The Study Of Soluble Fms-like Tyrosine Kinase-1,Placental Growth Factor,25-hydroxyvitamin D,D-dimer,Von Willebrand Factor,P-selectin In Preeclampsia

[Background]Hypertensive disorders complicating pregnancy(HDCP) is a common obstetric disease with high blood pressure. The incidence rate of HDCP of9.4%to10.4%reported in our country, and the incidence of preeclampsia was2%to7%.HDCP, the main reason for increased maternal and perinatal mortality, seriously affected the health of mother and infant. Preeclampsia is the second cause of maternal mortality in developing countries [1]. Preeclampsia is a severe stages of hypertensive disorders complicating pregnancy, it may occurred cerebral hemorrhage, cerebral edema, heart failure, pulmonary edema, liver rupture, placental abruption and disseminated intravascular coagulation in severe patients, and it can raise the long-term risk of cardiovascular and cerebrovascular diseases in those women after the postpartum. The effects on the fetus are that this disease can cause placental function decline, fetal growth restriction, fetal distress and iatrogenic premature delivery and so on. If the pregnant women with preeclampsia, especially early onset severe preeclampsia, there is no clear and effective clinical treatment in addition to termination of pregnancy. In recent years, studies have reported that the high-risk groups can significantly reduce the incidence of early-onset preeclampsia and perinatal mortality rates, if prophylactic use of low-dose aspirin before16weeks of gestation[2].Early prediction of preeclampsia are important for early prevention and treatment, especially for reducing the mortality of pregnant women and perinatal infant. It has always been the hotpot and difficult field of obstetrics. With high sensitivity, low false positive rate, non-invasive, simple, rapid, economic and repeatable characteristics, this is an ideal screening marker or method. But so far, there is no preeclampsia screening marker or method is effective, dependable and economic. Therefore, the exploration of higher sensitivity and specificity biomarkers and screening methods associated with preeclampsia which can be widely applied in clinical may very helpful for early screening of high-risk population of preeclampsia. Given reasonable and effective treatment before the clinical symptoms appear, it would effectively reduce the the mortality of pregnant women and perinatal infant and improve their outcomes.Therefore,the purpose of screening for preeclampsia is to early prevention and early detection of its occurrence and development,fighting for the prediction of preeclampsia and first prenatal work together,concerned about the occurrence of the disease for the individual patient trends and timely interventions,taking the appropriate preventive and control measures to reduce or delay preeclampsia and severe preeclampsia occur.Currently, it is not yet fully elucidated about the Etiology and pathogenesis of preeclampsia. The following theories have been generally accepted that recasting inadequate uterine spiral arteries, vascular endothelial injury, excessive activation of the immune inflammation, nutritional deficiency, genetic susceptibility and insulin resistance are the main cause of preeclampsia etiology[3]. The process of preeclampsia pathogenesis can be divided into two stages[4]. The first phase began with recasting obstacles uterine spiral arteries cause placental ischemia and hypoxia, which has led to a variety of factors in the release of the placenta.The second stage is the clinical symptoms:the consequence of the release of placental factors, which promote the activation of systemic inflammation and vascular endothelial injury in the maternal circulation, would cause the clinical symptoms. Therefore, it is good to predict preeclampsia before the clinical symptoms.In recent years, more and more evidence suggests that the damaged and dysfunction of vascular endothelial is the key to the pathological changes of preeclampsia[5].The following manifestations that increased permeability, hypoxia, hemoconcentration, elevated coagulation factors and reduced anticoagulant factors would be occured after vascular endothelial injury. The clinical symptoms with preeclampsia, which are caused by the consequence of unbalanced coagulation and fibrinolytic system and placental ischemia and hypoxia, are hypertension, protein urine and edema. Generally, the balance of coagulation system maintain blood flow.Once this balance is destroyed, it will lead to thrombosis or hemorrhage. It is important to change coagulation in the pathogenesis of preeclampsia. Thrombosis tends to be more in preeclampsia patients than in normal pregnant women with a relatively hypercoagulable state. The hypercoagulable state of severe preeclampsia in which there are many indicators can be detected, has been proven in many clinical studies[6].This is the result of coagulation and anticoagulation, fibrinolysis system, platelet activation and interaction of vascular endothelial injury. Thus, it is possible to predict preeclampsia by detecting the state of prothrombotic and change coagulation.Many domestic and foreign scholars have done a lot of researches in order to realize the early prediction, prevention, diagnosis and treatment of preeclampsia. With the development of etiology and pathogenesis of preeclampsia, it is recognized that the use of single marker or method is difficult to predict preeclampsia ideally. Therefore, a combined screening of the relevant marker or method is recommended in obstetric guidelines of American and Canada. In recent years,on the regularity of the biomarker prediction preeclampsia studies has gradually shifted from about20weeks to the first trimester of pregnancy.This is closer to the onset of preeclampsia initial stage to make an early prediction and prevention.More and more evidences indicated that the imbalance of angiogenesis factors in maternal blood circulation (such as placental growth factor) and anti-angiogenesis factors(such as soluble fms-like tyrosine kinase-1) was very relate to the pathogenesis of preeclampsia[7] sFlt-1/PIGF ratio reflects the functional status of the vascular endothelium,when sFlt-1/PIGF ratio to85as the boundary in the second trimester,the highest sensitivity to predict the occurrence of preeclampsia up to82%and a specificity of95%[8].In preeclampsia, the change levels of sFlt-land PIGF can be significantly advanced to13tol6weeks gestation[9].Studies suggest that25-hydroxyvitamin vitamin D activated form of1,25-dihydroxyvitamin vitamin D can significantly inhibit vascular endothelial growth factor(VEGF) induced endothelial cell sprouting and angiogenesis.It is presumed that the low level of maternal serum25-hydroxyvitamin vitamin D may be associated with reduction of VEGF.Maternal vitamin D deficiency during pregnancy may be an independent risk factor for preeclampsia[10].The change of prothrombotic and change coagulation is closely related to the occurrence of preeclampsia.The conventional coagulation checking such as prothrombin time, partial thromboplastin time, fibrinogen and platelets are not sensitive to reflect the prothrombotic state.D-dimer, von Willebrand factor and P-selectin is considered the most important biomarks to detect the state of prothrombotic and change coagulation[11]. Because there may be some correlation between biomarks, some biomarks combined screening can not increase the predictive value of preeclampsia. This suggests that all potential biomarks should be included in the same study in order to determine the best combination of screening. Currently, there is no study including these six biomarks at the same time.[Objective]All of the material sources were retrospectively analyzed from the hypertensive disorders complicating pregnancy patients delivery in Maternal and Child Health Hospital of Shenzhen from2009to2013.To investigate the incidence of hypertensive disorders complicating pregnancy and its impact on maternal and perinatal.To investigate the expression of soluble fins-like tyrosine kinase-1(sFlt-1), placental growth factor(PIGF),25-hydroxyvitamin D(25-OH-VD), D-dimer, von Willebrand factor(vWF) and P-selectin in maternal serum or plasma between10and13+6weeks of gestation. To discuss the effects of these biomarks in the pathogenesis of preeclampsia. To evaluate the predictive value with these indicators alone or together through using logistic regression and Receiver Operating Characteristic. To provide a reliable theoretical basis of early predicting, preventing and treatment of preeclampsia.[Materials and methods]1.Material source:All of the material sources were retrospectively analyzed from the hypertensive disorders complicating pregnancy patients delivery in Maternal and Child Health Hospital of Shenzhen from2009to2013.In this study,we recruited1081consecutive women who presented for antenatal care between10and13+6weeks of gestation from Shenzhen Maternity and Child Healthcare Hospital, from2013January to December. Of the1081pregnant women studied,50patients were diagnosed with preeclampsia. The age ranged from20to38years old, the average age was (30.00±4.61) and (29.70±4.34) years old, the average gestational weeks were (38.83±1.08) and (37.70±1.54) weeks. There are352normal pregnant women without complications as control group. The age ranged from20to39years old, the average age was (28.96±4.08) years old, the average gestational weeks were (39.37±0.96) weeks. The difference of mean age and pregnancy and birth history were compared between the three pregnant groups which is no significant difference(P>0.05). The difference of average gestational weeks and fetal birth weight were compared between normal group and severe preeclampsia group which is significant difference(P<0.05).The selected cases were singleton pregnancy, except for heart disease, diabetes, chronic hypertension, renal disease, connective tissue disease, hyperthyroidism, blood disease during pregnancy history, and no blood transfusion, immunotherapy and special drug history. Immediate extraction of elbow venous blood4ml into no anticoagulation glass tube when the person presented for antenatal care between10and13+6weeks of gestation, static30min, let it to self-curing, then3000g/min centrifuge10min, separate the serum in-80℃refrigerator and tested. Another4ml extract into sodium citrate anticoagulation glass tube, then3000g/min centrifuge10min, separate the plasma in-80℃refrigerator and tested. 2.Methods:①The incidence,general situation(age,hospital day and mode of delivery, etc.),clinical factors such as maternal and perinatal cases were analyzed.②Thawing at room temperature, then centrifuged at3000rpm for2minutes in4℃high-speed refrigerated centrifuge, the supernatant was used for experimental analysis. Using enzyme-linked immunesorbent assay(ELISA) to detect the peripheral serum or plasma sFlt-1, PIGF,25-hydroxyvitamin D, D-dimer, von Willebrand factor and P-selectin concentration in normal pregnancy group and preeclampsia group. Get the OD value of the samples from the Enzyme mark instrument.3. Statistical method:Using excel software and SPSS13.0statistical software for data processing. Using Chi-Square test to compare the count data.Using the mean±standard deviation(X±s) to express the measurement data, using two independent samples t test to compare the two samples, using single factor analysis of variance(One-way ANOVA) to many groups compares. The measurement data is converted into the multiple of the median(MoM).Using Logistic regression and ROC curve characteristics to evaluate the value of diagnosis preeclampsia for index alone or in combination. The inspection level is0.05, statistically significant differences in P<0.05.[Results]1. From2009to2013,there are a total of78,578cases of delivery in our hospital,including2486cases of hypertensive disorders complicating pregnancy patients, accounting for3.2%of the total number of delivery.With hypertensive disorders complicating pregnancy patients,the main mode of delivery is still cesarean section.It was not statistically significant differences in age and the mean hospital day.The proportion of mobile population is higher.The ratio of a serious complication of hypertensive disorders complicating pregnancy in descending order:postpartum hemorrhage, placental abruption, HELLP syndrome,heart failure,disseminated intravascular coagulation, acute renal failure, cerebrovascular accident.The ratio of perinatal serious complications in descending order:preterm birth,fetal growth restriction, neonatal asphyxia,stillbirth,abortion.2.1The levels of serum sFlt-1in normal group(n=352) and preeclampsia group(n=50) are (1004.10±456.15) pg/ml、(1456.12±253.34) pg/ml, the differences between the two group was statistically significant(P<0.05).2.2The levels of serum sFlt-1in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (1004.10±456.15) pg/ml、(1293.10±266.75) pg/ml、(1564.80±176.86) pg/ml, the integral comparison of differences between three groups was statistically significant(P<0.05).3.1The levels of serum PIGF in normal group(n=352) and preeclampsia group(n=50) are (56.37±11.10) pg/ml、(40.77±0.59)pg/ml, the differences between the two group was statistically significant(P<0.05).3.2The levels of serum PIGF in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (56.37±11.10) pg/ml、(48.18±10.85) pg/ml、(35.83±7.03) pg/ml, the differences between three groups was statistically significant(P<0.05).4.1The ratio of sFIt-1/PIGF in normal group(n=352) and preeclampsia group(n=50) are (21.20±20.75)、(38.39±13.71),the differences between the two group was statistically significant(P<0.05).4.2The ratio of sFlt-1/PIGF in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (21.20±20.75)、(27.32±4.66)、(45.78±12.75), the differences between three groups was statistically significant(P<0.05).5.1The levels of serum25-hydroxyvitamin D in normal group(n=352) and preeclampsia group(n=50) are (34.12±6.54) ng/ml、(22.86±9.71) ng/ml, the differences between the two group was statistically significant(P<0.05).5.2The levels of serum25-hydroxyvitamin D in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (34.12±6.54) ng/ml、(32.44±7.45) ng/ml、(16.48±4.21) ng/ml, the integral comparison of differences between three groups was statistically significant(P<0.05). 6.1The levels of plasma von Willebrand factor in normal group(n=352) and preeclampsia group(n=50) are (872.96±293.36) U/L、(1309.90±308.63) U/L, the differences between the two group was statistically significant(P<0.05).6.2The levels of plasma von Willebrand factor in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (872.96±293.36) U/L (1162.94±420.61) U/L、(1407.88±142.17)U/L, the differences between three groups was statistically significant(P<0.05).7.1The levels of plasma D-dimer in normal group(n=352) and preeclampsia group(n=50) are (275.52±173.79) ng/ml、(522.68±135.03) ng/ml, the differences between the two group was statistically significant(P<0.05).7.2The levels of plasma D-dimer in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (275.52±173.79) ng/ml、(452.42±184.75) ng/ml、(569.53±52.63) ng/ml, the integral comparison of differences between three groups was statistically significant(P<0.05).8.1The levels of plasma P-selectin in normal group(n=352) and preeclampsia group(n=50) are (93.21±40.47)ng/ml、(150.39±29.87)ng/ml, the differences between the two group was statistically significant(P<0.05).8.2The levels of plasma P-selectin in normal group(n=352), mild group(n=20) and severe preeclampsia group(n=30) are (93.21±40.47) ng/ml、(134.73±39.91) ng/ml、(160.83±13.51) ng/ml, the differences between three groups was statistically significant(P<0.05).9.ROC curve (AUC) of the multiple of the median(MoM) of serum sFlt-1,PIGF, sFlt-1/PIGF,25-hydroxyvitamin D and plasma von Willebrand factor, D-dimer, P-selectin were:0.813(95%CI,0.761±0.865, P<0.05);0.861(95%CI,0.804±0.919,P<0.05);0.884(95%CI,0.848±0.920, P<0.05);0.810(95%CI,0.740±0.881,P<0.05);0.837(95%CI,0.760±0.915, P<0.05);0.850(95%CI,0.782±0.917,P<0.05);0.851(95%CI,0.786±0.916, P<0.05)。 lO.The Logistic regression analysis showed that the characteristics of the ROC curve of the multiple of the median(MoM) of several indicators combined together:①Combining serum sFlt-1, PIGF and25-OH-VD using Logistic regression analysis get an area under the curve of0.866(95%CI,0.822～0.909, P<0.05), and a sensitivity of62%at a specificity of90.6%.②Combining serum sFlt-1/PIGF and25-OH-VD using Logistic regression analysis get an area under the curve of0.773(95%CI,0.683～0.863, P<0.05), and a sensitivity of64%at a specificity of93.7%.③Combining plasma vWF, D-dimer and P-selectin using Logistic regression analysis get an area under the curve of0.861(95%CI,0.800～0.922,P<0.05), and a sensitivity of64%at a specificity of90.6%.④Combining serum sFlt-1and plasma P-selectin using Logistic regression analysis get an area under the curve of0.923(95%CI,0.873～0.973,P<0.05), and a sensitivity of92%at a specificity of90.6%.⑤Combining serum sFlt-1/PIGF and plasma P-selectin using Logistic regression analysis get an area under the curve of0.919(95%CI,0.861～0.976,P<0.05), and a sensitivity of84%at a specificity of90.6%.⑥Combining serum sFlt-1/PIGF and plasma D-dimer, P-selectin using Logistic regression analysis get an area under the curve of0.919(95%CI,0.862～0.977, P<0.05), and a sensitivity of70%at a specificity of93.7%.⑦Combining serum sFlt-1、PIGF and plasma D-dimer, P-selectin using Logistic regression analysis get an area under the curve of0.942(95%CI,0.889～0.994,P<0.05), and a sensitivity of94%at a specificity of90.6%.⑧Combining serum sFlt-1, PIGF,25-OH-VD and plasma vWF, D-dimer, P-selectin using Logistic regression analysis get an area under the curve of0.959(95%CI,0.930～0.989,P<0.05), and a sensitivity of78%at a specificity of100%. ⑨Combining serum sFlt-1/PIGF,25-OH-VD and plasma vWF, D-dimer, P-selectin using Logistic regression analysis get an area under the curve of0.918(95%CI,0.869～0.967, P<0.05), and a sensitivity of92%at a specificity of84.4%.[Conclusion]1.Hypertensive disorders complicating pregnancy is still a threat to maternal and perinatal health.There is lower incidence in our hospital.The proportion of mobile population is higher.Cesarean delivery is still the main mode of delivery of HDCP patients.Remained a major complications with HDCP patients,it is a emphasis on postpartum hemorrhage, placental abruption, preterm birth and fetal growth restriction.The need for increased perinatal care,the need for explored an ideal screening marker or method,so as to get reasonable and effective treatments before the clinical symptoms appear and achieve improved maternal and perinatal outcomes.2. sFlt-1, PIGF,25-hydroxyvitamin D, D-dimer, von Willebrand factor and P-selectin maybe involved in the pathogenesis of preeclampsia. In addition to vWF, the level of other biomarkers are associated with the patient’s condition, therefore, this biomarkers can be well used as to monitor the changes of the patient’s condition.3.Vascular endothelial injury and the imbalance of coagulation and fibrinolytic system maybe involve in the pathogenesis of preeclampsia.4.The level of sFlt-1and the ratio of sFlt-1/PIGF in preeclampsia group is significant higher than the control group, the level of PIGF in preeclampsia group is significant lower than the control group. There may be a vicious cycle between the imbalance of sFlt-1and PIGF that may participate in the pathogenesis of preeclampsia.5. Lower maternal25-hydroxyvitamin D levels that maybe involve in the pathogenesis of preeclampsia at first-trimester are associated with an increased risk of preeclampsia.6.1n pregnancies that subsequently develop preeclampsia maternal plasma levels of D-dimer, von Willebrand factor and P-selectin at first-trimeste are altered. These may be promising biomarkers to increase the predictive value of preeclampsia. 7.Combining screening of different biomarkers could increase the predictive value of preeclampsia.