effective treatment.Under this condition,early and accurate diagnosis,effective intervention and treatments are critically important to delay the disease development and guarantee the relative safety of mother and baby.In order to accurately diagnose PE and determine treatment plans,a series of studies have been conducted on its pathogenesis,and it is preliminarily believed that related pathway factors such as vascular active substance imbalance,endothelial dysfunction,immune factors,inflammatory response and oxidative stress may be related to the occurrence and development of PE.In recent years,some scholars proposed the binary theory of PE pathogenesis: during the first stage of placenta formation,the trophoblast cells were insufficiently invasive,uterine spiral artery recasting disorder and placental shallow implantation,resulting in placental ischemia.In the second stage,due to placental ischemia and hypoxia metabolic disorder,soluble factors derived from placenta are released into maternal blood circulation in large quantities,causing extensive vascular endothelial injury of the mother and eventually causing systemic symptoms of the mother PE.During the process of PE,placenta is initially affected,followed by a series of clinical symptoms in the mother.It indicates that these two events are correlated,suggesting that some placental changes may be also reflected in the serum of PE patients.The research on clinical patients is influenced by many factors,such as social ethics,experimental observation objects,patient recognition,etc.,which restricts the clinical research progress of PE.The discovery of special markers that can lead to maternal comprehensive symptoms in the placenta and blood of PE patients has always been a focus of people's attention.The exploration of Protein Pathway Array(PPA)and Luminex technologies provide us with new tools to advance the clinical study of PE.This study was designed based on PE theory and the research was conducted on clinically diagnosed PE patients.PPA technology was used to screen the differentially expressed proteins in the placenta tissue when compared with normal pregnant women.Some related proteins with strong classification ability were pooled out among these proteins.Meanwhile,Luminex technology was employed to detect the changes of these related proteins in serum of mothers.Based on the clinical data and related laboratory tests,statistical analysis was done to explore the correlation between them.PE diagnosis model and severe PE prediction model are established,which may improve our ability of PE accurate diagnosis as well as to identify the patients at risk for PE complications.Methods:From July 2011 to June 2012,166 cases of pregnant women who underwent elective cesarean section in the First Hospital of Jilin University were selected,among which 101 cases were PE and 65 cases were normal control.The differentially expressed protein in placental tissue in these two groups were measured.A total of 172 patients were selected who gave birth in the First Hospital of Jilin University between April 2012 and August 2014,including 110 patients in PE group and 62 patients in the normal control group.The PE group was divided into two groups,among which 25 cases were in the preeclampsia with severe features(PS)group and 85 cases were in the preeclampsia without severe features(PNS)group without severe features,and the expression of differentially expressed proteins in the maternal serum was detected.Patients were excluded from this study if with multiple pregnancies,fetal malformations,fetal chromosomal abnormalities,maternal cardiovascular history,nephropathy history and other diseases related to hypertension,pre-pregnancy diabetes,taking anti-platelet agents,and non-steroidal anti-inflammatory drugs.In the first part,Protein Pathway Array(PPA)was used to detect relevant proteins in placental tissues of preeclampsia and normal pregnant women.We analyzed the differential expression protein,determined stronger classification ability of protein.Biometrics Research Branch(BRB)stratified cluster analysis was carried out for preeclampsia group and normotensive control group respectively using 12 proteins with strong classification ability selected above.PPA is a proteome detection technology,which is based on the multi-channel western-blot technology and integrates the proteome analysis.SAM(Significant Analysis of Microarray)Analysis and BRB-array Tools analysis can select and combine target protein antibodies by itself according to the needs.In the second part,our study focused on 11 potential maternal serum protein markers in preeclampsia using Luminex Technology as well as ELISA method.11 serological protein markers were firstly pooled out according to the result of the differences in protein expression in placenta tissue.The expression of these 11 serological proteins in PE group and the control group were measured via Luminex technology and ELISA.The clinical parameters of PS and PNS group,as well as the protein level were analyzed,and PE diagnosis model and severe preeclampsia prediction model are established.Luminex technology is a new generation of high throughput molecular diagnostic technology platform based on ELISA,which integrates the fluorescent microsphere coding,laser detection,fluidics,the latest high-speed digital signal and computer algorithms.Luminex has the advantages of simple operation and accurate measurement.And multiple proteins or nucleic acids can be detected in one experiment when compared to ELISA.SAM software was used to screen proteins with differential expression between PE group and normal control group.The cases were classified by using the k-fold cross validation(K=10)of BRB Array Tools v.3.3.0 software(HTTP://Linus.Nci.nih-gov/brb-arraytools.html)and the hierarchical cluster analysis method.The differentially expressed proteins were screened by student's t-test and the related clinical factors were analyzed by chi-square test.The data of non-normal distribution is converted to log2 for analysis.Proteins and clinical pathologic parameters were analyzed using univariate and multivariate logistic regression to produce independent predictors of 95% confidence intervals with regression coefficients,P values,odd ratio(OR values),and OR values,which were used to establish preeclampsia diagnostic models and preeclampsia predictive models with severe manifestations.The area under AUC curve was calculated using SPSS 17.0 software(SPSS v17.0 software,SPSS.,Chicago,IL),and P <0.05 was taken as statistically significant.Results:64(38.3%)proteins were positive expressed in placental tissues detected by PPA technique,among which 36(22%)proteins presented differential expression in PE group when compared with normal control group.Among the 36 proteins,19 proteins in PE present high expression in placenta tissues,including Stat1,p-PKC?,EGFR,AKT,HSP90,Calretinin,WT1,Factor XII I B,PSM,Endoglin,Nox4,PLT-1,Ebi3,Htr A,PAF,PBEF,Ptx3,PAI-1,TFPI2.17 proteins in PE present low expression in placenta tissues,including PLGF,glypican3,nrf2,IGFBP3,Annexin XI,TFIIHp89,PDEF,ICAM-1,after CREB,Bcl-6,NF?B p65,Bcl-2,14-3-3?,PCNA,Rap1,p-Stat3 and MAPK(Erk1/2).Calretinin,Annexin XI and Ebi3 were detected to be differentially expressed in the placenta tissue in PE,with Ebi3 being the first time to be reported in this study.Among the 36 differentially expressed proteins in PE group,12 proteins(Calretinin Htr A,Endoglin,Nox4,PAI-1,Ebi3,HSP90,Flt1,Ptx3,Annexin XI,PLGF,Bcl6)were found to have stronger classification ability,which can distinguish 89.5% PE accurately,with the sensitivity and speciality rate of 93.1% and 83.1% respectively.According to the result of the differences in protein expression in placenta tissues(s Endoglin,IL-27,PTX3 PLGF,PAI-1,s EGFR,s Flt-1),the existing antibodies list and related literature(cox-2,human prolactin,VEGF,NGAL),11 serological protein markers were selected and further measured with Luminex technology and ELISA.The results found that 7 proteins in PE group have significant differences,with VEGF and s Endoglin significantly increased,s Flt-1,Cox-2,PLGF,NGAL and s EGFR decreased significantly.SEndoglin and sflt-1 were significantly increased in PS group.Single factor,multiple factors regression analysis and the receiver-operating characteristic curve(ROC)analysis were done based on the difference between the placenta tissue and maternal serum protein changes,clinical data.VEGF,PLGF,s EGFR,s Endoglin or the combination of 4 proteins were served as either single or multiple analyzing factors.PE diagnosis model is established with these factors,and the area under the curve is 0.988,which possess a high diagnostic value.This model can be used to diagnose preeclampsia,especially for patients with normal blood pressure and silent onset,facilitating timely and accurately diagnose.On this basis,a predictive model with severe manifestations of preeclampsia was established,including serum creatinine,s Endoglin,and platelet count.This model has good predictive value for severe preeclampsia,with an area under the curve of 0.92.It is suggested that this model can be used to predict and evaluate whether PE patients will have severe maternal and infant complications,providing a new reference for determining clinical treatments,such as expectant treatment or termination of pregnancy.In the current study,for the first time we found that human serum level of s EGFR was significantly decreased in preeclampsia patients,comparing with normotensive pregnant women.Serum s EGFR level was one of the independent predictors for preeclampsia.It might serve as a biomarker for the diagnosis of this condition.Analysis of ROC curve to study the use of serum s EGFR concentration as a predictor of preeclampsia revealed cut-off points with the highest sum of specificity and sensitivity at 28.49 ng/m L,leading to specificity of 87% and sensitivity of 55% and yielding an AUC of 0.724.Conclusion:1.The use of PPA technology in PE placenta extract 36 differentially expressed proteins and there proteins are involved in angiogenesis,endothelial injury,inflammation,cell proliferation,immune regulation and etc.For the first time,calretinin,Annexin ?and Ebi3 in placenta tissue may be closely related with the occurrence of PE.2.According to the results of differential protein expression in placental tissues and related literature,Luminex technology and ELISA were used to detect the serum expression of there proteins in PE patients.It was found that VEGF,s Endoglin and sflt-1 were significantly increased,cox-2,PLGF,NGAL and s EGFR were significantly decreased.3.In this study it was found that s EGFR was significantly reduced in PE group,and s EGFR might be an independent predictor of PE,laying a foundation for further study on the pathophysiological mechanism of PE,as well as providing a basis for the assessment of PE risk.4.Using the indicators determined by Luminex technology,with the help of multivariate regression analysis and subject work characteristic curve(ROC)analysis,this study established four protein combination models of VEGF,PLGF,s EGFR and s Endoglin for accurate diagnosis of preeclampsia,and establish a risk coefficient model for the assessment and prediction of severe manifestations of preeclampsia using combination of serum creatinine,s Endoglin and platelet count.