| Objective: Preeclampsia(PE) is a specific syndrome in pregnancy associated with high blood pressure and proteinuria. It contributes significantly to maternal and perinatal morbidity and mortality. The pathogenesis of PE has not been fully understood, but it hypothesized that the progression of PE begins with shallow trophoblast invasion and disturbed remodeling of uterine spiral arteries to provide inadequate blood flow to placenta, subsequently leading increased oxidative stress and maternal endothelial dysfunction. It has been reported that increase in antiangiogenic factors such as soluble fms-like tyrosine kinase-1(s Flt-1) and soluble endoglin(s Eng) and decrease in angiogenic factors such as placental growth factor(PLGF) play important roles in predicting preeclampsia. Heme oxygenase(HO) catalyses the cleavage of heme to produce biliverdin(a potent anti-oxidant), iron, and carbon monoxide(CO). The three isoforms of HO have been identified HO-1, HO-2 and HO-3. HO-1 is the inducible of three HO isoforms, HO-2, on the contrary, is constitutively expressed, and the existence of a functionally relevant HO-3 isoform is still uncertain. Heme oxygenase enzymes(HO-1 and HO-2) and more specifically its catalytic by-products have been postulated to be involved in vasodilation, angiogenesis, regulation of hemodynamic control within placenta and fetal, regulation of the apoptotic and inflammatory cascades in trophoblast cells and maintenance of a balance of the oxidant-antioxidant status in healthy pregnancy. The aim of the present study was to evaluate the expression of HO in maternal serum, cord serum and placental tissues with preeclampsia and bilirubin levels in maternal serum to explore the correlation between HO and the pathogenesis of preeclampsia, providing a new theoretical basis for diagnosis and treatment of PE.Methods:1 Study groups: We included severe preeclampsia and normal pregnant women who visited the Department of Obstetrics and Gynecology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.Subjects were recruited between February 2013 and December 2014. A total of 98 singleton pregnancies were included, with 35 in each group of early onset preeclampsia(EPE), 30 consists late onset severe preeclampsia(LSPE) and 33 in the control group. All women provided their written informed consent to participate in the study. Definition and classification of PE was in accordance with “Obstetrics and Gynecology”, the eighth edition, People’s Medical Publishing House. All women delivered by cesarean section(CS) were included as cases.2 Serum and placental tissues collection and preparation: 6ml of blood samples was collected from the elbow vein into citrated vacutainer before any treatment. It remained at room temperature for 2 hours and blood samples were then centrifuged by 1000 rpm and 20 minutes. The supernatant was collected and stored at-70℃ HO-1, HO-2 and bilirubin concentration analysis. Immediately after the birth of the baby, the umbilical cord vein blood was collected, according to the maternal blood processing. All placental tissues were obtained from pregnancies delivered by cesarean section after delivery of the placenta. Full thickness pieces of the placenta were immediately dissected and cut into sizes of approximately 1cm×1cm×1cm within the umbilical cord root of 3cm in maternal surface. Immunohistochemical staining was carried out using the streptavidin perosidase method after the preparation that all placenta tissues were fixed in 10% neutral buffered formalin and embedded in paraffin.3 Measurement indexes: All of the subjects were measured the concentration of HO-1 and HO-2 in maternal blood and umbilical cord blood by enzyme-linked immunosorbent assay(ELISA). Distribution of HO-1, HO-2 expression was observed in the placenta by Immunohistochemical techniques(IHC) and image analysis for the mean optical density value(MOD) of HO-1, HO-2. Total bilirubin, Direct bilirubin and Indirect bilirubin levels in maternal serum were determined by Diazonium method.4 Statistical analyses: SPSS version 13.0 was applied for all statistical analyses. The clinical data and the results were expressed as the mean±SD, Comparisons among groups were tested by One-Way ANOVA analysis and Student-Newman-Keuls(SNK)test, Kruskal-Wallis H Test was used if the results were homogeneity of variance or non normal distribution. Linear correlation was assessed the linear relationship between two variables. For all tests performed, a P value <0.05 was considered statistically significant.Results:1 The serum concentrations of HO-1 were both significantly decreased in preeclampsia compared with normotensive pregnancy in maternal serum and umbilical cord serum(P<0.05, respectively), and HO-1 concentration was significantly lower in women with EPE than those with LSPE(P<0.05, respectively). And as the same with HO-1, HO-2 concentrations were also significantly decreased in preeclampsia compared with normotensive pregnancy in maternal serum and umbilical cord serum(P<0.05, respectively), and HO-1 concentration in EPE was reduced compared with LSPE(P<0.05, respectively).2 The concentration of HO-1 in the maternal serum was higher significantly compared to HO-2 in EPE, LSPE and normotensive pregnancy respectively(P<0.001, respectively). The same results for HO-1 compared to HO-2 in umbilical cord serum for all three groups were observed.3 HO-1 and HO-2 were both expressed in the placenta of the three groups(EPE, LSPE and normotensive pregnancy): Mean optical density(MOD) values for HO-1 and HO-2 in PE were significantly lower than the normotensive pregnancy(P<0.001, respectively). The expression of HO-2 in EPE was also significantly decreased compared to LSPE(P<0.05), whereas HO-1 did not differ between EPE group and LSPE group(P>0.05). The MOD of HO-1 in the placental tissues had no significant difference compared to HO-2 in EPE, LSPE and control groups respectively(P>0.05, respectively).4 HE staining: Placental villi matured well in normotensive pregnancy.. The blood vessels were rich and morphology was normal in placentas villi and stroma, and the nucleus of syncytiotrophoblast were arranged orderly on the surface of villi in normotensive pregnancy women. There were immature placental villi, arrangement in disorder of the nucleus of syncytiotrophoblasts, and the decrease, incompletion of elastic membrane, hyaline degeneration of arteriolar smooth muscle cells in LSPE. The number of placental villi became fewer in EPE, and there were obviously degeneration and necrosis in the villi and syncytiotrophoblast cell fusion largely on the surface of villi. In addition, placental thrombosis was also observed.5 Immunohistochemical staining: HO-1and HO-2 were expressed in the three groups included EPE, LSPE and the normotensive pregnancy group. Immunohistochemistry revealed positive staining of HO-1 in the syncytiotrophoblast layer with traces in the underlying cytotrophoblasts and fetal membrane, while HO-2 was also expressed in endothelial and smooth cells except the places the same as HO-1.6 HO-1 level was positively correlated with HO-2 in maternal serum, umbilical cord serum, and placental tissues(r=0.717, r=0.643, r=0.884; P<0.001, respectively).7 The concentrations of TBIL, DBIL and IBIL did not differ among the three groups included EPE, LSPE and the normotensive pregnancy group (P=0.914, P=0.134, P=0.434). Our results showed no correlation between HO-1 and TBIL, DBIL and IBIL respectively in maternal serum, umbilical cord serum and placental tissues(P>0.05). However, HO-2 level was negatively correlated with TBIL and IBIL in maternal serum(r=-0.412,P=0.026; r=-0.495, P=0.006), and had a positive correlation with IBIL in placental tissues(r=0.725, P<0.001). No correlation was observed between HO-2 and bilirubin in umbilical cord serum.Conclusion:1 The expression of HO-1 and HO-2 in maternal serum, umbilical cord serum and placental tissues with severe preeclampsia is downregulated, Furthermore, EPE group is lower than LSPE. Speculate that HO should be involved in the pathogenesis of PE, and correlated with severity of PE.2 The expression of HO-1 in maternal serum, cord serum with severe preeclampsia and healthy pregnancy women is higher than HO-2, While HO-1 and HO-2 expression in placental tissue have no difference.3 HO-1 and HO-2 are both expressed in the three groups included EPE, LSPE and the normotensive pregnancy group. HO-1 and HO-2 are mostly expressed in the syncytiotrophoblast layer and fetal membrane, HO-2 is also expressed in endothelial and smooth cells.4 The concentrations of bilirubin did not differ among the three groups. The bilirubin in maternal serum should not be involved in the pathogenesis of PE, and has no correlation with severity of PE.5 HO-2 level is negatively correlated with TBIL and IBIL in maternal serum and has a positive correlation with IBIL in placental tissues. There is no correlation between HO-1 and TBIL, DBIL and IBIL respectively in maternal serum, umbilical cord serum and placental tissues. The results suggested that the HO-2 would be closely related to bilirubin metabolism in pregnancy women. |
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