| Hypertension disorder complicating pregnancy(HDP) is a major disease which seriously endangers the safety of mother and fetal during pregnancy. The pathogenesis of HDP is not yet clear. Researchers have generally agreed that the occurrence of HDP is a multi-factor and multi-channel common disease, and its central pathogenesis is the systemic activation and injury of maternal endothelial cells which bring about a variety of clinical symptoms such as high blood pressure, protein urine, systemic inflammatory response and accumulation of anti-angiogenic factors. The mechanism of endothelial activation and injury is unknown, but some scholars have pointed out that the endothelial damage in HDP patients had existed during early pregnancy. The existence of endothelial activation prevent the invasion of trophoblast into uterine spiral arteries which leads to the shallow placental implantation, placental ischemia and hypoxia. These result in the synthesis and the release of too much "toxic factor" into the loop, directly or indirectly damage maternal endothelial cell function. The "toxic factor" which had been found out included tumor necrosis factor, fm-like tyrosine kinase 1 (sFlt-1) and endoglin (sEng), syncytiotrophoblast particles (STBM). A lot of studies had been done however anyone of them can predict the occurrence and development of disease. Therefore, we assume that if there is a way to assess the endothelial damage and determine the extent of disease and development, it is possible to give a reasonable prevention and treatment which is great significance to the clinical diagnosis and treatments of HDP.Neutrophil gelatinase-associated lipocalin (NGAL) is a newly discovered lipoprotein which can promote the growth and differentiation of many cells and proteolytic and degradation of collagen in organization. Studies have pointed out that NGAL is closely related with endothelial injury. The lever of NGAL had been elevated when the inflammation and tumor happened. Numerous studies confirm that NGAL is an early indicators of renal failure and acute inflammation, with a high degree of sensitivity and specificity. Furthermore, a related study found that the level of serum NGAL in the patients with hypertension is significant increase than the normal group. In view of both endothelial injury in pre-eclampsia, high blood pressure and kidney impairment characteristics, Italian scholar D'ANNA speculated that NGAL may be related to the pathogenesis of preeclampsia and the increase of NGAL in the blood during second trimester pregnancy as a sensitive indicator of early occurrence to predict preeclampsia by detecting serum NGAL levels in 48 patients with preeclampsia at 24 weeks-26 weeks in 2008. The result of this study revealed the relationship between NGAL and preeclampsia, however the expression of NGAL in patients with preeclampsia in third trimester, the level of NGAL in hypertensive pregnancy and the expression of NGAL in urine and placental tissue were still unknown.The aim of our study is to explore the expression of NGAL in third trimester of HDP patients by detecting NGAL in blood, urine and placental tissue and explore the relationship between NGAL and disease severity by combined with the clinical features. We hope to find an effective indicator which can determine the extent of disease fast and simply, so we can give the early stage of disease intervention and treatment, delay the occurrence and development of the disease, monitor the efficacy of treatment and reduce the incidence of high-risk pregnancies and improve the prognosis of mother and fetal.CHAPTER 1 THE EXPRESSION OF NGAL IN HDP PATIENTSOBJECTIVEThere were varying degrees of systemic inflammatory response and endothelial damage in HDP patients. we detected the expression of NGAL in their blood, urine and placental tissue in order to explore the relationship between NGAL and systemic endothelial damage and have a preliminary understanding of different level of NGAL in three types of HDP patients.METHODS1. Group divided:There are 38 HDP patients which selected from the hospitalization of pregnant women in the Southen Hospital and the Maternal and Child Health Hospital in Shenzhen from March to Decsember in 2009, including 9 cases of gestational hypertension(HP),7 cases of mild pre-eclampsia (MP),10 cases of severe pre-eclampsia group(SP) and 12 cases of early-onset preeclampsia (EOPE). The combination of MP group and SP group are called late-onset pre-eclampsia group (LOPE). There are 30 cases of normal pregnant women as control group. All groups were excepted for history of chronic hypertension, diabetes, infections, nephritis,lupus, spontaneous premature rupture of membranes, FGR, fetal malformation and assisted reproductive technology.2. Specimen collection:We need 3ml venous and 3ml urinary within 24 hours. The placenta we need was about 2.0cmx1.0cmx0.2cm which were taken from the maternal surface of placental and was cut into two parts. The venous, urinary and one part of placental were processed as required and then put into -80℃refrigerator. The other part was saved into the 4% formaldehyde in 4 degrees preservation.3. NGAL test:ELISA assay was used to detect the expression of blood and urine NGAL and immunohistochemical method and Westen Blot were used to detecte the expression of NGAL in human placenta.4. We use SPSS 13.0 software to analyse the results and p<0.05 is statistically significant difference.RESULTS1. The expression and comparison of serum and urine NGAL between two groups: The serum NGAL in HDP group was 567.3348±195.9752mg/L and the urine NGAL in HDP group was 113.9165±40.4284mg/L. The serum NGAL in normal group was 136.1140±28.5699mg/L and the urine NGAL in normal group was 75.1449±10.1471mg/L. There were significantly difference in serum NGAL (F=50.748, P=0.000) and urine NGAL (F=49.682, P=0.000) between two groups, the level of blood and urine NGAL in HDP group had positive correlation and the correlation coefficient was 0.8 (p=0.000)2. The expression and comparison of placenta between two groups: Immunohistochemical results showed that expression of NGAL in HP group was (+), the MP group was (+～++). the SP group was (+++) and the EOPE group was (+++). The expression of NGAL in the normal control group was negative. Westen blot results showed that there was a positive result in HDP group.CONCLUSIONCompared with the normal pregnant women, the HDP patients had a high level of NGAL in serum, urine and placenta. There was a positive relationship between serum and urine NGAL in case group. OBJECTIVEBecause NGAL was associated with endothelial injury, we detect the serum and urine NGAL levels of different types of HDP patients in order to explore whether the severity of illness was related to NGALMETHODS1. ELISA assay was used to detect the blood and urine NGAL level of HDP patients.2. Clinical features of patients were collected:(1) mothers age, gestational age, course of disease.(2) clinical symptoms:blood pressure, signs and symptoms. (3) auxiliary examination:blood routine, urine routine, liver and kidney function, 24-hour urine protein.3. Statistical analysis:SPSS 13.0 was used to analyse. Multiple comparison was used in each two groups and pearson correlation was used to analyse the HDP clinical characteristics and NGAL levels. Spearman rho was used to analyse the NGAL level and the severity of disease in HDP patients.RESULTS1. The expression and comparison of serum and urine NGAL in three different stages of HDP patients:The serum NGAL in HP group was 377.0352±50.2905mg/L and the urine NGAL in HP group was 89.3235±10.6441mg/L. The serum NGAL in MP group was 373.1072±51.2086mg/L and the urine NGAL in MP was 88.7220±6.0632mg/L. The serum NGAL in SP group was 600.0151±30.5950mg/L and the urine NGAL in SP was 105.2433±32.7259mg/L.The serum NGAL was significant different in three groups except the comparison of Hp group and MP group (P>0.05).The urine NGAL was not different among groups (P>0.05) 2. The relationship between the NGAL and the clinic feature:The serum NGAL levels was significant interrelated to high blood pressure and urine protein in HDP group. The correlation coefficient of systolic blood pressure and serum NGAL was 0.751 (p=0.000) and the correlation coefficient of diastolic blood pressure and serum NGAL was 0.737 (p=0.000). The correlation coefficient of random urine protein and serum NGAL was 0.554 (p=0.000) and the correlation coefficient of 24-hour total urinary protein and serum NGAL was 0.472 (p=0.004). The urine NGAL levels was also interrelated to high blood pressure and urine protein in HDP group. The correlation coefficient of systolic blood pressure and urine NGAL was 0.471 (p=0.000) and the correlation coefficient of diastolic blood pressure and urine NGAL was 0.466 (p=0.000). The correlation coefficient of random urine protein and urine NGAL was 0.524 (p=0.001) and the correlation coefficient of 24-hour total urinary protein and urine NGAL was 0.386 (p=0.022)3. The relationship between the NGAL and the severity of disease:Through the comparison of NGAL level in HDP patients we find that NGAL changes along with the increased severity of the disease. The correlation coefficient of serum NGAL and disease severity was 0.934 (p=0.000) and the correlation coefficient of urine NGAL and disease severity was 0.693 (p=0.000). The serum and urine NGAL were associated with disease severity and the serum NGAL was better.CONCLUSIONThe expression of NGAL in serum and placental tissue in different types of HDP patients is different. NGAL was related to the elevated blood pressure and urine protein, so there was a significant important correlation between the NGAL and severity of the disease. It is a good way to detect the serum NGAL to predict the occurrence and development of the disease, monitoring disease severity and treatment effect. So the patients can get their treatment as earlier as possible.CHAPTER 3 THE CORRELATION ANALYSIS OF NGAL AND THE CLINIC FEATURE OF EOPE PATIENTSOBJECTIVEEOPE is a special type of HDP, because of its earlier onset, more severe clinic feture and complications and higher mortality, the main treatment of EOPE is forced termination of pregnancy as a result of blood pressure and urine protein in progressive increase. We want to detect the NGAL in EOPE patients and explore the relation between the occurrence and development of EOPE and NGAL expression in order to find a reliable indicator to monitor disease progression and therapeutic effect.METHODS1. Group divided:All the pre-eclampsia patients were selected from the hospitalization of pregnant women in the Southen Hospital and the Maternal and Child Health Hospital in Shenzhen from March to Decsember in 2009. We divided the group on the basis of the onset time of disease. The onset of disease was less than 34 weeks belongs to the EOPE group(n=12) and the other belongs to the LOPE group(n=17). There are 30 cases of normal pregnant women as control group. All groups were excepted for history of chronic hypertension, diabetes, infections, nephritis,lupus, spontaneous premature rupture of membranes, FGR, fetal malformation and assisted reproductive technology.2. The specimen collection just like as chapter one.3. NGAL test:ELISA assay was used to detect the expression of blood and urine NGAL and immunohistochemical method and Westen Blot were used to detecte the expression of NGAL in human placenta. RESULTS1. The expression and comparison of serum and urine NGAL in EOPE and LOPE patients:The serum NGAL in EOPE group was 796.1254±115.9243mg/L and the urine NGAL in EOPE group was 154.2859±42.1187mg/L. The serum NGAL in LOPE group was 567.3348±195.9752mg/L and the urine NGAL in LOPE was 113.9165±40.4284mg/L. The serum and urine NGAL was significant increase in EOPE group (p=0.000)2. The expression of NGAL in placenta of EOPE patients had been significantly increased in HDP patients.CONCLUSIONThe abnormally high expression of NGAL in EOPE patients is consistent with abnormally clinical characteristics of high blood pressure and urine protein. It may prompt that there was serious dysfunction of endothelial cells and the systemic inflammatory response in EOPE patients. Therefore, NGAL is also important to identify disease severity in EOPE patients.CHAPTER 4 THE APPLICATION OF NGAL IN CLINICAL DIAGNOSIS AND TREATMENT OF HYPERTENSIVE DISORDERS IN PREGNANCYOBJECTIVEThe early study have found that NGAL was overexpression and related to the severity of the disease in hypertensive disorders in pregnancy patients. The next step we intend to quantify the role of NGAL in the development of hypertensive diseases during pregnancy can improve a reliable basis for predicting, monitoring disease progress and treatment.METHODS1. ROC curve analysis. 2. Establishing disease severity prediction formula by stepwise discriminant analysis3. Three test are used to verify the validity of the formula.RESULTS1. ROC results:Area under the curve of blood NGAL as 1 (p=0.000,95% CI 1.0 to 1.0) and the cutting plot was 88.582mg/L; Area under the curve of urine NGAL as 0.874 (p=0.000,95% CI 0.791～0.957) and the cutting plot was 52.512mg/L.2. Disease severity prediction formulaFirst step:Function 1=X·(-0.028)+Y·0.021+(-2.680) Function 2=X·0.103+Y·0.008+(-30.280)△X(days) is the first time of disease onset and Y(mg/L) is the serum NGAL.Second step:HP-Function 1<0, Function 2<0. MP-Function 1<0, Function 2>0. SP-Function 1>0.2. TestThe accuracy of original test is 100%. The accuracy of cross-validated test is 96.2%. The accuracy of forward-looking test is 66.67%CONCLUSIONThe NGAL is good for diagnosis of hypertensive disorders in pregnancy. The establishment of discriminant formula which was based on the clinical characteristics and NGAL was used to assess the severity of disease and monitor the progression of disease and clinical therapy.SUMMARY1. NGAL exists in the blood, urine, and placental tissues in the third trimester of HDP patients. Compared to the normal pregnant women the expression of NGAL is significantly increased in HDP patients. There is a significant relationship between the serum NGAL and urine NGAL. 2. The NGAL level in different stages of HDP patients is significant different. NGAL was related to the elevated blood pressure and urine protein, so there was a significant important correlation between the NGAL and severity of the disease.3. The abnormally high expression of NGAL in EOPE patients is consistent with abnormally clinical characteristics of high blood pressure and urine protein. It may prompt that there was serious dysfunction of endothelial cells and the systemic inflammatory response in EOPE patients. The detecting of serum NGAL can predict the disease severity and kidney injury of EOPE patients.4. The NGAL is good for diagnosis of hypertensive disorders in pregnancy. The establishment of discriminant formula which was based on the clinical characteristics and NGAL was used to assess the severity of disease and monitor the progression of disease and clinical therapy.5. NGAL plays an important role to the occurrence and development of HDP. The serum NGAL can screen and predict the severity of the disease. The urine NGAL can help diagnose renal dysfunction in pregnancy as a reference factors. The NGAL in placenta may be associated with the occurrence of preeclampsia and can be used as identification in chronic hypertension/chronic nephritis complicated with preeclampsia.
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