Study The Placenta Tissue Protein Of Selective Intrauterine Growth Restriction In Monochorionic Twins

BackgroundThe incidence of twin pregnancy continues to rise, which has been linked largely to the increased utilization of artificial reproductive technologies and, to a lesser extent, the increase in mean maternal age over this period of time. Twin pregnancies are more prone to complications than single pregnancies, with an increased rate of preterm birth, low birth weight, malformations, gestational hypertension, gestational diabetes and unique complications such as twin-twin transfusion syndrome (TTTs), intrauterine growth restriction (IUGR). In order to design potential strategies to reduce these high risks, more knowledge is needed about the natural course of twin pregnancies.Twin pregnancies are classified according to either zygosity or chorionicity, and chorionicity rather than zygosity determines the outcome. During pregnancy, twins are classified as dichorionic(DC) or monochorionic(MC) based on placentation. MC twins have an increased perinatal mortality rate than DC twins, this can be explained by servering the individual placental anastomoses. To establish the chorionicity, the placenta and dividing membrane between twins were examined by the ultrasound between11-14gestational weeks. Ultrasound criteria used for the determination of chorionicity in the first and early second trimester were the number of placental sites, the lambda or "T" sign.Selective intrauterine growth restriction (sIUGR) is a common condition associated with twin pregnancy. The term’selective intrauterine growth restriction’ in twin pregnancies is applicable to cases where the estimated fetal weight (EFW) of the small fetus falls below the10th percentile. Significant fetal weight discordance is an important element of the clinical picture, which will often accompany this condition, but is not necessary for diagnosis. It is increasingly considered to be an important complication of MC twins, with potentially significant risks of intrauterine fetal demise (IUFD) or neurological adverse outcome for both twins. In recent years the pathophysiological insight of sIUGR has been substantially improved, although the ability to reliably predict the clinical outcome remains elusive-this will probably remain the case, as the clinical presentation and outcome seem to depend on a combination of multiple factors. The most feared complication of sIUGR in MC twins was the intrauterine demise of the growth-restricted twin, which carries an associated postmortem risk of acute feto-fetal transfusion from the normal to the deceased IUGR fetus. Indeed, death of one MC twin has been reported to be accompanied by concomitant death of the larger twin in25-30%of cases and neurological damage in30%of survivors. The principle cause for the development of sIUGR in MC twins is inadequate placental sharing. The relationship between unequal placental territory and birth weight discordance has recently been revealed in several studies showing that discordance increases with increased placental territory discordance. Extremely asymmetric distribution of placental territories is often associated with very eccentric or velamentous cord insertion, although it is unclear whether velamentous insertion is a mere consequence of the asymmetric displacement of the vascular equator or whether it has any implications in the pathophysiology of growth restriction. Aside from placental territory discordance, a second factor largely influencing fetal weight discordance and the natural history of sIUGR in MC twins is the presence of vascular anastomoses in the MC placenta. To manage twin growth disorders, it is important to detect the high-risk factors and prevent the neonatal complications. Thus, the aim of this study was to investigate the maternal risk factors for sIUGR firstly, and then using two dimensional difference gel electrophoresis (2D-DIGE) to identify novel proteins associated with the restricted(smaller) and the other normal(bigger) fetus’s placenta in monochorionic selective intrauterine growth restriction (sIUGR) twins.The placenta plays a vital role in the regulation of fetal growth and development during pregnancy. Roles include nutrient supply to the fetus, removal from the fetus of metabolic waste and hormone production. There have reports about abortion, pre-eclampsia, preterm, assistant reproductive technology using proteomics technology, but there has no research studied for sIUGR yet.2D-DIGE is a method which label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye DIGE dye multiple labeling method and DeCyder difference2D analysis software. Base on the advantages of DIGE technology, great opportunities comes to us to discover the information of differential proteome contained in sample of the patients with sIUGR.Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot was performed to validate representative findings from the2D-DIGE analysis. Objective1. To evaluate the impact of chorionicity on the perinatal outcomes of twin pregnancies. And to investigate the high-risk factors in monochorionic sIUGR and dichorinic sIUGR.2. To compare the differences of protein expression of the restricted(smaller) and normal(bigger) fetus’s placenta in monochorionic sIUGR in hopes of finding associatied proteins of sIUGR by using2D-DIGE, so as to elucidate the pathogenic mechanism of sIUGR.3. Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot analysis was performed to validate representative findings from the2D-DIGE analysis. Methods1. In total,1264pairs of twins were enrolled from March2009to June2012.According to the chorionicity,1144pairs of twins were divided into monochorionic twins and dichorionic twins. Maternal characteristics, complications, and perinatal outcomes were analyzed among the two groups. Logistic regression was performed to identify the interrelationships between independent maternal risk factors for sIUGR in a multivariable model.2. The total proteins extracted from placenta tissues of the smaller and bigger fetuses were separated by means of2D-DIGE. The first dimension was carried by IPG PH gradient isoelectric focusing system, the second dimension was separated by vertical SDS-PAGE.3. Differentially expressed proteins spots in2D-DIGE maps were chosed and incised after in-gel by trypsin, and the candidate proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). The peptide mass spectrometry maps and acid sequences were searched from SEQUEST databases. And representative findings from the2D-DIGE analysis were confirmed by western blot analysis. Results1. The overall twin pregnancy incidence was2.55%. Monochorionic twin pregnancies was more earlier in maternal age and delivery age than dichorionic twin pregnancies (P<0.05). And monochorionic twin pregnancies had more sIUGR, discordance growth, intrauterine death and neonatal asphyxia than dichorionic twin pregnancies (P<0.05). The co-twins’birth weights of monochorionic twin pregnancies were lower than that of dichorionic twin pregnancies (P<0.05). Gravidity and mode of conception were protective factors, hypertensive disorders of pregnancy and delivery age were high risk factors for all sIUGR. Gravidity was the protective factors for the MC sIUGR. Whereas hypertensive disorders of pregnancy, delivery age and fetal malformation were high risk factors for the DC sIUGR.2. Compared with the bigger fetus’ placenta,45protein spots with1.5folds of difference statistically in smaller fetus’placenta were found.3.20proteins were identified by MALDI-TOF/TOF, including13up-regulated and7down-regulated proteins spots. Among them, Fibrinogen beta chain, Fibrinogen gamma chain, Keratin(type I cytoskeletal10), Keratin(type II cytoskeletal8), Glucocorticoid-Inducible protein5, Alpha-1-antitrypsin, HHIP-like protein1were down regulated in restricted fetuses’ placentas compared with normal fetuses’ placentas, and Elongation factor Tu, Keratin(type I cytoskeletal19), Tropomyosin beta chain, Tropomyosin alpha-3Chain, Glutamate receptor Subunit epsilon-2, Heat shock protein beta-1, Thioredoxin-dependent peroxide reductase, Chorionic somatomammotropin hormone, ATP synthase subunit d, Myosin regulatory light polypeptide9, Myosin regulatory light chain12B, Calmodulin, Long palate,lung and nasal epithelium carcinoma-associated were up regulated in restricted fetuses’ placentas. Western blot analysis of calmodulin and PRDXIII confirmed that these two proteins were significantly up-regulated in smaller fetuses’s placentas. Conclusion1. There was a trend towards worse outcomes in MC pregnancies compared with DC pregnancies. The gravity, mode of conception, delivery age, hypertensive disorders of pregnancy and fetal malformation were related with the sIUGR. It is recommended that the chorionicity should be examined by the ultrasound between11-14gestational weeks every twin pregnancy.2. Proteomic analysis of placenta reveals differential expression of several proteins between fetuses of monochorionic sIUGR.3. These proteins are implicated in a variety of cellular functions such as cell survival and proliferation, cell apoptosis regulation, cytoskeletal structure, oxidative stress and coagulation.4. Western blot analysis of calmodulin and PRDXIII confirmed the findings from the2D-DIGE.