VEGF及其可溶性受体sFlt-1与复发性流产的相关性研究
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摘要
研究背景
原因不明复发性流产(Unexplained Recurrent Spontaneous Abortion, URSA)是指与同一性伴侣连续发生2次或2次以上的自然流产,且不存在染色体、解剖、内分泌和自身免疫功能异常以及生殖道感染等病因。URSA的发生机制非常复杂,是妇产科领域一个很有临床价值的研究课题。近年来研究表明,许多不明原因复发性流产的发生与胎盘组织中血管增生平衡和胎儿血液供应不足有密切关系,血管内皮生长因子(vascular endothelial factor, VEGF)是最主要的促进血管形成的血管形成增生调节因子,在保持蜕膜组织中血管密度、有效血流量和维持胎儿营养供应上起重要作用。成功的妊娠依赖于胎盘良好的发育和生长,在胎盘植入和胎盘形成的过程中,血管的生成对于成功妊娠是非常关键的。sFlt (Soluble fms-like tyrosine kinase)为血管内皮生长因子受体的一种,由于它仅有胞外配体结合区,保留了与VEGF高亲和力的特性,其与VEGF结合后抑制其促血管生长作用。sFlt-1在先兆子痫、肿瘤等疾病中的作用已有较多研究成果,但在复发性流产的研究罕见。本研究探讨VEGF及sFlt-1与不明原因复发性流产发病的相关性及研究VEGF基因多态性与广西地区壮族人群复发性流产的相关发病风险,进一步阐明URSA发病机制,为URSA诊治提供理论基础及治疗前景。
第一部分VEGF及其可溶性受体sflt-1与妊娠相关性研究
目的检测正常未孕妇女及正常早孕妇女,不明原因复发性流产未孕妇女及不明原因复发性流产早孕妇女外周血中的sFlt-1及VEGF水平,探讨sFlt-1及VEGF与妊娠相关性；动态检测VEGF及sFlt-1在正常孕早期不同孕周外周血浓度,探讨sFlt-1及VEGF在孕早期不同孕周的浓度变化。
方法选择正常未孕妇女、不明原因复发性流产未孕妇女及正常早孕妇女各50例,应用ELISA法检测各组外周血sFlt-1及VEGF,探讨sFlt-1及VEGF与妊娠相关性。正常妊娠妇女妊娠确诊后分别抽取孕4-6周；孕7-8周；9-10周；211-12周外周血,应用ELISA法监测不同孕周sFlt-1和VEGF水平浓度,探讨sFlt-1及VEGF在孕早期不同孕周浓度变化。
结果
1.不明原因复发性流产未孕组VEGF及sFlt-1水平分别为242.8±236.52026及878.56±837.1065,正常未孕组VEGF及sFlt-1水平分别为253.8277±137.0413及1587.001±1678.2486。两组VEGF及sFlt-1水平无显著性差异,p>0.05。
2.正常早孕组与正常未孕组sFlt-1水平分别为3631.742±3000.0891；1587.001±1678.2486,正常怀孕组sflt水平高于正常未孕组,两者间差异有显著性差异(P<0.01),；正常早孕组与正常未孕组VEGF水平分别为969.64±696.9974；253.8277±137.0413,正常怀孕组VEGF水平高于正常未孕组,两者间差异有非常显著性差异(P<0.01)。
3.正常孕早期,VEGF及sFlt-1在孕4-6周血清浓度高,特别是孕4周(2256.50±1995.09ng/L；11796.67±9916.08ng/L),随后下降波动在一定水平。
结论
VEGF及sFlt-1与妊娠有相关性,妊娠确诊即能检测到VEGF.sflt-1的表达,说明在绒毛发育早期,VEGF及sflt-1即可发挥作用,对整个妊娠进行调节,妊娠后两者水平升高；在孕早期,胚胎刚着床滋养细胞侵入浅,相对缺氧,导致sFlt-1及VEGF水平升高,VEGF及sFlt-1在孕4-6周血清浓度高,随着妊娠周数增加,滋养细胞逐渐侵入,相对缺氧现象逐渐改善,sFlt-1及VEGF水平下降至一定水平。
第二部分VEGF及其可溶性受体sflt-1与不明原因复发性流产相关性研究
目的研究URSA患者中VEGF及其可溶性受体sflt一1血清浓度及在绒毛组织中蛋白及基因表达情况,探讨VEGF及其可溶性受体sflt-1与复发性流产的相关性。
方法前瞻性观察1124例不明原因复发性流产再孕患者,有35例URSA组患者胚胎停育,以35例正常早孕行人工流产术妇女的血清及绒毛作为对照组,应用ELISA法及免疫组织化学方法观察35例URSA患者胚胎刚停育(胚胎停育组)血清及绒毛中VEGF及sflt-1的蛋白表达情况；应用RT-PCR技术检测30例不明原因复发性流产患者及30例正常早孕妇女绒毛组织中的sFlt-1mRNA及VEGF-mRNA表达水平。
结果
1.对照组外周血中VEGF的水平为982.67±675.47ng/L:URSA组外周血中VEGF的水平为2637.50±2145.09ng/L.外周血中VEGF水平URSA组较对照组高,两者间差异有非常显著性差异(P<0.01)。
2.对照组外周血中sFlt-1的水平为3540.67±2989.80ng/L:URSA组外周血中sFlt-1的水平为13796.67±11917.08ng/L,外周血中sFlt-1水平URSA组较对照组高,两者间差异有非常显著性差异(P<0.01)。
3.不明原因复发性流产胎停育组绒毛组织中VEGF及sflt-1阳性染色(分别为81.25%和84.38%)较正常早孕组(分别为22%和28%)强,URSA组较正常早孕绒毛组织中VEGF及sflt-1蛋白表达水平高,两者间差异有显著性(P<0.05)。
4.不明原因复发性自然流产组和正常早孕组绒毛组织中VEGF mRNA及sFlt-1mRNA表达的检测,不明原因复发性自然流产组较正常早孕组绒毛组织中sFlt-1mRNA表达高(分别为38.83±2.64及28.53±1.70),两者间差异有显著性差异(P<0.01)；不明原因复发性自然流产组较正常早孕组绒毛组织中sFlt-1mRNA表达高(分别为38.83±2.64及28.53±1.70),两者间差异有显著性差异(P<0.01)；
结论
1. URSA的发生与VEGF.sfl t-1具有相关性,VEGF.sfl t-1的升高导致流产发生。
2.复发性自然流产患者血清及绒毛组织可溶性VEGF受体sFlt-1含量升高可能与早孕期滋养层绒毛发育过程中缺氧有关,孕早期缺氧,循环中sFlt-1的表达增加,造成循环中游离态VEGF水平降低,另外,sFlt-1高表达会拮抗VEGF促进血管生成的作用,影响滋养细胞增殖和内皮细胞功能,导致流产发生。
第三部分VEGF基因多态性与广西地区壮族人群复发性流产的相关性研究
目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)-2578C/A、-54G/A、-634G/A、-460C/T和+936C/T位点单核甘酸多态性(single nucleotide polymorphisms,SNP)与广西地区壮族URSA人群发性流产发病的关联性。
方法应用PCR-限制性片段长度多态性技术(PCR-RFLP)检测URSA组120例和健康对照组120例的VEGF5个SNP位点基因型频率分布情况。
结果VEGF-2578C/A、-1154G/A、-634G/C、-460C/T和+936C/T的SNP位点基因型和等位基因频率分布在病例组和对照组间差异均无统计学意义(P>0.05)。
结论
1.VEGF-2578C/A、-1154G/A、-634G/C、-460C/T和+936C/T位点的基因多态性可能与广西壮族人群不明原因复发性流产发病风险不相关。
2.由于基因多态性位点在不同种族,不同人群中的作用不一致所致及VEGF基因的高度多态性,需要进一步加大样本量对不同种族人群进一步研究,从而全面了解广西人群中VEGF基因SNPs在URSA的易感性。
Background
The modern definition of recurrent spontaneous abortion (RSA) is the spontaneous loss of two or more consecutive pregnancies before20weeks of gestation. It is a common clinical problem that affects1%of couples who attempt to have children. Various factors have been identified that are thought to play a role in about50%of cases of RSA including genetic, endocrine, anatomical or autoimmune issues and infections. However the mechanism involved in the other50%of RSAs remains unexplained (Unexplained RSA). Recent studies have indicated that placental ischemia/hypoxia and endothelial dysfunction may contribute to cases of RSA. Yet the pathogenesis has not been fully elucidated. It is reported that growth and development of the fetus are critically dependent on the placental function and on vascular development. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that is produced by a variety of cell types, including the placenta. Fms-like tyrosine kinase-1(Flt-1), a transmembranous tyrosine-kinase, is a receptor for VEGF. Flt-1mRNA produces a soluble form of Flt-1by alternative splicing (sFlt-1). Soluble Flt-1(sFlt-1) is a splice variant of the Flt-1that lacks the transmembrane and cytoplasmic domains. It is a good antagonist of VEGF and can competitively inhibit the proangiogenic role of VEGF. Although sFlt-1has been demonstrated to inhibit VEGF activity, the association between sFlt-1and human disease remains unclear. Several recent experimental studies have demonstrated that VEGF and sFlt-1, contribute to the endothelial dysfunction observed in these pregnancy complications, such as hypertension, fetal growth restriction, and preeclampsia. In this study, we planned to evaluate the association between the VEGF and its soluble receptor-1, sFlt-1, in cases of recurrent spontaneous abortion. To further explose the pathogenesis of URSA. and to provide theories for the clinical prevention and treatment
Part One Association study between Vascular Endothelial Factor and its Soluble Receptor-1in pregnancy women
Objective To explores the connection between the Vascular Endothelial Growth Factor (VEGF) and its Soluble Receptor-1(sFlt-1) in pregnancy women. And to explore serum levels of VEGF and sFlt-1in diferrence early pregnancy weeks.
Methods
1. Case-contrl study:During the study period,50cases of no-pregnant women,50cases of normal pregnant women;50cases of URSA no-pregnant women,50cases of URSA early pregnant women were included in this study. Immunoassay for sFlt-1and VEGF serum levels of each group, respectively.
2. Following up study:50cases of normal pregnant women were included in this study. Immunoassay for sFlt-1and VEGF serum levels of pregnancy4to6weeks;7to8weeks;9to10weeks;11to12weeks, respectively.
Results
1. The mean sFlt-1level was higher in normal pregnancy group compared to the non-pregnant group (3631.742±3000.0891pg/ml vs.1587.001±1678.2486pg/ml, P<0.05). The mean VEGF level was significantly higher in the normal pregnancy compared to the non-pregnant group (969.6429±696.9974pg/ml vs.253.8277±137.0413pg/ml, P<0.001).
2. The mean sFlt1concentration in non-pregnancy women with RSA compared to the women with normal non-pregnancy (878.56±837.1065pg/ml vs.1587.001±1678.2486pg/ml, P>0.05). There is no significant difference between the two groups; The mean VEGF levels in the non-pregnancy women with RSA compared to the non-pregnant womem. There is no difference between the two groups.(242.8±236.52026pg/ml vs.253.8277±137.0413pg/ml, P>0.05).
3. In the first trimester pregnancies, the mean sFltl and VEGF concentration were highest in4to6weeks, especially in the4weeks. Then decreased and reach to certain level.
Conelusions
There is a correlation of sFlt-1and VEGF with pregnancy. In the early stages of specific placental proteins are regulated by sFlt-1and VEGF. The mean concentration of sFltl and VEGF are higher in pregnancy compared to non-pregnancy women. There is the highest level in4-6weeks in the the first trimester pregnancies, which indicates that embryo implantation and early pregnancy development occur in a relatively hypoxic environment.
Part Two The Correlation Study on Vascular Endothelial Factor and its Soluble Receptor-1in Patients with Unexplained Recurrent Spontaneous Abortion
Objective
To explores the connection between the Vascular Endothelial Growth Factor (VEGF) and its Soluble Receptor-1(sFlt-1) in pregnancy women with Unexplained Recurrent Spontaneous Abortion.
Methods
1. A total of35URSA patients, that subsequently miscarried and35healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The sFlt-1and VEGF serum levels of each group were deteced by immunoassay.
2. A total of35URSA patients, that subsequently miscarried and35healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The expression of VEGF and sFlt-1proteins in chorionic villus tissues were deteced by immunohistochemistry.
3. A total of30URSA patients, that subsequently miscarried and30healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The expression of expression of sFlt-1mRNA and VEGF mRNA in chorionic villus tissues were deteced by reverse transcription-polymerase chain reaction (RT-PCR).
Results
1. Serum level of VEGF in the URSA patients who subsequently miscarried was significantly higher than in the healthy controls (2637.5±2145.09ng/L vs.982.67±675.47ng/L P<0.001)。Serum level of sflt-1in the patients of URSA who subsequently miscarried was significantly higher than in the healthy controls (13796.67±11917.08ng/L vs.3540.67±2989.80ng/L, P<0.001)
2. The expression level of VEGF protein in chorionic villus of URSA embryo growth arrest patients were significantly higher than that in normal early pregnant women (P<0.05).
3. The expression level of sflt-1protein in chorionic villus of URSA embryo growth arrest patients were significantly higher than that in normal early pregnant women (P<0.05).
4. The expression of sFlt-1mRNA in URSA group was found significantly increase compared with control group (38.83±2.64vs.28.53±1.70, P<0.005,). A significant increase in VEGF mRNA levels has been also detected in URSA patients when compared with control group (74.34±4.03vs.21.20±1.65, P<0.0001).
Conclusions
In this prospective study we found a high level of expression of VEGF and sFlt-1in various tissues and in the serum of URSA patients that subsequently miscarried, compared to controls. This indicates that there is a relationship between early URSA and VEGF and sFlt-1and suggests that over-expression and high levels of the sFlt-1and VEGF may be associated with the pathogenesis of URSA.
Part Three The Association of VEGF Single Nucleotide Polymorphisms with the Risk of Recurrent Spontaneous Abortion of Zhuang Nationality in Guangxi
Objective To investigate the association of single nucleotide polymorphisms (SNPs) in VEGF gene with the risk of recurrent spontaneous abortion of Zhuang nationality in Guangxi province.
Methods
110Zhuang nationality patients in Guangxi suffering recurrent spontaneous abortion and110controls were enrolled in this study. SNP of VEGF were measured by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.
Results
The study show there is no significant difference in allele and genotype distributions of the five VEGF SNP(-2578C/A、-1154G/A、-634G/C、-460C/T and+936C/T) between URSA and controls(p>0.05). The analysis show there is no evidence of assoeiation between these polymorphisms and URSA
Conclusion
(1) No significant evidence of assoeiation or linkage was found at any of the markers tested, indicating that the SNP of VEGF(-2578C/A、-1154G/A、-634G/C、-460C/T and+936C/T) is unlikely to play a major role in the etiology of URSA in Zhuang nationality in Guangxi.(2)Because of the highly Polymorphic of VEGF molecule, further investigation are needed to address the contribution of other SNPs and several Polymorphisms in the form of haplotypes.
原因不明复发性流产(Unexplained Recurrent Spontaneous Abortion, URSA)是指与同一性伴侣连续发生2次或2次以上的自然流产,且不存在染色体、解剖、内分泌和自身免疫功能异常以及生殖道感染等病因。URSA的发生机制非常复杂,是妇产科领域一个很有临床价值的研究课题。近年来研究表明,许多不明原因复发性流产的发生与胎盘组织中血管增生平衡和胎儿血液供应不足有密切关系,血管内皮生长因子(vascular endothelial factor, VEGF)是最主要的促进血管形成的血管形成增生调节因子,在保持蜕膜组织中血管密度、有效血流量和维持胎儿营养供应上起重要作用。成功的妊娠依赖于胎盘良好的发育和生长,在胎盘植入和胎盘形成的过程中,血管的生成对于成功妊娠是非常关键的。sFlt (Soluble fms-like tyrosine kinase)为血管内皮生长因子受体的一种,由于它仅有胞外配体结合区,保留了与VEGF高亲和力的特性,其与VEGF结合后抑制其促血管生长作用。sFlt-1在先兆子痫、肿瘤等疾病中的作用已有较多研究成果,但在复发性流产的研究罕见。本研究探讨VEGF及sFlt-1与不明原因复发性流产发病的相关性及研究VEGF基因多态性与广西地区壮族人群复发性流产的相关发病风险,进一步阐明URSA发病机制,为URSA诊治提供理论基础及治疗前景。
第一部分VEGF及其可溶性受体sflt-1与妊娠相关性研究
目的检测正常未孕妇女及正常早孕妇女,不明原因复发性流产未孕妇女及不明原因复发性流产早孕妇女外周血中的sFlt-1及VEGF水平,探讨sFlt-1及VEGF与妊娠相关性；动态检测VEGF及sFlt-1在正常孕早期不同孕周外周血浓度,探讨sFlt-1及VEGF在孕早期不同孕周的浓度变化。
方法选择正常未孕妇女、不明原因复发性流产未孕妇女及正常早孕妇女各50例,应用ELISA法检测各组外周血sFlt-1及VEGF,探讨sFlt-1及VEGF与妊娠相关性。正常妊娠妇女妊娠确诊后分别抽取孕4-6周；孕7-8周；9-10周；211-12周外周血,应用ELISA法监测不同孕周sFlt-1和VEGF水平浓度,探讨sFlt-1及VEGF在孕早期不同孕周浓度变化。
结果
1.不明原因复发性流产未孕组VEGF及sFlt-1水平分别为242.8±236.52026及878.56±837.1065,正常未孕组VEGF及sFlt-1水平分别为253.8277±137.0413及1587.001±1678.2486。两组VEGF及sFlt-1水平无显著性差异,p>0.05。
2.正常早孕组与正常未孕组sFlt-1水平分别为3631.742±3000.0891；1587.001±1678.2486,正常怀孕组sflt水平高于正常未孕组,两者间差异有显著性差异(P<0.01),；正常早孕组与正常未孕组VEGF水平分别为969.64±696.9974；253.8277±137.0413,正常怀孕组VEGF水平高于正常未孕组,两者间差异有非常显著性差异(P<0.01)。
3.正常孕早期,VEGF及sFlt-1在孕4-6周血清浓度高,特别是孕4周(2256.50±1995.09ng/L；11796.67±9916.08ng/L),随后下降波动在一定水平。
结论
VEGF及sFlt-1与妊娠有相关性,妊娠确诊即能检测到VEGF.sflt-1的表达,说明在绒毛发育早期,VEGF及sflt-1即可发挥作用,对整个妊娠进行调节,妊娠后两者水平升高；在孕早期,胚胎刚着床滋养细胞侵入浅,相对缺氧,导致sFlt-1及VEGF水平升高,VEGF及sFlt-1在孕4-6周血清浓度高,随着妊娠周数增加,滋养细胞逐渐侵入,相对缺氧现象逐渐改善,sFlt-1及VEGF水平下降至一定水平。
第二部分VEGF及其可溶性受体sflt-1与不明原因复发性流产相关性研究
目的研究URSA患者中VEGF及其可溶性受体sflt一1血清浓度及在绒毛组织中蛋白及基因表达情况,探讨VEGF及其可溶性受体sflt-1与复发性流产的相关性。
方法前瞻性观察1124例不明原因复发性流产再孕患者,有35例URSA组患者胚胎停育,以35例正常早孕行人工流产术妇女的血清及绒毛作为对照组,应用ELISA法及免疫组织化学方法观察35例URSA患者胚胎刚停育(胚胎停育组)血清及绒毛中VEGF及sflt-1的蛋白表达情况；应用RT-PCR技术检测30例不明原因复发性流产患者及30例正常早孕妇女绒毛组织中的sFlt-1mRNA及VEGF-mRNA表达水平。
结果
1.对照组外周血中VEGF的水平为982.67±675.47ng/L:URSA组外周血中VEGF的水平为2637.50±2145.09ng/L.外周血中VEGF水平URSA组较对照组高,两者间差异有非常显著性差异(P<0.01)。
2.对照组外周血中sFlt-1的水平为3540.67±2989.80ng/L:URSA组外周血中sFlt-1的水平为13796.67±11917.08ng/L,外周血中sFlt-1水平URSA组较对照组高,两者间差异有非常显著性差异(P<0.01)。
3.不明原因复发性流产胎停育组绒毛组织中VEGF及sflt-1阳性染色(分别为81.25%和84.38%)较正常早孕组(分别为22%和28%)强,URSA组较正常早孕绒毛组织中VEGF及sflt-1蛋白表达水平高,两者间差异有显著性(P<0.05)。
4.不明原因复发性自然流产组和正常早孕组绒毛组织中VEGF mRNA及sFlt-1mRNA表达的检测,不明原因复发性自然流产组较正常早孕组绒毛组织中sFlt-1mRNA表达高(分别为38.83±2.64及28.53±1.70),两者间差异有显著性差异(P<0.01)；不明原因复发性自然流产组较正常早孕组绒毛组织中sFlt-1mRNA表达高(分别为38.83±2.64及28.53±1.70),两者间差异有显著性差异(P<0.01)；
结论
1. URSA的发生与VEGF.sfl t-1具有相关性,VEGF.sfl t-1的升高导致流产发生。
2.复发性自然流产患者血清及绒毛组织可溶性VEGF受体sFlt-1含量升高可能与早孕期滋养层绒毛发育过程中缺氧有关,孕早期缺氧,循环中sFlt-1的表达增加,造成循环中游离态VEGF水平降低,另外,sFlt-1高表达会拮抗VEGF促进血管生成的作用,影响滋养细胞增殖和内皮细胞功能,导致流产发生。
第三部分VEGF基因多态性与广西地区壮族人群复发性流产的相关性研究
目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)-2578C/A、-54G/A、-634G/A、-460C/T和+936C/T位点单核甘酸多态性(single nucleotide polymorphisms,SNP)与广西地区壮族URSA人群发性流产发病的关联性。
方法应用PCR-限制性片段长度多态性技术(PCR-RFLP)检测URSA组120例和健康对照组120例的VEGF5个SNP位点基因型频率分布情况。
结果VEGF-2578C/A、-1154G/A、-634G/C、-460C/T和+936C/T的SNP位点基因型和等位基因频率分布在病例组和对照组间差异均无统计学意义(P>0.05)。
结论
1.VEGF-2578C/A、-1154G/A、-634G/C、-460C/T和+936C/T位点的基因多态性可能与广西壮族人群不明原因复发性流产发病风险不相关。
2.由于基因多态性位点在不同种族,不同人群中的作用不一致所致及VEGF基因的高度多态性,需要进一步加大样本量对不同种族人群进一步研究,从而全面了解广西人群中VEGF基因SNPs在URSA的易感性。
Background
The modern definition of recurrent spontaneous abortion (RSA) is the spontaneous loss of two or more consecutive pregnancies before20weeks of gestation. It is a common clinical problem that affects1%of couples who attempt to have children. Various factors have been identified that are thought to play a role in about50%of cases of RSA including genetic, endocrine, anatomical or autoimmune issues and infections. However the mechanism involved in the other50%of RSAs remains unexplained (Unexplained RSA). Recent studies have indicated that placental ischemia/hypoxia and endothelial dysfunction may contribute to cases of RSA. Yet the pathogenesis has not been fully elucidated. It is reported that growth and development of the fetus are critically dependent on the placental function and on vascular development. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that is produced by a variety of cell types, including the placenta. Fms-like tyrosine kinase-1(Flt-1), a transmembranous tyrosine-kinase, is a receptor for VEGF. Flt-1mRNA produces a soluble form of Flt-1by alternative splicing (sFlt-1). Soluble Flt-1(sFlt-1) is a splice variant of the Flt-1that lacks the transmembrane and cytoplasmic domains. It is a good antagonist of VEGF and can competitively inhibit the proangiogenic role of VEGF. Although sFlt-1has been demonstrated to inhibit VEGF activity, the association between sFlt-1and human disease remains unclear. Several recent experimental studies have demonstrated that VEGF and sFlt-1, contribute to the endothelial dysfunction observed in these pregnancy complications, such as hypertension, fetal growth restriction, and preeclampsia. In this study, we planned to evaluate the association between the VEGF and its soluble receptor-1, sFlt-1, in cases of recurrent spontaneous abortion. To further explose the pathogenesis of URSA. and to provide theories for the clinical prevention and treatment
Part One Association study between Vascular Endothelial Factor and its Soluble Receptor-1in pregnancy women
Objective To explores the connection between the Vascular Endothelial Growth Factor (VEGF) and its Soluble Receptor-1(sFlt-1) in pregnancy women. And to explore serum levels of VEGF and sFlt-1in diferrence early pregnancy weeks.
Methods
1. Case-contrl study:During the study period,50cases of no-pregnant women,50cases of normal pregnant women;50cases of URSA no-pregnant women,50cases of URSA early pregnant women were included in this study. Immunoassay for sFlt-1and VEGF serum levels of each group, respectively.
2. Following up study:50cases of normal pregnant women were included in this study. Immunoassay for sFlt-1and VEGF serum levels of pregnancy4to6weeks;7to8weeks;9to10weeks;11to12weeks, respectively.
Results
1. The mean sFlt-1level was higher in normal pregnancy group compared to the non-pregnant group (3631.742±3000.0891pg/ml vs.1587.001±1678.2486pg/ml, P<0.05). The mean VEGF level was significantly higher in the normal pregnancy compared to the non-pregnant group (969.6429±696.9974pg/ml vs.253.8277±137.0413pg/ml, P<0.001).
2. The mean sFlt1concentration in non-pregnancy women with RSA compared to the women with normal non-pregnancy (878.56±837.1065pg/ml vs.1587.001±1678.2486pg/ml, P>0.05). There is no significant difference between the two groups; The mean VEGF levels in the non-pregnancy women with RSA compared to the non-pregnant womem. There is no difference between the two groups.(242.8±236.52026pg/ml vs.253.8277±137.0413pg/ml, P>0.05).
3. In the first trimester pregnancies, the mean sFltl and VEGF concentration were highest in4to6weeks, especially in the4weeks. Then decreased and reach to certain level.
Conelusions
There is a correlation of sFlt-1and VEGF with pregnancy. In the early stages of specific placental proteins are regulated by sFlt-1and VEGF. The mean concentration of sFltl and VEGF are higher in pregnancy compared to non-pregnancy women. There is the highest level in4-6weeks in the the first trimester pregnancies, which indicates that embryo implantation and early pregnancy development occur in a relatively hypoxic environment.
Part Two The Correlation Study on Vascular Endothelial Factor and its Soluble Receptor-1in Patients with Unexplained Recurrent Spontaneous Abortion
Objective
To explores the connection between the Vascular Endothelial Growth Factor (VEGF) and its Soluble Receptor-1(sFlt-1) in pregnancy women with Unexplained Recurrent Spontaneous Abortion.
Methods
1. A total of35URSA patients, that subsequently miscarried and35healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The sFlt-1and VEGF serum levels of each group were deteced by immunoassay.
2. A total of35URSA patients, that subsequently miscarried and35healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The expression of VEGF and sFlt-1proteins in chorionic villus tissues were deteced by immunohistochemistry.
3. A total of30URSA patients, that subsequently miscarried and30healthy normal pregnancy women who induced abortions between weeks6and12of pregnancy were included in this study. The expression of expression of sFlt-1mRNA and VEGF mRNA in chorionic villus tissues were deteced by reverse transcription-polymerase chain reaction (RT-PCR).
Results
1. Serum level of VEGF in the URSA patients who subsequently miscarried was significantly higher than in the healthy controls (2637.5±2145.09ng/L vs.982.67±675.47ng/L P<0.001)。Serum level of sflt-1in the patients of URSA who subsequently miscarried was significantly higher than in the healthy controls (13796.67±11917.08ng/L vs.3540.67±2989.80ng/L, P<0.001)
2. The expression level of VEGF protein in chorionic villus of URSA embryo growth arrest patients were significantly higher than that in normal early pregnant women (P<0.05).
3. The expression level of sflt-1protein in chorionic villus of URSA embryo growth arrest patients were significantly higher than that in normal early pregnant women (P<0.05).
4. The expression of sFlt-1mRNA in URSA group was found significantly increase compared with control group (38.83±2.64vs.28.53±1.70, P<0.005,). A significant increase in VEGF mRNA levels has been also detected in URSA patients when compared with control group (74.34±4.03vs.21.20±1.65, P<0.0001).
Conclusions
In this prospective study we found a high level of expression of VEGF and sFlt-1in various tissues and in the serum of URSA patients that subsequently miscarried, compared to controls. This indicates that there is a relationship between early URSA and VEGF and sFlt-1and suggests that over-expression and high levels of the sFlt-1and VEGF may be associated with the pathogenesis of URSA.
Part Three The Association of VEGF Single Nucleotide Polymorphisms with the Risk of Recurrent Spontaneous Abortion of Zhuang Nationality in Guangxi
Objective To investigate the association of single nucleotide polymorphisms (SNPs) in VEGF gene with the risk of recurrent spontaneous abortion of Zhuang nationality in Guangxi province.
Methods
110Zhuang nationality patients in Guangxi suffering recurrent spontaneous abortion and110controls were enrolled in this study. SNP of VEGF were measured by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.
Results
The study show there is no significant difference in allele and genotype distributions of the five VEGF SNP(-2578C/A、-1154G/A、-634G/C、-460C/T and+936C/T) between URSA and controls(p>0.05). The analysis show there is no evidence of assoeiation between these polymorphisms and URSA
Conclusion
(1) No significant evidence of assoeiation or linkage was found at any of the markers tested, indicating that the SNP of VEGF(-2578C/A、-1154G/A、-634G/C、-460C/T and+936C/T) is unlikely to play a major role in the etiology of URSA in Zhuang nationality in Guangxi.(2)Because of the highly Polymorphic of VEGF molecule, further investigation are needed to address the contribution of other SNPs and several Polymorphisms in the form of haplotypes.
引文
1.庞丽红；杨冬梅；马燕等.血清VEGF及其可溶性受体sFlt-1与原因不明复发性流产.中国妇幼保健.2010,25(29)：4258-4259
2.郑颖,左常婷,王国华.VEGF与复发性流产的研究进展.中国优生与遗传杂志,2008.16(9)：10-11.
3.习惯性流产治疗的最新进展.日本医学介绍,1997(10)：41-42.
4.庞丽红,孙燕.VEGF可溶性受体sFlt-1在复发性流产的作用研究进展.中国妇幼保健.2009-11-20
5. Rai, R., L. Regan, Recurrent miscarriage. Lancet,2006.368(9535):601-611.
6. Ferrara, N., Vascular endothelial growth factor:basic science and clinical progress. Endocr Rev,2004.25(4):p.581-611.
7.王宋平,钱桂生,李玉英等.人可溶性血管内皮生长因子受体-1基因的克隆与鉴定.重庆医学.2008-03-15
8. Nakatsukasa H,Masuyama H, Takamoto N, et al. Circulating leptin and angiogenic factors in preeclampsia patients [J]. Endocr J,2008,55:565-573
9. Hua Lu, Xiao-Ou Shu, Yong Cui, et al. Association of Genetic Polymorphisms in the VEGF Gene with Breast Cancer Survival [J]. Cancer Research,2005, 65(12):5015-5019.
10. Acobs E, Feigelson H, Bain E, et al. Polymorphisms in the vascular endothelial growth factor gene and breast cancer in the Cancer Prevention Study Ⅱ cohort [J].Breast Cancer Res,2006,8(2):22.
11.刘青,张华,李琰等.VEGF基因多态性与子宫内膜异位症发病风险的相关性研究.中国优生与遗传杂志,2010-04-25
12.庞丽红；李敏清；杨冬梅等.可溶性血管内皮生长因子受体-1(sFlt-1)与不明原因复发性流产的相关性研究.生殖与避孕,2011,31(1)：58-59
13.谭兵兵,王乾兴,谭晓珊,刘华庆.胎盘绒毛血管生成状况及VEGF、MMP-2和MMP-9表达与早期自然流产的关系.生殖与避孕.2006,26(8)：477-482
14. BeckL, Damore PA. Vascular development:cellular and molecular regulation [J]. FASEB J,1997,11:356-373
15. Krussel JB, Behr B, Milki AA, et al. Vascular endothelial growth factor Mrna splice variants are diferentially expressed in human blastocysis[J]. Mol Hum Reprod,2001.7(1):57-63.
16. Groker IP, Strachan BK, Lash GE, et al.Vascular endothelial growth factor but not placental growth factor promotes trophoblats syncytialization in vitro [J]. J Soc Gynecol Investig,2001,8(6):341-346
17. Nagamatsu T, Fujii T, Kusumi M, et al.Cytotrophoblasts up-regulate soluble fms-like tyrosine kinase-1 expression under reduced oxygen:an implication for the placental vascular development and the pathophysiology of preeclampsia. Endocrinology,2004,145(11):4
18. Hefler L, Obermair A, Husslein P et al1 Vascular endothelial growth factor serum levels in pregnancy and preeclampsia [J]. Acta Obstet Gynecol Scand, 2000,79(1):79
19. Roker IP, Strachan BK, Lash GE, et al.Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro. J Soc Gynecol Investig.2001.8:341-346.
20. Shiraishi, S., et al. Immunohistochemical localization of vascular endothelial growth factor in the human placenta. Placenta,1996.17(2-3):111-21.
21. Ahmad S, Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Cire Res,2004,95(9): 884
22.韦舟玲；庞丽红；杨冬梅等.VEGF在早期自然流产患者血清及绒毛组织中的表达及临床意义.微创医学.－2012-12-25
23.郑颖；左常婷；刘爱兰.VEGF在复发性流产患者绒毛及蜕膜中的表达, 中国妇幼保健-2009-02-20
24.韦红华,庞丽红,李敏清等.可溶性血管内皮生长因子受体在复发性流产绒毛组织及外周血中的表达及意义.中国妇幼保健,2011,26(16)：2510-2512
25. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science,2005,308(5 728):1592-4.
26. Merviel P, Carvillon L, Challier JC, el al. pathophysiology of preeclampsia: links with implantation disorders. European J Obstet Gynecol Reprod Biol, 2004,115(2):134-47.
27. Jakeman, L.B., et al., Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology,1993.133(2):p. 848-59.
28. Kaitu'u-Lino TJ, Whitehead CL, Ngian G-L, Permezel M, Tong S. Serum(2012) Concentrations of Soluble Flt-1 Are Decreased among Women with a Viable Fetus and No Symptoms of Miscarriage Destined for Pregnancy Loss. PLoS ONE 7:e32509.
29.VEGF基因多态性与子宫内膜异位症发病风险的相关性研究刘青；张华；李琰；康山；-《中国优生与遗传杂志》-2010-04-25
30. Hyun Haing Lee, Seung Ho Hong,Seung Ju Shin,et al.Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertility and Sterility,2010,93 (4),1244-1247.
31.Shagun Aggarwal, Farah Parveen, Rehan Mujeeb Faridi, et al.Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages-Reproductive Bio-Medicine Online,2011,22,59-64.
32. H.Samli,B.C.Demir,A. Ozgoz, et al.Vascular endothelial growth factor gene 1154 G/A,2578 C/A,460 C/T,936C/T polymorphisms and association with recurrent pregnancy losses.Genet. Mol. Res,2012,11 (4),4739-4745.
33. Xiuye Xing, Junhao Yan, Zi-Jiang, et al.Association of Vascular Endothelial Growth Factor Gene Polymorphisms with Recurrent Spontaneous Abortion in Chinese Han Women.Am J Reprod Immunol,2011,65(5),521-5.
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41. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science,2005,308(5728):1592-4.
42. Merviel P, Carvillon L, Challier JC, el al. pathophysiology of preeclampsia: links with implantation disorders. European J Obstet Gynecol Reprod Biol, 2004,115(2):134-47.
43. Nagamatsu T, Fujii T, Kusumi M, et al. Cytotrophoblasts up-regulate soluble fins-like tyrosine kinase-1 expression under reduced oxygen:an implication for the placental vascular development and the pathophysiology of preeclampsia. Endocrinology,2004,145(11):4838-45.
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2.郑颖,左常婷,王国华.VEGF与复发性流产的研究进展.中国优生与遗传杂志,2008.16(9)：10-11.
3.习惯性流产治疗的最新进展.日本医学介绍,1997(10)：41-42.
4.庞丽红,孙燕.VEGF可溶性受体sFlt-1在复发性流产的作用研究进展.中国妇幼保健.2009-11-20
5. Rai, R., L. Regan, Recurrent miscarriage. Lancet,2006.368(9535):601-611.
6. Ferrara, N., Vascular endothelial growth factor:basic science and clinical progress. Endocr Rev,2004.25(4):p.581-611.
7.王宋平,钱桂生,李玉英等.人可溶性血管内皮生长因子受体-1基因的克隆与鉴定.重庆医学.2008-03-15
8. Nakatsukasa H,Masuyama H, Takamoto N, et al. Circulating leptin and angiogenic factors in preeclampsia patients [J]. Endocr J,2008,55:565-573
9. Hua Lu, Xiao-Ou Shu, Yong Cui, et al. Association of Genetic Polymorphisms in the VEGF Gene with Breast Cancer Survival [J]. Cancer Research,2005, 65(12):5015-5019.
10. Acobs E, Feigelson H, Bain E, et al. Polymorphisms in the vascular endothelial growth factor gene and breast cancer in the Cancer Prevention Study Ⅱ cohort [J].Breast Cancer Res,2006,8(2):22.
11.刘青,张华,李琰等.VEGF基因多态性与子宫内膜异位症发病风险的相关性研究.中国优生与遗传杂志,2010-04-25
12.庞丽红；李敏清；杨冬梅等.可溶性血管内皮生长因子受体-1(sFlt-1)与不明原因复发性流产的相关性研究.生殖与避孕,2011,31(1)：58-59
13.谭兵兵,王乾兴,谭晓珊,刘华庆.胎盘绒毛血管生成状况及VEGF、MMP-2和MMP-9表达与早期自然流产的关系.生殖与避孕.2006,26(8)：477-482
14. BeckL, Damore PA. Vascular development:cellular and molecular regulation [J]. FASEB J,1997,11:356-373
15. Krussel JB, Behr B, Milki AA, et al. Vascular endothelial growth factor Mrna splice variants are diferentially expressed in human blastocysis[J]. Mol Hum Reprod,2001.7(1):57-63.
16. Groker IP, Strachan BK, Lash GE, et al.Vascular endothelial growth factor but not placental growth factor promotes trophoblats syncytialization in vitro [J]. J Soc Gynecol Investig,2001,8(6):341-346
17. Nagamatsu T, Fujii T, Kusumi M, et al.Cytotrophoblasts up-regulate soluble fms-like tyrosine kinase-1 expression under reduced oxygen:an implication for the placental vascular development and the pathophysiology of preeclampsia. Endocrinology,2004,145(11):4
18. Hefler L, Obermair A, Husslein P et al1 Vascular endothelial growth factor serum levels in pregnancy and preeclampsia [J]. Acta Obstet Gynecol Scand, 2000,79(1):79
19. Roker IP, Strachan BK, Lash GE, et al.Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro. J Soc Gynecol Investig.2001.8:341-346.
20. Shiraishi, S., et al. Immunohistochemical localization of vascular endothelial growth factor in the human placenta. Placenta,1996.17(2-3):111-21.
21. Ahmad S, Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Cire Res,2004,95(9): 884
22.韦舟玲；庞丽红；杨冬梅等.VEGF在早期自然流产患者血清及绒毛组织中的表达及临床意义.微创医学.－2012-12-25
23.郑颖；左常婷；刘爱兰.VEGF在复发性流产患者绒毛及蜕膜中的表达, 中国妇幼保健-2009-02-20
24.韦红华,庞丽红,李敏清等.可溶性血管内皮生长因子受体在复发性流产绒毛组织及外周血中的表达及意义.中国妇幼保健,2011,26(16)：2510-2512
25. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science,2005,308(5 728):1592-4.
26. Merviel P, Carvillon L, Challier JC, el al. pathophysiology of preeclampsia: links with implantation disorders. European J Obstet Gynecol Reprod Biol, 2004,115(2):134-47.
27. Jakeman, L.B., et al., Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology,1993.133(2):p. 848-59.
28. Kaitu'u-Lino TJ, Whitehead CL, Ngian G-L, Permezel M, Tong S. Serum(2012) Concentrations of Soluble Flt-1 Are Decreased among Women with a Viable Fetus and No Symptoms of Miscarriage Destined for Pregnancy Loss. PLoS ONE 7:e32509.
29.VEGF基因多态性与子宫内膜异位症发病风险的相关性研究刘青；张华；李琰；康山；-《中国优生与遗传杂志》-2010-04-25
30. Hyun Haing Lee, Seung Ho Hong,Seung Ju Shin,et al.Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertility and Sterility,2010,93 (4),1244-1247.
31.Shagun Aggarwal, Farah Parveen, Rehan Mujeeb Faridi, et al.Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages-Reproductive Bio-Medicine Online,2011,22,59-64.
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