Association Study Of Vascular Endothelial Growth Factor Gene And Polymorphisms Of Its Gene With Ectopic Pregnancy

BackgroundEctopic pregnancy (ectopic pregnancy, EP) is refers to the fertilized egg in the uterine cavity outside implantation pregnancy, belongs to the department of obstetrics and gynecology common acute abdomen illness. According to the different implantation site, it is could be divided into the fallopian tube pregnancy, ovarian pregnancy, abdominal pregnancy, cervical pregnancy and pregnancy in rudimentary horn, wherein the tubal pregnancy is the most common, accounting for more than 90%. Salpingitis, tubal operation history, IUD, assisted reproductive technology all can induce ectopic pregnancy, in recent years the incidence rate is rising up, the current occurrence rate is about 2% of the total number of pregnancy. Once the ruptured ectopic pregnancy, that can lead to intra-abdominal bleeding, directly threat to the patient’s health and life, which is the one of the important causes of maternal morbidity and mortality, early mortality accounted for about 9-13%. It Directly affects the prognosis of patients with ectopic pregnancy, about 1/3 of the patients with the first ectopic pregnancy were induced to infertility, another 1/3 can be induced to ectopic pregnancy, so early diagnosis of ectopic pregnancy is particularly important.At present, the diagnostic methods of ectopic pregnancy have the patients clinical manifestation, laboratory examination and auxiliary examination. The following concrete diagnostic method:ultrasonic examination after the patients serum P-HCG detection, the patients serum β-HCG detection after ultrasound examination, patients with blood progesterone, progesterone administered after detection of the blood of the patients with ultrasonography and serum P-HCG detection in patients with serum β-HCG after detection, ultrasonic examination after the patients serum β-HCG and blood progesterone, detection clinical examinations were repeated ultrasound examination, etc. Studies have shown that, after ultrasonic examination given serum β-HCG and patients with progesterone is the most effective, with high diagnostic accuracy. So that these methods most likely cause the missed diagnosis and misdiagnosis, patients from intrauterine pregnancy error scraping the possibility of very low, at the same time, examination time is very short, can save a lot of time for follow-up treatment of patients, help to improve the prognosis of patients. Li Cuimei proved that the accuracy rate of diagnosis of ectopic, detection of blood serum β-HCG, progesterone, detection of ultrasonographic endometrial thickness of pregnancy were 48.28%,72.41%,81.03%, and combined diagnosis detection, progesterone serum P-HCG detection, ultrasonographic endometrial thickness accuracy rate of 98-28%. Ruan Baohua et al research shows that detection, progesterone serum P-HCG detection, transvaginal ultrasonography package block type, is to determine the comprehensive index of the ectopic pregnancy conservative treatment is successful and has a high clinical value. The serum P-HCG detectionfor long time, sharp abdominal pain and vaginal bleeding disorders patients, non diagnostic method of choice, can be used as the preferred scheme of ultrasound diagnosis of diseases, the purpose is toguarantee the life safety of patients, it has higher clinical significance. The traditional diagnostic methods, laparoscopy and the postoperative pathological examination is the gold standard in the diagnosis of ectopic pregnancy by laparoscopy, diagnostic curettage and pathological tissue, is one of the effective methods in the diagnosis of ectopic pregnancy, but will cause significant trauma on patients. At present only in the diagnosis of spontaneous abortion and ectopic pregnancy will be used, diagnostic curettage is the most effective method to diagnose the disease, can make up for the short comings of the serological test. Abdominal CT and MRI examination while also can be used for the diagnosis of ectopic pregnancy, but the cost is expensive, not easily to accept for patients, so will not be the first choice in the diagnosis of asectopic pregnancy. Therefore,commonly used in clinical examination means for the gynecological B ultrasound and serum β-HCG detection to aid in the diagnosis of ectopic pregnancy, but the two examination have some limitations, such as urine human chorionic gonadotrop in (HCG) or serum human chorionic gonadotropin beta subunit (β-HCG) can only determine the pregnancy, but cannot determine the parts of pregnancy. In addition, because of individual differences from the last menstrual period to have anatomical markers reflect on ultrasonographic images will have a period of time interval has not yet formed ultrasound visible gestational sac, prone to stretch the blind area, neither is certainly cannot rule out intrauterine pregnancy, ectopic pregnancy. Especially for does not appear menopause, abdominal pain, vaginal bleeding and shock the common clinical manifestations of atypical ectopic pregnancy, more difficult to identify. So the most fatal ectopic pregnancy is due to delay in diagnosis and improper inspectionby. Despite the high resolution analysis of transvaginal ultrasonography and serum high sensitivity β-HCG quantitative diagnosis of the diseaserate is gradually increased, but there are still some patients with atypical symptoms and be missed, still early 40%-50% ectopic pregnancy may be misdiagnosed, severe cases can lead to patients in shock or even death. Because of this, it is particularly important to the early diagnosis of ectopic pregnancy. Therefore, early for auxiliary diagnosis index is simple, stable, high reliability becomes the current research on the inevitable.Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) also known as vascular permeability factor (vascular permeability factor, VPF) in 1989 by Ferrara in bovine pituitary follicular stellate cell culture medium by ammonium sulfate precipitation, heparin Sepharose affinity chromatography and two reverse phase high performance liquid chromatography a glycoprotein purified. VEGF was first isolated from tumor cells, the molecular weight of 35-45KD, is aheparin binding two mer its glycoprotein, glycoprotein monomers with two sulfur bond into two dimers have activity, is the role of tyrosine kinase signaling pathway. It is a specific mitogen of endothelial cells, is also an effective angiogenesis and vascular permeability inducing factor. The human VEGF gene is located in chromo some 6p21.3, length 14KB encoding VEGF gene, composed of 8 exons and 7 intronscomposed of alternating. VEGF gene transcription level after shear, canproduce 5 different transcripts, namely 5 kinds of isomers (VEGF206, VEGF189, VEGF165, VEGF123, VEGF121). In many isomers, VEGF 121, VEGF 145 and VEGF 165 could induce the proliferation an dangiogenesis of vascular endothelial cells to form, but with the highest activity of VEGF 165. VEGF exerts its biological effects by binding to its receptor. At present in the endothelial cell membrane were detected in two with combined with VEGF highly specific receptors:Fltl (fms like lyrosine kinase) and Flkl (fetal liver kinase, also known as KDR), which belongs to the type Ⅲ receptor tyrosine, Flt4 has also been confirmed as a VEGF receptor, its transmembrane protein extracellular domain has 7 similar to immunoglobulin functional areas, but the VEGF is not high on Flt4 specific.The biological role of VEGF:One, the role of cytoplasmic calcium accumulation, the direct interaction of VEGF selectively, on two kinds of Fltl and Flkl receptor, the biological effects of first see is increased cytoplasmic calcium ion, a few seconds can make the calcium ion concentration increases more than 4 times. Two, promote endothelialcell proliferation, VEGF is an endothelial cell specific mitogen, selectively, direct effects on two kinds of Fltl and Flkl receptors andcan induce the growth of monocyte. This is mainly VEGF chemotaxisinduced by stimulation and on vascular endothelial growth. Three, the induction of angiogenesis, angiogenesis is a process of primitive vascular plexus or existing vessels by sprouting or other means to form a new tube. VEGF is a specific promotion of vascular endothelial cells with mitosis, regulation of angiogenesis factor and angiogenesis induced by alone. Four, increased vascular permeability, VEGF has very strongly increasing vascular permeability, increased permeability ofvein and postcapillary venules, is found in one of the most intensesubstance increased vascular permeability. Research shows that the increased vascular permeability is achieved through the gap is increased by endothelial cells. Five,vascular protective effects of antithrombotic effect (1):VEGF can maintain the integrity of endothelial cells, to maintain the normal physiological function of endothelial cells,thus avoiding triggering endogenous or exogenous coagulation pathway; inhibition of platelet aggregation and adhesion, and thus playthe role of anti thrombosis. (2) the inhibition of vascular smooth muscle cells (SMC) of the excessive growth of protecting endothelial cells. (3)the anti injury and apoptosis of endothelial cells. (4) anti-inflammatory:inflammation is a defensive reaction to the position of circulating white blood cells and plasma protein extravasation into tissue damage as the characteristic, and leukocyte adhesion to the endothelial surface is thechemotactic migration, through the vessel wall and into the initial step ofthe inflammatory lesion. VEGF can make between leukocytes and endothelial function is abate, has anti-inflammatory effect. (5) the change of extracellular matrix (ECM):VEGF can induce endothelial cellexpression of plasminogen activator and plasminogen activator inhibitor-1 expression can also induce tissue factor, stromal collagenase inendothelial cells, stimulate Ⅷ factors released from endothelial cells.These effects can change the extracellular matrix, is conducive to the growth of blood vessels from sprouting around.As everyone knows, vascular endothelial growth factor (VEGF) is associated with angiogenesis, is a kind of important angiogenesis factor, is mainly produced by vascular endothelial cells. It is the specific effects on vascular endothelial cells, mitosis promoting factor, the main regulator of blood vessel growth, increase the uterine endometrium, decidua and trophoblast cell permeability, and prompted the embryonic development of the formation of blood vessels, play a key role in the formation of placenta and the implantation process, from fertilization, embryo implantation, placental formation, formation and fetal growth and development, its role throughout the. Early pregnancyac companied by the establishment of embryonic implantation and vascular between implantation, embryo and endometrium, vascular endothelial growth factor produced in great quantities. Vascular endothelial growth factor secretion and expression is not only induced by growth factors and cytokines, but also according to the condition of the time, such as hypoxia, studies have shown that can increase cellvascular endothelial growth factor produced under hypoxic conditions. Fallopian tube is the most common site of occurrence of ectopic pregnancy, uterine cavity due to structure different from the uterus, can not form a complete embryo implantation, decidualization, full communication and maternal blood can not, embryonic development in hypoxia, so the ectopic pregnancy increase vascular endothelial growthfactor production and secretion. While the trophoblastic tissueimplanted into the oviduct wall may damage the function of the fallopian tube, or by changing the ciliated epithelial cells, or caused by inflammatory reaction of the wall layer of the muscle tissue structure disorder, increased risk of ectopic pregnancy. It is believed that the function of the oviduct damage depends on trophoblast invasion of the tubal wall degree. And vascular endothelial growth factor concentration is high, may be more conducive to the trophoblastic tissue invasion of the tubal wall depth. Prospective study of Felemban shows, the serum VEGF with 200ng/L as cutoff point, the distinction between normal intrauterine pregnancy and ectopic pregnancy sensitivity of 88.10%, specificity of 100%, positive predictive value of 100%; the difference inectopic pregnancy with abnormal intrauterine pregnancy, the sensitivity was 87.15%, specificity of 75.10%, positive predictive value of 77.18%. Triple VEGF combined with pregnancy associated plasma protein A and P analysis can clearly distinguish the normal pregnancy and ectopic pregnancy. Torry DS et al found that, the expression of VEGF were detected from the oocyte to the blastocyst stage cells, and the implantation of the blastocyst in the prophase cells were expressed, suggesting that it may be related to the development and. In the VEGF and its receptor in early pregnancy is mainly expressed in cells and syncytiotrophoblast of trophoblast of leaf cells, indicating that VEGF infiltration, in trophoblast cell differentiation plays an important role in the. The embryo through the promotion of VEGF production led to the formation of femaleembryos between blood vessels in the implantation site. Shifren JL et al stimulated villous trophoblast with VEGF, found the villous trophoblast cell proliferation capacity was significantly strengthened, by the stimulation of VEGF cells on microporous membrane penetration to study the erosion and erosion forceextravillous trophoblast cells found also strengthened.Ectopic pregnancy is a common acute abdomen in Department of gynecology and obstetrics, the treatment includes expectant treatment, drug treatment and operation treatment. Early diagnosis creates conditions for conservative treatment, the current treatment tend to be conservative treatment. Some scholars believe that in the future, conservative treatment will become the preferred method of treatment of ectopic pregnancy, operation treatment will only consider as a remedy for conservative treatment at the time of failure. Natale et al believe that the higher activity of trophoblast cells, the easier to penetrate the wall of muscle, the opportunities of the fallopian tube rupture more increased. Normal pregnancy from implantation to form the placenta and the blood vessels are related to newborn. VEGF is a potent angiogenic factor, secreted by vascular endothelial cells, trophoblast cells and inflammatory macrophages, may be involved in the placenta formation of normal pregnancy and the invasion of the trophoblast cells, in early pregnancy, VEGF and its receptors mainly expressed on syncytiotrophoblast cells and trophoblast cells, which indicate that VEGF plays an important role in the trophoblast invasion and cell differentiation. The embryo lead to the formation of blood vessels between female and embryo through the promotion of VEGF production in the implantation site. Athanassiades et al found that the villous trophoblast cell proliferation capacity was improved obviously through VEGF stimulated villous trophoblast cells, also found the erosion force of villous trophoblast cells strengthened by VEGF stimulated cells on microporous membrane penetration. The of role VEGF 165 and VEGF121 is the same, this effect is blocked anti VEGF antibody, it suggested that VEGF could promote the dual role of extravillous trophoblast cells and the proliferation of erosion.With the development of molecular biology and genetics, people gradually realize that gene polymorphism in various diseases role. Gene polymorphism refers to the presence of more than two kinds ofallele in a population in which the gene frequency, lowest than mutationcan sustain high (i.e. frequency is greater than 1%). Its essence iscaused by various reasons of chromosome DNA in nucleotide sequence change, the change of protein expression may exist up or down effect,with enhanced or reduced role on the cytokines in the control of gene,its in explaining human on disease risk plays an important role in. The study found that there are some polymorphisms in the VEGF gene,which can affect differences in genetic predisposition to disease of the individual through the transcriptional level and protein changes in the expression of VEGF. VEGF gene is located on chromosome 6p21.3,14KB long, and composed of 8 exons and 7 introns composed of alternating, there are at least 30 single nucleotide polymorphisms. The gene that encodes human VEGF has been cloned and mapped to chromosome 6 and comprises a 14-kb coding region with eight exons.17 Many polymorphisms have been reported in the promoter and the 5’- and 3’-untranslated regions (UTRs) of the VEGF gene. These polymorphisms are associated with various diseases, including recurrent pregnancy loss, pre-eclampsia, and preterm delivery. There are several observations that support the participation of the VEGF in the development of ectopic pregnancy, besides the fact that several patients do not present any risk factor for this disease. Our intention was to study if the functional polymorphism within the promoter and UTR of the VEGF gene may influence the risk of ectopic pregnancy.Serum VEGF level in patients with ectopic pregnancy is higher than that in normal pregnancy, but serum VEGF level in patients with ectopic pregnancy whether is helpful in differentiating ectopic pregnancy abortion or diapause, whether it can predict the occurrence of rupture of ectopic pregnancy, abortive ectopic pregnancy whether was adopted in expectant treatment or conservative treatment of on monitoring condition of chorionic gonadotropin and VEGF, So far there is no relevant reports. According to the appropriate clinical indicators, diagnosis, treatment, prevention of ectopic pregnancy has became the future direction of development of reproductive health, that also is an important problem to be resolved by the obstetrician.The purpose of this study is to investigate the association study of vascular endothelial growth factor gene and polymorphisms of its gene with ectopic pregnancy, and provide guidance for ectopic pregnancy diagnosis, treatment, prevention and other existing problems. This study is divided into diagnosis, treatment, prevention of three chapters:the clinical value of vascular endothelial growth factor in the diagnosis of ectopic pregnancy (the first chapter); vascular endothelial growth factor and P-human chorionic gonadotropin are associated with trophoblastic invasion into the tubal wall in ectopic pregnancy (the second chapter); association study of vascular endothelial growth factor gene polymorphisms with ectopic pregnancy (the third chapter).The first chapter The clinical value of vascular endothelial growth factor in the diagnosis of ectopic pregnancyObjective Clinically, conventional serum β-HCG, progesterone and B ultrasound examination were used in diagnosing ectopic pregnancy. But the two examinations all have certain limitation. Early 40%-50% ectopic pregnancy still may be misdiagnosed. VEGF plays an important role in fertilization, implantation, placental angiogenesis and fetal development. Ectopic pregnancy patients with serum VEGFconcentration was higher than the normal intrauterine pregnancy. In this study, by comparing the differences between ectopic pregnancy and normal pregnancy women in serum VEGF, P-HCG and progesterone levels, to explore the clinical value of vascular endothelial growth factor in the diagnosis of early ectopic pregnancy. Method Normal pregnancy group includes 186 cases, ectopic pregnancy includes 192 cases. Serum P-HCG and P was determined by double antibody sandwich photochemical, ELISA method was used to determine the concentration of serum VEGF. The data were analyzed and made ROC curve by using SPSS 13.0 software packet.Result1、The serum concentrations of β-HCG and P in normal intrauterine pregnancy were higher than in ectopic pregnancy, there was the significant difference(P<0.01). The serum concentrations of VEGF in ectopic pregnancy were higher than in normal intrauterine pregnancy, there was the significant difference(P<0.01).2、The concentration of serum VEGF and β-HCG、P concentrations were negatively correlated, the correlation coefficients were -0.556 and-0.532 (P<0.01); there is a positive correlation of between serum β-HCG concentration and P concentration, the correlation coefficient was 0.735(P<0.01).3、In the ROC curve of diagnosis tubal pregnancy and normal intrauterine pregnancy, the area under the curve about VEGF, β-HCG, P were 0.955,0.866,0.835, area under the curve is greater about VEGF than β-HCG and P, which suggested that serum VEGF in the differential diagnosis of tubal pregnancy with normal intrauterine pregnancy may be superior to P-HCG and p.4、When the concentration of serum VEGF was 160Pg/ml, Youden index 0.79 was the maximum value, the sensitivity was 84.90%, specificity was 94.09%, the positive and negative likelihood ratio were 14.37 and 0.16. The optimal operating point of serum VEGF in differential diagnosis of ectopic pregnancy was 160Pg/ml.Conclusion The serum concentrations of β-HCG and P in ectopic pregnancy were lower than in normal intrauterine pregnancy, but the serum concentrations of VEGF in ectopic pregnancy were higher than in normal intrauterine pregnancy. Serum VEGF has a certain value, the distinction between ectopic pregnancy and normal intrauterine pregnancy in the early diagnosis of ectopic pregnancy, that is expected to become a new auxiliary index detection of ectopic pregnancy. The sensitivity, specificity and accuracy of combined detection of β-HCG, VEGF and progester increased significantly, the detection effect is better than that of single index.The second chapter Vascular endothelial growth factor and β-human chorionic gonadotropin are associated with trophoblastic invasion into the tubal wall in ectopic pregnancyObjective Ectopic pregnancy is a common acute abdomen in the department of gynecology and obstetrics, the treatment includes expectant treatment, drug treatment and operation treatment. Early diagnosis creates conditions for conservative treatment, the current treatment tend to be conservative treatment. Some scholars believe that in the future, conservative treatment will become the preferred method of treatment of ectopic pregnancy, operation treatment will only consider as a remedy for conservative treatment at the time of failure. Natale et al believe that the higher activity of trophoblast cells, the easier to penetrate the wall of muscle, the opportunities of the fallopian tube rupture more increased. It is believed that impairment of tubal function depends on the degree of invasion of the trophoblast into the tubal wall. There are still no adequate criteria for the prediction of the depth of invasion of trophoblastic tissue into the tubal wall. Maternal serum β-HCG concentration seems to be related to the depth of trophoblastic penetration into the tubal wall in ampullary pregnancies. Higher VEGF concentrations may permit deeper invasion of trophoblastic tissue into the tubal wall. The aim of the present study was to assess the association between the depth of trophoblastic penetration into the tubal wall and serum concentrations of both VEGF and p-HCG in patients affected by ampullary pregnancy.Method This was a prospective study. Patients with a diagnosis of tubal pregnancy in the ampullary region with radical surgical treatment (salpingectomy), and measurement of serum VEGF and serum β-HCG on the day of surgery, which were 80 cases. Cases in which there was no agreement regarding the location of the tubal pregnancy upon surgical description and histological analysis were excluded. A total 5ml of blood was withdrawn and the blood samples were centrifuged and lml double antibody sandwich photochemical was mearsured for P-HCG. The rest were stored at -86 ℃ until VEGF was measured by enxyme-linked immunosorbent assay (ELISA). The fallopian tubes were fixed in 10% formalin and sectioned serially for light-microscopic analysis. An average of 10 sections stained with hematoxylin-eosin was analyzed. To facilitate the identification of tissue invaded by the trophoblast, histological material was also stained with Masson’s trichrome to identify muscular fibers. Immunohistochemical staining for human placental lactogen (hPL) was performed to identify intermediate trophoblast and determine the depth of trophoblastic invasion into the tubal wall. Ampullary pregnancies were classified histologically according to the depth of trophoblastic infiltration into the tubal wall: stage I, trophoblastic infiltration limited to the tubal mucosa; stage II, trophoblastic infiltration extended to the tubal muscularis; stage III, complete tubal wall infiltration with or without rupture of the serosa. The data were analyzed and made ROC curve by using SPSS 13.0 software packet. P<0.05 was considered statistically significant.Result1、The age of the patients ranged from 19 to 41 years (29.1± 5.8 years), and there was no significant difference in mean maternal ages among the three histological groups (stage Ⅰ,29.2±5.9; stage Ⅱ,29.7±5.6; stage Ⅲ,28.5±6.0; P=0.774). Histological analysis showed that 27 patients (33.7%) had stage Ⅰ tubal infiltration,24 (30.0%) had stage Ⅱ, and 29 (36.3%) had stage Ⅲ. The gestational age ranged from 41 to 55 days (48.0±3.9 days), and there was no significant difference in mean gestational ages among the three histological groups (stage Ⅰ,48.3±4.3; stage Ⅱ,47.9±3.6; stage Ⅲ,47.7 ±4.0; P=0.848).2、One-way ANOVA revealed a significant difference in the VEGF concentrations (stage Ⅰ,218.77±67.37; stage Ⅱ,392.50±43.62; stage Ⅲ, 452.50±56.67; P<0.001). The mean value of serum VEGF in patients with stage Ⅰ tubal infiltration was significantly lower than that in stage Ⅱ (P<0.001), which was significantly lower than that in stage Ⅲ (P<0.001).3、One-way ANOVA revealed a significant difference in the β-HCG concentrations (stage Ⅰ,2139.9±1058.8; stage Ⅱ,9103.1±4126.3; stage Ⅲ, 14336.4±4116.6; P<0.001). The mean value of serum β-HCG in patients with stage Ⅰ tubal infiltration was significantly lower than that in stage Ⅱ (P<0.001), which was significantly lower than that in stage Ⅲ (P<0.001).4、Using the ROC curve, the serum VEGF level that best differentiated stage Ⅰ from stage Ⅱ trophoblastic invasion was 308.6 pg/mL. The positive likelihood ratios were 13.5, accuracies were 96.1%, sensitivities were 100.0%, specificities were 92.6%, positive predictive value (PPV) were 92.3%, and negative predictive value (NPV) were 100.0% according to the cutoff scores; a serum VEGF level of 431.9 pg/mL best differentiated stage Ⅱ from stage Ⅲ infiltration. The positive likelihood ratios were 3.8, accuracies were 79.3%, sensitivities were 79.3%, specificities were 79.2%, positive predictive value (PPV) were 82.2%, and negative predictive value (NPV) were 76.0% according to the cutoff scores.5% Using the ROC curve, the serum P-HCG level that best differentiated stage I from stage II trophoblastic invasion was 2509.6mIU/mL. The positive likelihood ratios were 5.0, accuracies were 86.3%, sensitivities were 91.7%, specificities were 81.5%, positive predictive value (PPV) were 81.5%, and negative predictive value (NPV) were 91.7% according to the cutoff scores; a serum P-HCG level of 13142.6mIU/mL best differentiated stage Ⅱ from stage III infiltration. The positive likelihood ratios were 17.2, accuracies were 83.0%, sensitivities were 72.4%, specificities were 95.8%, positive predictive value (PPV) were 95.4%, and negative predictive value (NPV) were 74.2% according to the cutoff scores.Conclusion The depth of trophoblastic penetration into the tubal wall is associated with maternal serum VEGF and β-HCG concentration. VEGF and β-HCG concentration is more higher, trophoblastic tissue infiltration of fallopian tube wall is more deeper.The third chapter association study of vascular endothelial growth factor gene polymorphisms with ectopic pregnancyObjective Vascular endothelial growth factor involved in the formation and development of ectopic pregnancy, ectopic pregnancy patients with and without any of these risk factors. We hypothesized thatvascular endothelial growth factor gene polymorphism may be associated with ectopic pregnancy risk related. At present, there are few domestic research reports, this area therefore, our objective was toinvestigate the expression of vascular endothelial growth factor (-460C/T, -1154G/A,-2578C/A,+936C/T) genetic polymorphisms in riskand ectopic pregnancy and provide experimental basis at molecular level for the prevention and treatment of ectopic pregnancy.Method This was a case-control study wherein 192 women with a history of ectopic pregnancy were compared to 210 post-menopausal controls with two pregnancies and no ectopic pregnancy for the genotyping of VEGF polymorphisms. Five ml of venous blood from each subject was drawn in vacutainer tubes. The white blood cell DNA was extracted by using proteinase K-digestion followed by a salting out procedure. Genotyping of the VEGF gene polymorphisms at -460C/T,-1154G/A,-2578C/A and +936C/T were performed by polymerase chain reaction-restriction fragment length polymorphism. Statistical analysis was performed using the Statistical Package for the Social Sciences software package (version13.0; SPSS, Chicago, IL, USA). Hardy-Weinberg equilibrium (HWE) analysis was performed to compare the observed and expected genotype frequencies using the Chi-square test. The data of age and amenorrhea weeks in the case group were presented as mean±SD. Comparison of the VEGF -460C/T,-1154G/A,-2578C/A and +936C/T genotype distributions in the study groups was performed by means of two-sided contingency tables using Chi-square test. The VEGF -460C/T,-1154G/A,-2578C/A and +936C/T haplotype frequencies and linkage disequilibrium coefficient were estimated using the EH linkage software (version 1.2, Rockefeller University, New York) and 2LD program, respectively. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model. A probability level of 5% was considered significant.Result1、The distributions of the VEGF -460C/T,-1154G/A,-2578C/A and +936C/T genotypes in the control groups did not significantly deviate from that expected for a Hardy-Weinberg equilibrium (all P value> 0.05).2、The frequencies of the VEGF -460C/T C and T allele among endometriosis patients and healthy controls were 21.6%,21.2% and 78.4%,78.8%, respectively; No significant difference in the -460C/T allele distribution was shown between ectopic pregnancy patients and controls (P>0.05). The distribution of the C/C, C/T and T/T genotypes between ectopic pregnancy patients (3.6%,35.9% and 60.4%, respectively) and controls (5.2%,31.9% and 62.9%, respectively) also had no significant difference (P>0.05). Compared with the T/T genotypes, the genotypes of -460C/C+C/T did not significantly influence the risk of EP (OR=1.11,95%CI= 0.74-1.66).3-. The frequencies of the VEGF +936C/T C and T allele among endometriosis patients and healthy controls were 81.0%,83.8% and 19.0%,16.2%, respectively; No significant difference in the +936C/T allele distribution was shown between ectopic pregnancy patients and controls (P>0.05). The distribution of the C/C, C/T and T/T genotypes between ectopic pregnancy patients (65.1%,31.8% and 3.1%, respectively) and controls (69.5%,28.6% and 1.9%, respectively) also had no significant difference (P>0.05). Compared with the C/C genotypes, the genotypes of +936C/T+T/T did not significantly influence the risk of EP (OR=1.22,95%CI=0.81-1.86).4^ The genotype frequencies of the VEGF-1154 A/A, G/A and G/G in cases and controls were 2.1%/6.2%,26.6%/33.8% and 71.4%/60.0%, respectively; the A and G allele frequencies in the two groups were 15.4%/23.1% and 84.6%/76.9%, respectively. There was a significant difference in genotype and allele distributions of the VEGF-1154G/A between two groups (P=0.021 and 0.006, respectively). Compared with the G/G genotype, the A/A+G/A genotype could significantly decrease the risk of developing EP (OR=0.61, 95%CI=0.42-0.87).5^ The genotype frequencies of the VEGF -2578 A/A, C/A and C/C in cases and controls were 3.6%/9.1%,31.3%/35.7% and 65.1%/55.2%, respectively; the A and C allele frequencies in the two groups were 19.4%/26.9% and 80.6%/73.1%, respectively. There was a significant difference in genotype and allele distributions of the VEGF -2578C/A between the two groups (P=0.034 and 0.010, respectively) (Table II). Compared with the C/C genotype,...