Study On Correlation Between TLR3, TLR4, TNF-α And Idiopathic Fetal Growth Restriction

The fetal growth restriction (FGR) which is one of the significant complications of pregnancy is usually defined as a birth weight less than 2500g at term. The FGR is not only closely related to the perinatal mortality rate, but also the adulthood cardiovascular disease and the endocrine metabolic disease. The etiology of FGR is complicated. About 40% causes are unknown at recently, which is called idiopathic fetal growth restriction (IFGR). One of the IFGR is defined as the symmetric IFGR which the fetal weight, the head circumference and the length are subjected to restriction. The other has the general head circumference and the length but lower weight, which is called the asymmetric IFGR. The studies suggested that the disordered mother's innate immune maternal-fetal interface may affect fetal growth. The toll-like receptors family (TLRs) identify the different types of pathogenic mocroorganisms and altered self-components. To detect the human placenta we find that TLR1-10 of the mRNA were expressed, including TLR3, TLR4 which is expressed stronger. TLR3 recognizes double-stranded RNA (dsRNA) molecules and plays an important role in the anti-viral immunity. TLR4 can recognize invading bacterial pathogens and altered self-components. Both of them start the innate immune response, ensure normal fetal growth and against the disadvantage from the outside world. If this balance is broken, it is bound to impact on pregnancy outcome and occur some of the fetal growth related cytokines disordered, such as tumor necrosis factor-a (TNF-a). This is probably one of the pathogenesis of IFGR. The study on correlation between TLR3, TLR4, TNF-a and IFGR are rarely reported in china and abroad. The aim of these study is to analysis the perspective of the immune pathogenesis of IFGR.ObjectiveTo investigate the expression and the significance of TLR3 and TLR4 in placenta, TNF-a in the maternal blood and the cord blood of the symmetric IFGR and the asymmetric IFGR; To explore the expression of correlation between TLR3, TLR4 in placenta and TNF-αin the maternal blood and the cord blood in IFGR; The aim is to prospect for clinical use of the drug.Methods1. Study Object:From April 2008 to April 2009,42 primiparae of singleton pregnancy and perinatal new-borns suffering from IFGR were chosen from patients in the Third Hospital Affiliated to Zhengzhou University to enroll in this study, all of whom were terminated by cesarean section in term delivery. The diagnostic criteria of IFGR is according to the sixth edition of Obstetrics and Gynecology which the chief editor is Le Jie and is published by the public health people publishing company in Beijing.The symmetric IFGR (n=20) and the asymmetric IFGR (n=22) were divided according to the PI typing approach. Another 42 non-IFGR pairs were randomlysampled as the control group.2. Method:2.1 Placental Specimen Collection:All the placenta samples were immediately taken from the site which was 1cm×1cm×1cm within 15 mintues after cesarean section.The samples which had been cleaned by normal sodium were fixed in buffered formalin, followed by routine paraffin embeding, cutting (3μm), dring overnight at 60℃, then waited to be detect.2.2 Blood Specimen Collection:All the maternal blood samples were collected two hours before cesarean section and the cord blood were immediately taken from the umbilical veins which were closed to the placenta, then all the blood samples were injected to the non-anticoagulant test tube. After putting them respective aside at room temperature, centrifuging 15 minutes at 4000 rev./min, Separating the Serum, located in the Low temperature environment, the samples were prepared to detect.2.3 Experimental Method:The Laboratory Personnel was commissioned to sort the whole three groups of samples under the premise of the detector unaware of these. One of the section preparations of each group were stained with hematoxylin and eosin (HE) for morphology observation.non-biotin polymerized horseradish peroxidase (HRP) immunohistochemical method were used to detected TLR3 and TLR4 leveles by two of the other section preparations. HRP immunohistoche-mistry masculine cell diagnosed standard is like that:syncytiotrophoblast cells and hofbauer cells was presented buffy or brown. Both of TLR3 and TLR4 in syncytiotrophoblast cells were collected by computer image analysis system of Biosens Digital Imaging System and analyzed by the software of leica Qwin, and the average of masculine hofbauer cells were calculated. The enzyme linked-immuno-sorbent assay (ELISA) were used to detect TNF-alevels in each group of maternal blood and the cord blood samples.3. The consequent had been completed by one individual. Statistical analysis was performed with SPSS 13.0 software, numerical data was demonstrated as mean±standard deviation(x±s). Group comparisons were using analysis of variance and the least significant difference (LSD). The linear correlation (Pearson) analysis was used to determine the relationship. Statistically significant level was considered as "alpha equals 0.05".Results1. Comparison of Clinical Data:Comparing the clinical data of Three groups of pregnant women, the general information on the onset time, the symmetric group is longer than the asymmetric group which has statistically significant (P<0.05). Neonatal basic information, the two groups IFGR children with birth weight, birth length were lower than those of normal. According to the length, the symmetric group was significantly lower than the asymmetric group children (P<0.01).2. Morphology observation of placenta by HE staining among the three groups. For the two IFGR groups, the stucture of placental villus presented obviously changes as follow, interstitial fibrosis in villus, placental infarction and syncytiotrophoblast cell nodule increased. Compared to the control group which had statistically significant (P<0.01). For the symmetric group and the asymmetric group, interstitial fibrosis in villus has almost not different to the asymmetric group (P>0.05), but the placental infarction and the syncytiotrophoblast cell nodule were substantially lower than that of the asymmetric group (P<0.01).3. The comparison between the leveles of TLR3 and TLR4 in maternal placenta.3.1 For the symmetric group and the asymmetric group, the levels of TLR3 in placenta were substantially lower than that of the corresponding control groups, and the difference is statistically significant (P<0.01). In the symmetric group, the number of cells with positive TRL3 expression in Hofbauer cells were substantially lower than that in the control groups, and the difference is statistically significant(P<0.01). In the asymmetric group, the number of such cells were substantially higher than that in the control groups, and the difference is statistically significant (P<0.01).3.2 For the symmetric group and the asymmetric group, the levels of TLR3 in placenta were substantially higher than that of the corresponding control groups, and the difference is statistically significant (P<0.01). The number of cells with positive TRL3 expression in Hofbauer cells were almost not differences in the three groups (P>0.05).4. The correlation between the maternal blood and the cord blood which expressed the levels of TNF-a in IFGR.For the symmetric group and the asymmetric group, the levels of TNF-a in the maternal blood were 90.3±9.6μg/L and 85.5±10.7μg/L respectively, while in the cord blood the levels were 91.7±11.6μg/L and 95.9±7.8μg/L respectively, which were all substantially higher than that of the control groups (P<0.01). For the symmetric IFGR, the TNF-aexpression in the maternal blood was almost not correlated with that in the cord blood (P>0.05).5. The correlation between the expression of TNF-a in the maternal blood, the cord blood and the expression of TLR3,TLR4 in maternal placentaFor the symmetric IFGR and the asymmetric IFGR, the expression of TNF-a in the maternal blood was not correlated with TLR3 expression in the placenta. But for the symmetric IFGR, the expression of TNF-a in cord blood was correlated with TLR3 expression in Hofbauer cells, which was significant (P<0.05). expression of TNF-αin the maternal blood, cord blood were related to the causes of the two types of IFGR.2 The reasons for the symmetric IFGR were possibly related to the decreased expression of TLR3 in Hofbauer cells, and the causes of the asymmetric IFGR were possibly related to the increased expression of TLR3 in Hofbauer cells.