Expression Of Ox-AAT And NE In Maternal Peripheral Blood, Umbilical Cord Blood And Fetal Membranes In Premature Rupture Of Membranes And Correlation With Histologic Chorioamnionitis

Premature rupture of membrane(PROM),which was defined as rupture of fetalmembranes before birth.PROM is common complication of obstetrics, and theincidence is rising in recent years.About the etiology and pathogenesis of PROM isnot fully clear.Most research found that PROM is closely related to infection.PROMcan be characterized by clinical chorionic amniotic membrane inflammation andsubclinical infection,which also knows as Histologic chorionic amniotic membraneinflammation(HCA).HCA have no obvious manifestation of early, while the placentatissue pathological results after giving birth suggest for inflammatory infiltration.PROM combine with HCA can lead to serious complications of pregnant women andfetus,even death.There are great significance of diagnosis PROM merger of HCAtimely.In recent years, many researchers pay attention on diagnose of HCA at early, buthave yet to find indicator of high sensitivity and specificity.Oxidation type of Alpha1-antitrypsin(ox-AAT)is an inactive form of Alpha1-antitrypsin(AAT)ox-AAT is a production of the inflammatory response,and playing an important role not only increasing but enlarging inflammation.Free radicalsreleased by inflammatory cells can effect the active site of AAT,and AAT have beenoxidized into ox-AAT,which lose the function of inhibit protease andanti-inflammatory.Neutrophil elastase(NE)mainly released by the polymorphhonuclears(PMN), which is one of the members of super family of serine protease.When inflammation occurs,PMN chemotaxis towards the inflammation parts andremove pathogenic,promote the repair of tissue damage.However,excessiveinflammation can lead to increased NE release, and lead to tissue injury andamplification phenomenon of inflammation.ObjectiveThis study by testing expression level of ox-AAT and NE in peripheral blood、umbilical cord blood and fetal membranes of pregnant woman with PROM,as well asthe correlation between them.To discuss the relationship between ox-AAT、NE andPROM combine with HCA.Expect to find laboratory predictor which can earlydiagnose HCA.Materials and methods1Object:Choose PROM pregnant women delivery in the third affiliated hospital ofZhengzhou university between September2012to March2013.The diagnosis ofPROM reference Xie-xing effusion of editor in chief of the eighth edition of theobstetrics and gynecology.Exclude patients diagnosed clinical chorionic amniotic membraneinflammation(pregnant women T>37.8℃、 P>120times/min、 fetus HR>160times/min, with one of the following two criteria: uterine tenderness or amnioticfluid is smelly), infectious diseases such as hepatitis、nephritis、upper respiratory tractinfection and60case finally selected into the study.60PROM pregnant women divided into HCA group(PROM pregnant womencombine with HCA,22of60) and control group(PROM pregnant women without HCA,38of60)according to the postpartum placental tissue pathology results(HCAdiagnosed by inflammatory cells number>5at each high magnification)。Thediagnosis of neonatal infectious diseases will be subject to neonatology departmentdiagnosis.All pregnant women after admission record vital signs, detection of CRP andwhite blood cells (WBC)count,and informed consent obtained before samples aremade to be included.2Methods2.1Serum specimen collection: Blood serum samples5ml came from suckingpregnant women before use of antibiotics,and umbilicus vein blood5ml after placentadeliveried. vein blood were saved at-80℃after centrifuge.2.2Fetal membranes and placental specimen collection: Take membranes breach tothe edge of placenta,about1.0cm×4.0cm,flushed by cold saline and make the rollshape,fixed with a pin,divided into two parts,then tube into the cryopreserved andquickly put into liquid nitrogen,while the other part fixed by10%formaldehydesolutionrainsed.while placenta tissue collected about2.0cm×2.0cm from the center ofthe placenta and avoiding bleeding and calcify region. All the placenta samples fixedby10%formaldehyde solutionrainsed, then were examined by the microscope.2.3The enzyme-linked immunoadsordent assay (ELISA) was used to detect thelevels of ox-AAT and NE in serum of pregnant women,and the levels of ox-AAT andNE in umbilical cord serum.2.4The reverse transcription-polymerase chain reaction(RT-PCR)was used to detectthe mRNA levels of ox-AAT and NE in tissue of membranes of pregnant women.2.5Immunohistochemical method used to detect the ox-AAT、 NE in fetalmembranes and placenta.3Statistics methodsSPSS13.0statistical analysis was employed to analyse all the data.Data wereshowed by using mean±standard(X S)of measurement groups,comparisonbetween groups using independent sample t test or rank inspection.And data wereshowed by rate of enumerationdata groups,comparison between groups using2test.Pearson linear was used to analyze the relativity analysis between two factors.“α”=0.05as the inspection level.Results1The comparison of the general data between the HCA group and the controlgroup:The difference between two groups of pregnant women in their ages,gestationalweeks and pragnante degrees have no statistic meaning(P>0.05).The differencebetween the two groups of pregnant women in their time from rupture to fetalbirth,count of WBC and level of CRP have no statistic meaning(P>0.05)although thelevel of HCA group higher than the level of control group.2The expression of ox-AAT and NE in serm of pregnant women and newbornof the HCA group and the control group:The levels of ox-AAT in maternal serum(2.328±0.014)and umbilical cordserum(2.920±0.182)of HCA group are higher than that of control group (1.503±0.12、2.433±0.004), and the levels of NE in maternal serum(0.407±0.082)and umbilicalcord serum(0.744±0.065)are higher in HCA group than that of controlgroup(0.260±0.090、0.415±0.010),and all the differences were statistically significant(P <0.05).3The expression of ox-AAT mRNA and NE mRNA in membranes of pregnantwomen of the HCA group and the control group:The expression of ox-AAT mRNA(0.960±0.03)and NE mRNA(0.670±0.075)inmembranes in HCA group is higher than that in control group(0.532±0.01、0.271±0.09),and the differences were statistically significant (P <0.05).4The expression of ox-AAT and NE in membranes of pregnant women of theHCA group and the control group:The expression of ox-AAT(0.023±0.005)and NE(0.019±0.002)in fetalmembranes in HCA group is higher than that in control group(0.015±0.002、0.005±0.001),and the differences were statistically significant(P <0.05).5The expression of ox-AAT and NE in placenta of pregnant women of theHCA group and the control group: The expression of ox-AAT(0.182±0.001)and NE(0.261±0.003)in placenta inHCA group is higher than that in control group(0.04±0.008、0.055±0.009),and thedifferences were statistically significant (P <0.05).6The correlation analysis6.1The correlation analysis of ox-AAT, NE in maternal serum and their expressionin umbilical cord serumThe expression of ox-AAT in maternal serum were positively correlated to thelevels in umbilical cord serum of HCA group(r=0.825,P<0.05); The expression ofNE in maternal serum were positively correlated to umbilical cord serum in HCAgroup (r=0.847,P<0.05).The control group has no relevance respectively.6.2The correlation analysis of ox-AAT, NE in maternal serum and their expressionin fetal membranes of HCA groupThe expression of ox-AAT、NE in maternal serum were positively correlated tothe levels in fetal membranes in HCA group(ox-AAT: r=0.836，P<0.05;NE:r=0.867，P<0.05).6.3The correlation analysis of ox-AAT and NE in HCA groupThe levels of ox-AAT in maternal serum were positively correlated to the levelsof NE in maternal serum(r=0.885,P=0.000); The expression level of ox-AAT inumbilical cord serum were positively correlated to the levels of NE in umbilical cordserum(r=0.756,P=0.000); The expression ox-AAT in membranes were positivelycorrelated to the levels of NE in membranes in HCA group(r=0.876,P=0.000).7The comparison of two groups of maternal and neonatal outcomeThere without a case of60cases of pregnant women combine with postpartumhemorrhage、puerperal infection and placental abruption.18of22pregnant of HCAgroup delivery by cesarean,while9of38pregnant of control group,and thedifferences were statistically significant (2=12.59，P<0.05).In cases of60newborn,1case death from sepsis of HCA group, there no happen of neonatal omphalitis andnecrotizing enteritis.But there are15neonatal combine with pneumonia of HCAgroup compare to the control group of13, and the differences were statisticallysignificant (2=6.461, P <0.05). Conclusions1．The higher level of ox-AAT and NE in maternal serum、umbilical cord serum andmembranes may related to the pathogenesis of Histologic chorioamnionitis.2．Monitoring the ox-AAT and NE in serum is expected to diagnose Histologicchorioamnionitis early.