Histological Analysis Of Fetal Amniotic Fluid In Patients With Simple Ventricular Septal Defect And Its Influencing Factors

Ventricular septal defect (VSD) is one of the commonest congenital heart malformations, accounting for up to 40% of all cardiac anomalies. The incidence of VSD is approximately 1.35 to 17.3 per 1000 live births, which caused a heavy psychological and economic burden. Due to the progress of molecular biologic techniques, the past decade have witnessed spectacular development in elucidation of the molecular mechanisms of heart formation. In particular, several transcription factors such as NKX2.5, GATA4, TBX5 and Homez were proved to be essential for heart development. Protein is the final performance of genetic information and can characterize post-translational modifications. Peptidomics is an emerging branch of proteomics that investigates endogenously produced protein fragments. Endogenous peptides can interact directly with the cellular targets, producing a functional effect. Peptidomics can present a dynamic view of the health status. Nowadays, cardiovascular proteomics dramatically broaden our knowledge on the complex myocardial physiology and pathophysiological states, and rapidly advance the identification of disease mechanisms.With the development of fetal echocardiography techniques, we can diagnose and localize VSD in fetus and infancy. VSD is usually asymptomatic and can often close spontaneously. The spontaneous closure rate varies from 6% to 71% due to differences between study population, diagnostic standards, testing methods, time span of follow-up study and type of VSD. This study utilized fetal echocardiography and follow-up information to evaluate the spontaneous closure rate of VSD in eastern China. We followed up our patients from fetal period and summarized a prediction formula including factors influencing spontaneous closure. We hope that our data may provide useful information to aid prenatal counseling and prognosis assessing.Part 1Peptidomic analysis of amniotic fluid for identification of putative bioactive peptides in ventricular septal defectObjectives:1) To gain insight into the mechanisms of protein and peptides in cardiovascular development, we construct a comparative peptidomic profiling of human amniotic fluid between normal and VSD fetuses.2) Screening out peptides associated with abnormal heart development, providing the basis for subsequent functional studies.Methods:1) We construct a comparative peptidomic profiling of human amniotic fluid between normal and VSD fetuses using a stable isotope labelled profiling strategy using tandem mass tag reagents (TMT), followed by nano liquid chromatography tandem mass spectrometry (LC-MS/MS). After removing the redundancy data, we conducted a statistical analysis, more than 1.5 fold change were considered statistically significant (P<0.05). In this way, differentially expressed peptides were screened out.2) We analyzed the physical and chemical properties, cleavage sites of differentially expressed peptides and checked whether these peptides were located within the functional domains of the precursor protein. For corresponding precursor proteins, we applied IPA pathway analysis, GO analysis, and screening out peptides associated with heart development;3) Peptides satisfied the above requirements were selected as a target peptide, we traced them in the peripheral blood serum of corresponding mothers with VSD and normal control fetuses through high-resolution multiple reaction monitoring system using (HR-MRM) to valid and find correlation with amniotic fluid data.4) Based on the sequence conservation, tissue-specific expression and some other standards, we screened peptides that were closely related to cardiac development. These peptides were bioinformatic analyzed to predict potential enzymatic function changes.Results:1) We identified and quantified 692 non-redundant peptides,183 of which were differentially expressed in the amniotic fluid of healthy and VSD fetuses; 69 peptides were up regulated and 114 peptides were down regulated.35 peptides were related to heart development and were also located in functional domain structure of precursor proteins;2) 12 peptides showed the same expression trends in fetal amniotic fluid and their mothers’peripheral blood serum by HR-MRM;3) Peptides "YSGPTCH" from NOTC4 and PGAPLLPPLLL from C05A1 demonstrate high specificity in cardiac tissue/cardiomyocytes; and both of them have good species conservation;4) Bioinformatics analysis showed that NOTC4 (precursor protein of "YSGPTCH") was involved in the Notch signaling pathway which is closely related to embryonic heart development; and YSGPTCH cleavage sites showed production of this peptides may be related to activity changes of matrix metalloproteinase-2 and 9.Conclusion:1) Compared with normal controls, peptides expression in amniotic fluid of VSD fetuses were significant changed, indicating that peptides were involved in the regulation of heart development process.2) By validation through HR-MRM, we concluded that mass spectrometry results of amniotic fluid peptides were reliable and can be used for subsequent analysis.Part 2Forecast of spontaneous closure of isolated ventricular septal defects in utero and postnatal lifeObjectives:1) By conducting a follow-up survey in children who were diagnosed with simple ventricular septal defect in their mothers’second trimester through fetal echocardiography, we hope to describe the outcome of the fetus with ventricular septal defect after the birth in Jiangsu Province, and to identify factors contributing to spontaneous closure (SC) of VSD.2) We could provide a reference for prenatal counseling and clinical decision making, reduce birth defects and improve the quality of the population and predict the prognosis of children with VSD.Methods:1) A total of 423 fetal patients examination who had been diagnosed with isolated VSD by fetal echocardiographic in their mother’s second trimester were enrolled in this retrospective study between January 2011 and December 2013.Fetal echocardiography was performed by two skilled sonographers using ultrasound system Acuson Sequoia 512 (Siemens, USA).2) Data in ultrasound record contained gestational weeks, mother’s age, fetal heart rate, width of aorta and pulmonary artery, location and diameter of the defect, direction of the shunt. Parental consents were obtained from the parents of each neonate.3) Questions in the interview contained:whether the defect was closed, specific time of the SC, current treatment for those who were not SC, gender, birth weight of the infancy, whether premature birth existed, whether there were infection or metabolic disease during pregnancy, with or without a family history of heart disease.4) The differences among the three groups were assessed using analysis of variance (ANOVA) for continuous variables. The comparison between group 1 plus 2 and group 3 was done using Chi-square test for categorical variables. P< 0.05 was considered statistically significant. Clinical and echocardiographic variables that were identified as statistically significant through the above analysis were subsequently evaluated simultaneously by binary logistic regression analysis to identify independent predictors of outcome. A receiver operating characteristic (ROC) analysis was performed to assess the sensitivity and specificity of the diameter of the defect for predicting SC of VSD.5) Verification of the predicted formula:comparing the actual results of the followed up study from January to March,2014 and P values calculated by binary logistic regression prediction equation.Results:1) Initially, the number of follow-up patients was 423 people, and effective follow-up was 257 people,44 cases received termination of pregnancy,213 infants were born.8 cases died after birth,205 cases survived. At the time of follow-up,19 cases underwent clinical surgery,24 cases were still not closed, spontaneous closure occurred in 110 children (49 closed during pregnancy, and 61 closed postpartum).2) The post-natal death, children underwent surgery and group of children whose defects were unclosed were classified group 1, along with defects closed postpartum and during pregnancy were classified as group 2 and 3 respectively. The comparison of echocardiography and post-natal follow-up results among the three groups showed significant differences (P<0.05) in the following data: defect diameter (3.422±0.972,2.426±0.599,2.292±0.479mm), birth weight (3.095±0.774,3.174±0.535,3.499±0.532kg), defect location, direction of blood flow through the defect. The average closing time was 7.31±6.073 months. When SC is defined as a state variable and the defect diameter is defined as test variables, we can get a receiver operating characteristic curve: area under the curve is 0.842, P value:O.OOO.Cut-off value is 2.55mm. In this case, sensitivity= 0.800, specificity= 0.718.3) Binary logistic regression analysis showed that after removing the impact of the defect location, birth weight is a protective factor, while defect diameter is a risk factor for SC. The probability of spontaneous closure was described by the equation:probability= (1+exp [-2.125-.176*birth weight+1.393*diameter])-1. We examine children with VSD from January to March in 2014 according to the probability formula in the binary logistic regression to predict SC,19 out of 23 children were in line with forecasts. Maternal age, fetal aortic diameter, the diameter of the pulmonary artery, the fetal heart rate and maternal morbidity during pregnancy situation were not statistically significant.Conclusion:1) By using statistical analysis, cut-off value of the defect diameter to predict the rate of SC in children with VSD who were diagnosed in their mothers’ second trimester was 2.55mm, the smaller the defect was, the more likely SC will happen.2) Greater birth weight has higher probability of SC;3) Male fetuses have higher probability of SC; muscle defect has higher probability of SC; defect without bloodstream detection were easier to close; full-term infants have a higher probability of healing.