The Expressions And Roles Of BFGF, Bax, Bcl-2 And MMP-9 In The Amniochorion Of The Patients With Premature Rupture Of The Fetal Membranes

Background and ObjectivesPremature rupture of the fetal membranes (PROM) is defined as spontaneous rupture of the membranes before the onset of labor. Premature rupture of the membranes occurring before 37 weeks' gestation is usually referred to as preterm premature rupture of the fetal membranes (pPROM) . Term premature rupture of the fetal membranes (tPROM ) is defined as rupture of the membranes occurring after 37 completed weeks' gestation. Now, about 10 percent of pregnant women present with term premature rupture of the membranes in China. Preterm premature rupture of membranes occurs at 2.0 to 3.5 percent of pregnancies and is responsible for approximately 30 to 40 percent of all preterm births. And 25 percent of preterm premature rupture of membranes occurs before 26 weeks. The treatment of preterm premature rupture of the membranes is in a dilemma because of the constitutive leakage of the amnion fluid and the consequential complications of neonatal morbidity and mortality. And creative approaches are in urgent need of clinical treatment.The exact cause of premature rupture of membranes is obscure, and the mechanism of this problem is still unclear. It might be the result of multiple factors. The human amniochorion membranes are composed of the amnion and the chorion and the collagenous extracellular matrix. The integrity of the amnion and collagen content in the extracellular matrix (ECM) maintains its tensile strength throughout the pregnancy. More recent evidence suggests that membranes rupture is also related to biochemical process and structural damagement.The degredation of collagen within the extracellular matrix of the amnion and the chorion is mediated primarily by matrix metalloproteinases (MMPs) , which are inhibited by specific tissue inhibitors of matrix metalloproteinases (TIMPs ) and other protease inhibitors. Matrix metalloproteinases is a family of enzymes produced by various types of cells that hydrolyze at least one component of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9 ) proteolytically degradates the typeⅣcollagen, which forms the basement membrane, the next layer of the amnion. Matrix metalloproteinase-9 might play an important role in the membranes rupture.The programmed cell death in the fetal membranes is associated with the premature rupture of the membranes. Although apoptotic changes have been identified in fetal membranes, the mechanisms regulating apoptosis and the subsequent effects on the tensile strength of fetal membranes have yet be elucidated. Bcl-2 associated X protein (Bax ) and B cell lymphoma / leukemia-2 protein (Bcl-2), known as the pro-apoptotic gene products Bax and anti-apoptotic gene products Bcl-2, are homologous proteins that belong to the same family of Bcl-2. A homodimer of Bcl-2 will promote cell proliferation, whereas a homodimer of Bax will promote apoptosis, Normal cell functioning is maintained by the presence of heterodimeric Bax/Bcl-2 complexes.Basic fibroblast growth factor (bFGF) is a kind of polypeptide, which can promote the proliferation, migration and growth of types of cells derived from mesoblastema and neuroectoderm, especially endothelium, epithelium and fibroblast. Evidences have suggested the growth and migration of the amnion epithelium induced by basic fibroblast growth factor in vitro.So we studied the expressions of bFGF, Bax, Bcl-2 and MMP-9 in the fetal membranes, and evaluated the roles and correlations of the four factors in the pathogenetic mechanisms of premature rupture of the membranes so as to help us prevent PROM and find a novel way to treat with it.Materials and Methods1. Materials The study population consisted of patients from the Third Affiliated Hospital of ZhengZhou University from October 2005 to June 2006 (gestational age is from 32 to 40 week) . 36 pregnant women with PROM were randomly enrolled in this study , and divided into 16 patients with pPROM (average age was 27.8±2.86 years old, average gestational age was 34.9±1.47 weeks ) , 20 patients with tPROM (average age was 28.1±2.91 years old, average gestational age was 38.9±1.05 weeks ) according to gestational age; according to chorioamnionitis, the pPROM group and tPROM group were divided into two groups: chorioamnionitis group and non-chorioamnionitis group. 20 pregnant women without PROM (average age was 28.2±2.86 years old, average gestational age was 38.8±0.94 weeks ) were recruited as control group. All the amniochorions (1×1cm~2) were immediately taken from the rupture site and 10 cm far away from the rupture site after cesarean section. The samples were fixed in buffered formalin, followed by routine paraffin embedding, cutting, and staining with hematoxylin and eosin. 2. Methods Chorioamnionitis was histopathologically confirmed by H&E stain; The levels of bFGF, Bax, Bcl-2 and MMP-9 expression in the amniochorion were detected by immunohistochemistry and image analysis by the software of Biosens Digital Imaging System, and compared between preterm PROM, term PROM and control group. All cases had no other complications of pregnancy. All cases had no regular uterus contraction or did not use antibiotics before taking the fresh amniochorions, body temperature was below 38.0℃, the pulse rate of pregnant women was below 100 beats per minute, white blood cell count was below 1.5×10~4/L. Statistical analysis was performed with SPSS 10.0 software, using analysis of variance, linear regression analysis, and X~2 test. Statistically significant level was considered as "alpha equals 0.05".Results1 Morphological changes:1.1 The structural changes of the amniochorin with premature rupture of the fetal mmbranes were particularly marked in the amnion and included delamination and loss of amniotic epithelial cells from the amniotic basement membrane, and a dramatic loss of fibrillar collagen from the amniotic extracellular matrix. Laminar necrosis was marked in the chorion, especially in the chorion with chorioamnionitis, in which the neutrophilic infiltrate accompanied necrotic foci.1.2 The rate of chorioamnionitis in the amniochorion with pPROM, tPROM and PROM was 43.75%, 30% and 36.11%, respectively. The rate of medium and severe chorioamnionitis in the amniochorion with pPROM was 12.5%, and the one of light chorioamnionitis was 31.25%. The rate of medium and severe chorioamnionitis in the amniochorion with tPROM was 5%, and the one of light chorioamnionitis was 25%.2 Immunohistochemistry results:2.1 The expression of Bax protein was observed in the cytoplasm of the amniotic epithelial cells, fibroblast and trophoblast cells. The levels of Bax expression in the amniochorion with pPROM and tPROM with chorioamnionitis were higher than the ones of pPROM and tPROM without chorioamnionitis (P<0.05) , were significantly higher in four groups of patients with PROM than those of the control group (P<0.05) ; there were no statistically differences between the rupture site and the remote site of the four study groups (P>0.05 ) .2.2 The expression of Bcl-2 protein was observed in the cytoplasm of the amniotic epithelial cells, fibroblast and trophoblast cells. There were no statistically difference among groups of the levels of Bcl-2 expression in the amniochorion with pPROM and tPROM with chorioamnionitis and pPROM and tPROM without chorioamnionitis and the one of control group (P>0.05) ; there were no statistically differences between the rupture site and the remote site of the four study groups(P>0.05) .2.3 The ratio of Bax/Bcl-2 in the amniochorion with pPROM and tPROM with chorioamnionitis were higher than the ones of pPROM and tPROM without chorioamnionitis (P<0.05) , were significantly higher in four groups of patients with PROM than those of the control group (P<0.05) ; there were no statistically differences between the rupture site and the remote site of the four study groups(P>0.05) .2.4 The expression of MMP-9 protein was observed in the cytoplasm of the lymphocytes, neutrocytes, fibroblast, and the amniotic epithelial basement membrane. The levels of MMP-9 expression in the amniochorion with pPROM and tPROM with chorioamnionitis were higher than the ones of pPROM and tPROM without chorioamnionitis (P<0.05) , were significantly higher in four groups of patients with PROM than those of the control group (P<0.05) ; there were no statistically differences between the rupture site and the remote site of the four study groups(P>0.05) . 2.5 The expression of bFGF protein was observed in the cytoplasm of the lymphocytes, neutrocytes, fibroblast and trophoblast cells, and the amniotic epithelial basement membrane. The levels of bFGF expression in the amniochorion with pPROM and tPROM with chorioamnionitis were higher than the ones of pPROM and tPROM without chorioamnionitis (P<0.05) , were significantly higher in four groups of patients with PROM than those of the control group (P<0.05) ; there were no statistically differences between the rupture site and the remote site of the four study groups (P>0.05) .3 Correlation results:3.1 Bax in the study group of PROM had positive correlation with MMP-9, r=0.878, P<0.01.3.2 bFGF in the study group of PROM had positive correlation with Bax and MMP-9, r=0.904, P<0.01 and r=0.885, P<0.01 respectively.Conclusions1 The increased expression of Bax and no clearly variation of Bcl-2 expression in the amniochorion with PROM, which cause the imbalance of heterodimeric Bax/Bcl-2 complexes, may be one of the important mechanisms in the pathogenesis of PROM; and apoptosis can activate matrix metalloproteinases.2 The expression of MMP-9 in the amniochorion with PROM is increased, which can overdegradate the collagen in the extracellular matrix of the amnion.3 The increased expression of bFGF in the amniochorion with PROM suggests that intrinsic bFGF may play a role of repair in the stress mechanism in human fetal membranes with PROM, which can be as a theory to direct the clinical treatment for PROM.