| Premature Rupture of Membrane (PROM) refers to rupture of membranehappens before labor, which severely affects pregnant women and newborns.Premature rupture of membrane can cause infection, placental abruption, postpartumhemorrhage, premature birth, compression of umbilical cord and various diseases thatcan endanger mother and child’ health and life. There are many factors which cancause premature rupture of membrane during gestation period, but the most basicpathological changes are inflammation and the structure deficiency of fetal membraneitself. In recent years, scholars at home and abroad study the causes of the occurrenceof premature rupture of membrane mainly form fetal membrane structure changes,mechanics of machinery, infection factors, and immune and so on, they have madegreat progresses. Infection is recognized by scholars as one of the closest relevantfactors to the occurrence of premature rupture of membrane. In clinic, there are fairlyparts of pregnant women who have no obvious clinical manifestations. Before andafter premature rupture of membrane, the pregnant women stay at subclinicalinfection status. Therefore, early diagnosis for premature rupture of membraneconcurrent infection is especially important. Among them, bacterial infection,mycoplasma and chlamydia infection are the common factors resulting in genital tractinflammation. Toll like receptors (TLRs) are a type of ancient protein family participating in innate immunity, TLRs play an important role in cell signaltransduction, they are the gate receptors activating the related cells to secretinflammatory medium, of which, TLR-2is one of the molecules which have thewidest expression range, can recognize the most pathogenic microorganism and thetypes of products. Pathological study results also prove that the characteristic ofpremature rupture of membrane is the low level expression of extracellular matrix(ECM) protein. Matrix metalloproteinases (MMPs) almost can degrade allcomponents of ECM, of which MMP-2keeps to express during the entire gestationperiod. But the concrete pathogenesis of PROM has not been completely explained,there are still not ideal prevention methods now. Through study the expression ofTLR-2and MMP-2in the placenta and fetal membrane of the patients with prematurerupture of membrane and the normal pregnant women at late trimester of pregnancyand their relation to further reveal its pathogenesis, this paper may provide researchdirection for the prevention and treatment of PROM.ObjectiveThrough study if the expressions of TLR-2mRNA, MMP-2mRNA and protein inthe fetal membrane tissue of PROM group and normal late pregnancy group hasdifferences, locate TLR-2and MMP-2by immunohistochemistry in placenta and fetalmembrane tissue of two groups, this paper aims at investigating the function andrelation of TLR-2and MMP-2in the occurrence and process of premature rupture ofmembrane.Materials and Methods1. Study Object:30pregnant women with full term premature rupture of membrane endedpregnancy by caesarean section hospitalized in the third affiliated hospital ofZhengzhou University due to premature rupture of membrane from September of2012to January of2013were randomly selected as experiment group (PROM group),the diagnosis for the patients with premature rupture of membrane is based on the seventh edition textbook Obstetrics and Gynecology edited by Le Jie. Another30pregnant women of the normal late pregnancy and the unbroken fetal membraneended pregnancy by caesarean section at the same term were randomly selected ascontrol group. The patients in two groups all had not matrix diseases and obstetricscomplications and so on. The pregnant women in two groups areprimiparity,single-live-birth, caesarean section indications are patients’ requirementsand contracted pelvis, etc.2.Methods:The pregnant women in two groups are informed and consent, All specimenswere collected in20min after placenta delivery, matrix placenta tissue adhere toumbilical cord was selected. Two tissues (about2cm×2cm×2cm size) were verticallycut, avoiding infarct placenta surface, calcification, hemorrhage and other abnormallocation. Two tissues of fetal membrane with2cm×2cm around crevasse of thepatients with premature rupture of membrane was cut; pathological examination wasperformed under optical microscope to diagnose chorioamnionitis.10%formaldehyde was used to fix it and then sent it to pathological examination.0.9%sodium chloride solution was used to rinse the obtained tissue cleanly; sterility filterpaper was used to remove the blood on the tissue, one tissue was kept in formaldehydesolution and paraffin was used to embeding the section, the histology result ofconventional haematoxylin-eosin staining indicated they were chorioamnionitispatients. Another tissue was kept in sterility cryopreserved tube in-80℃forextracting mRNA. Real-time PCR method was used to detect the expressions ofTLR-2mRNA and MMP-2mRNA in fetal membrane tissues of pregnant women inexperiment group and control group. Immunohistochemical Streptomycesavidin一peroxidase connection method (SP method) was used to detect the locationand expression of TLR-2mRNA and MMP-2mRNA in placenta and fetal membranetissues of30patients of full term PROM and30pregnant women in normal group.3Statistical Methods:SPSS17.0statistical software was used to make statistical analysis. Thestatistical result of measurement data was expressed by mean±standard deviation(x s), t test was used to compare the two groups independent samples, one-way analysis of variance was used to compare many groups, and LSD-t method was usedto compare between groups.Pearson correlation analysis was used to analyze thecorrelation of two variables. α=0.05was chosen as inspection level.Results1. Comparison of general clinical data conditions of pregnant women in PROMgroup and control groupIn PROM group and control group, their ages were among23-35years old,theaverage age of pregnant women in PROM group was25.341±5.484years old;gestational week was among37-41weeks, average gestational weekwas38.516±2.124weeks. The normal pregnant women at the same term as controlgroup, average age was25.841±3.334years old, gestational week was among37-41weeks, average gestational week was38.433±1.764weeks. BMI respectively was29.023±3.841,29.534±3.681during pregnancy, the age, BMI during pregnancy,gestational week of two group were compared, the differences had no statisticaldifferences (P>0.05).2. Expressions of TLR-2mRNA and MMP-2mRNA in fetal membrane tissue atlate pregnancy of PROM group and normal control groupThe expression of TLR-2mRNA,MMP-2mRNA can be detected in the twomaternal fetal membranes, relative expression levels of TLR-2mRNA,MMP-2mRNA in PROM group were0.581±0.142,0.616±0.164, respectively, in normalcontrol group were0.353±0.191，0.348±0.123; The expression of TLR-2mRNA,MMP-2mRNA in fetal membranes tissue of the experimental group weresignificantly higher than the control group, and the differences were statisticallysignificant (P <0.001).3. Location and expression of TLR-2and MMP-2protein in placenta and fetalmembrane tissue of PROM group and normal control group at late pregnantperiodFigure2was placenta and fetal membrane tissue negative control and HEstaining result, negative control: the result of using PBS to replace primary antibody in the process of immunohistochemistry; the result of immunohistochemistry showed:TLR-2and MMP-2protein all expressed in placenta and fetal membrane tissue ofPROM group and normal control group, the staining strength in placenta and fetalmembrane tissue of PROM group was higher than that of normal control group, twogroups were compared, the differences had statistical significance (P<0.001).In the placenta tissue of PROM group, TLR-2mainly expressed in the andmembranes cytoplasm of syncytiotrophoblast, MMP-2mainly expressed in the andcytoplasm of syncytiotrophoblast,showing claybank, particle and deposition; Inaddition, there was also TLR-2expressing in placental villus and deciduamesenchyme, there was a little MMP-2expressing in placental villus vascularendothelial cell and mesenchymal cell, TLR-2and MMP-2expressed in amnioticepithelial cells of fetal membrane and cytotrophoblast of chorion in PROM group, theexpression and location of TLR-2and MMP-2were basically same in experimentgroup and control group, but claybank particles could be seen in cytoplasm of mostcells in experiment group. Pathology image analysis system was applied to measureTLR-2and MMP-2average optical density value, the measurement results as follow:TLR-2and MMP-2average optical density values in PROM group were respectively0.141±0.011and0.186±0.015, TLR-2and MMP-2average optical density values innormal pregnancy group were respectively0.083±0.004and0.118±0.007, thedifferences of PROM group and normal pregnancy group had significance (bothP<0.001). TLR-2and MMP-2average optical density values in fetal membrane tissueof PROM group were respectively0.318±0.007and0.486±0.015, TLR-2and MMP-2average optical density values in fetal membrane tissue of normal full term groupwere respectively0.283±0.004and341±0.011, the differences of PROM group andnormal full term group had significance (both P<0.001). The correlation of TLR-2and MMP-2protein expression quantity in fetal membrane tissue:there was nocorrelation between the two in normal pregnancy group;there was significantcorrelation between the two in full term PROM group.(r=0.166，P>0.05；r=0.434，P<0.05)4. TLR-2and MMP-2protein level changes in fetal membrane tissue of thepatients with chorioamnionitis The fetal membrane pathological examination results of the pregnant women intwo groups showed that there were15patients who had chorioamnionitis, amongthem, there were13patients who had chorioamnionitis coming from PROM group,the infection rate was21%; There were2patients chorioamnionitis coming fromcontrol group, the infection rate was3.3%. The TLR-2and MMP-2levels in the fetalmembrane of the patients who had chorioamnionitis were higher than that of thepatients who had no chorioamnionitis, the two were compared, the differences hadstatistical significance (P<0.001and P<0.001).5.Different Apgar scores of TLR-2and MMP-2protein levels in fetalmembrane tissue of newborns in PROM groupApgar scores of newborns≤7in PROM group, their TLR-2and MMP-2levels in fetalmembrane tissue were obviously higher than that of Apgar scores of newborns≥8.Different Apgar scores of TLR-2and MMP-2protein levels in fetal membrane tissueof newborns in PROM group were compared, the differences had statisticalsignificance (P<0.001and P<0.001).6. TLR-2and MMP-2protein level changes in fetal membrane tissue of thepatients in PROM group at different membrane rupture timesWith membrane rupture time prolonging, TLR-2and MMP-2protein level in fetalmembrane tissue of patients in PROM group rose, especially, the membrane rupturetime exceeded24hours, the rising was more obvious. TLR-2and MMP-2proteinlevels at different membrane rupture times in PROM group and control group werecompared, the differences had statistical significance (P <0.001and P <0.001).ConclusionThe increase of TLR-2and MMP-2expression in placenta and fetal membranetissue could promote the occurrence of full term premature rupture of membrane.Theexpressions of TLR-2and MMP-2in full term PROM group had significant positivecorrelation, which shows that the two jointly participate and regulate the process of membrane rupture through certain mechanism. |
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