孕妇血浆中胎儿DNA的数量变化的研究

目的探讨孕妇血浆中胎儿游离DNA的数量变化的规律。方法提取68例孕妇血浆中的DNA,用实时荧光定量聚合酶链式反应(FQ-PCR)技术检测其胎儿SRY基因,并对其中怀男胎孕妇外周血浆中的胎儿DNA的数量进行动态分析。结果在怀男胎的孕妇外周血中均检测到了SRY基因,而在怀女胎的孕妇中未检测到。胎儿游离DNA最早出现的时间平均为7.7周,SRY基因拷贝数平均为5.53copies/ml。随孕期的增加,母血浆中胎儿DNA的含量在逐渐增加,并得出各孕周的标准参考值。在分娩后母血中胎儿DNA的含量就显著下降,到分娩后第二天就完全不能检测到。结论孕妇外周血浆中游离DNA的含量变化具有一定的规律,可以利用其进行无创伤性产前诊断。     

孕妇外周血胎儿游离DNA的检测及应用研究

从1997年首次在孕妇外周血中发现胎儿DNA以来,众多学者致力于将其应用于无创性的产前诊断。荧光定量PCR实现了胎儿游离DNA浓度的检测,目前研究发现,正常孕妇血浆胎儿DNA浓度稳定于某一水平,有望成为产前诊断和孕期检测的一项新指标。有学者认为,子痫前期母循环中胎儿DNA可有异常增高,而且子痫前期的孕妇在有临床症状前,孕妇血浆胎儿DNA水平异常增高,这与较多胎儿DNA释放到母血中,和肝肾功能异常导致胎儿DNA在母血中清除紊乱有关。此外许多妊娠合并症和并发症都伴有母外周血DNA水平的异常增高,例如:性连锁疾病、Rh血型不合、父系遗传性疾病、染色体非整倍体胎儿、早产等。现就孕妇外周血胎儿游离DNA的检测及临床应用作一综述。     

孕妇外周血中胎儿细胞及胎儿DNA的检测

目的　建立从孕妇外周血中分离有核红细胞 (NRBC)及DNA的可靠方法 ,并鉴定其来源。方法　对88例孕妇外周血分别通过密度梯度离心法 ,流式细胞术 (FCM )和荧光激活细胞分离技术 (FACS)分离NRBCs。应用套式PCR技术对 6 5例孕妇血浆DNA进行正常男性SRY基因检测。结果　①NRBCs:2 7例样品经密度梯度离心 ,其中 14例分选到 1～ 10个NRBCs。FACS技术对 6 1例样品进行转铁蛋白受体阳性细胞 (CD71+ )分选 ,所有样品均有CD71+ 细胞 ,其浓度为 (0 35± 0 2 5 )× 10 -2 。②胎儿DNA :4 6例怀男胎孕妇血浆DNA中SRY基因检测出率为 6 5 2 2 % (30 / 4 6 ) ,怀女胎的 19例样品SRY基因未检出率为 94 74 % (18/ 19)。结论　从孕妇外周血中分选胎儿NRBCs和血浆中胎儿DNA的方法已取得较大进展。利用母血中胎儿细胞及DNA诊断遗传性疾病可望成为最佳非创伤性产前诊断技术     

孕妇外周血中胎儿DNA水平检测在子痫前期诊断中的应用

目的探讨孕妇外周血中胎儿DNA水平检测在子痫前期诊断中的应用价值。方法选择30例子痫前期孕妇为子痫前期组(其中轻度18例,重度12例),另选择30例正常孕妇作为对照组,两组孕妇分别于孕20周、孕晚期(子痫前期组孕33周+3、对照组孕34周+3)、分娩后1、3、6 h取外周血,采用荧光定量PCR检测外周血中Y染色体上的性别决定基因(SRY基因)胎儿DNA水平(B超确定两组孕妇所妊娠的胎儿均为男性);放射免疫法检测两组孕妇孕晚期内皮素水平。结果(1)子痫前期组孕20周时的胎儿DNA水平为(316±61)copy/m l,其中轻度、重度患者分别为(266±79)、(396±91)copy/m l;对照组孕妇为(165±43)copy/m l,子痫前期组及轻度、重度患者明显高于对照组,两组比较,差异有统计学意义(P<0.01)。(2)子痫前期组孕晚期胎儿DNA水平为(970±413)copy/m l,其中轻度、重度患者分别为(758±357)、(1285±573)copy/m l,对照组孕妇为(319±99)copy/m l,子痫前期组及轻度、重度患者明显高于对照组,两组比较,差异有统计学意义(P<0.01)。(3)子痫前期组产后1、3、6 h胎儿DNA水平分别为(139±45)、(76±31)、(44±13)copy/m l,其中轻度患者分别为(102±42)、(57±25)、(36±12)copy/m l,重度患者分别为(209±51)、(97±40)、(52±17)copy/m l;对照组分别为(33±13)、(9±5)、0 copy/m l。子痫前期组及轻度、重度患者明显高于对照组,两组比较,差异有统计学意义(P<0.01)。(4)子痫前期组内皮素水平为(80±18)ng/L,其中轻度患者为(74±14)ng/L,重度患者为(89±32)ng/L;对照组为(50±11)ng/L,子痫前期组及轻度、重度患者明显高于对照组,两组比较,差异有统计学意义(P<0.01)。(5)子痫前期组胎儿DNA水平与内皮素水平呈正相关关系(r=0.748,P<0.01)。结论孕妇外周血胎儿DNA水平变化可以作为预测和诊断子痫前期发病与疾病程度的一个指标。     

两种胎儿特异性mRNA在母血中的表达研究

目的:检测孕妇外周血中ε血红蛋白基因和PLAC4基因mRNA表达水平,比较其特异性及实用性。方法:选取55例正常妊娠孕妇和21例健康非孕妇女为研究对象,提取全血总RNA,采用实时定量PCR法检测ε血红蛋白基因和PLAC4基因mRNA表达水平,并探讨其与孕龄的相关性。结果:55例孕妇外周血中14例扩增出胎儿ε基因片段,31例为阴性;胎儿ε血红蛋白基因mRNA质量浓度最小为0.571×103copies/ml,最大为1.812×103copies/ml,中位数为1.15×103copies/ml,在孕7~10周该基因浓度与孕龄呈负相关(P0.05)。55例正常妊娠孕妇外周血均扩增出PLAC4基因片段,最小值为1.853×103copies/ml,最大值为15.221×103copies/ml,中位数为7.836×103copies/ml,表达水平与孕龄无相关性(P0.05)。21例健康非孕妇女外周血均未扩增出ε血红蛋白基因和PLAC4基因片段。结论:与ε血红蛋白基因mRNA相比较,PLAC4基因mRNA具有特异性更强、与孕龄无关、阳性率更高等优点,更适用于21三体的无创性产前诊断。     

孕妇血浆中胎儿DNA含量与早产关系的研究

目的:研究早产和先兆早产孕妇血浆胎儿DNA的含量以及临床意义.方法:选择孕满28周至不足37周出现自发性规律宫缩的孕妇(单男胎)51例,其中23例孕周<37周分娩为早产组;28例出现有威胁的早产宫缩但经抑制宫缩治疗后足月产为先兆早产组,另选择正常妊娠的孕妇25例为正常对照组.采用实时荧光定量PER方法测定孕妇血浆中总DNA和胎儿DNA的量,非参数统计方法进行数据分析.结果:①早产组孕妇血浆总DNA量中位数7639.0拷贝/ml高于正常对照组6931.8拷贝/ml,差异有统计学意义(P<0.05);②早产组孕妇血浆胎儿DNA中位数为386.6拷贝/ml,先兆早产组为312.9拷贝/ml,均高于正常对照组230.5拷贝/ml,差异均有统计学意义(P<0.05);③以正常对照组孕妇血浆胎儿DNA量的第90百分位作为阳性预测值,早产组的阳性预测率为82.6%,先兆早产组为46.4%,差异有统计学意义(P<0.05).结论:早产孕妇血浆中胎儿DNA水平升高,观察孕妇血浆中胎儿DNA变化可有助于发现存在早产的可能,便于及时干预和处理.     

定量检测孕妇血浆中胎儿DNA在产前诊断的应用

目的探讨应用荧光定量PCR技术检测孕妇血浆中胎儿DNA量，了解胎儿DNA和母体DNA随孕周增长的变化，为将来应用于非创性产前诊断做临床前期研究。方法选择妊娠7～42周，B超确诊为单胎的孕妇58例，使用DNA试剂盒提取孕妇血浆中的胎儿DNA，用荧光定量PCR（FQ-PCR）技术测定血浆中β-actin基因和SRY基因的量。结果58例正常孕妇，有37例男性胎儿，检出率为100％。胎儿DNA的量在早孕组为9．08拷贝／ml（3．5～12．8），中孕组为45．41拷贝／ml（14．38～76．5），晚孕组为300．95拷贝／ml（84～840）。21例女性胎儿均未检出SRY基因。结论胎儿DNA随着孕周的增加而增长，母体血浆是进行无创性产前诊断的非常有价值的底物。     

妊娠期糖尿病或胰岛素依赖性（1型）糖尿病妇女母系和父系家族糖尿病史

有研究表明先兆子痫孕妇血浆中其胎儿循环的DNA总量升高。为证实母体循环中DNA量的变化进行测定。 先兆子痫诊断标准为妊娠20周后间隔4 h 2次血压≥18.7/12 kPa(140/90 mmHg),或舒张压≥14.7 kPa(110mmHg),并有蛋白尿≥300 mg/24h,严重先兆子痫至少有下列标准之一:2次血压>21.3/14.7 kPa(160/110 mmHg),蛋白尿>5g/24h,子痫HELLP综合征,持续右上头痛或胎儿宫内生长<5个百分点;共取先兆子痫孕妇血浆44例,其中12例有严重体征;妊娠期血压正常的53例作对照,均单胎,妊娠男胎39例,     

母血中胎儿DNA预测前置胎盘合并粘连的探讨

目的:通过实时荧光定量PCR方法测定孕妇外周血中胎儿DNA量,初步探讨前置胎盘合并胎盘粘连孕妇外周血中胎儿DNA量与正常对照组有无统计学差异;母血中胎儿DNA定量能否预测前置胎盘合并胎盘粘连。方法:收集35例孕28~34周且胎儿为男性,经我院B超诊断为前置胎盘的单胎孕妇作为研究组,其中合并胎盘粘连8例经手术确诊,称为前置胎盘粘连组,未合并胎盘粘连27例称为前置胎盘未粘连组。收集30例孕28~34周,且胎儿为男性,正常单胎妊娠孕妇作为对照组,通过实时荧光定量PCR方法测定3组孕妇外周血中胎儿DNA量,比较3组结果的异同。结果:前置胎盘粘连组孕妇外周血中胎儿DNA量高于对照组,差异有统计学意义(P<0.05)。前置胎盘未粘连组孕妇外周血中胎儿DNA量与对照组差异无统计学意义(P>0.05)。前置胎盘粘连组孕妇外周血中胎儿DNA量高于未合并胎盘粘连组,差异有统计学意义(P<0.05)。孕妇外周血中胎儿DNA量与前置胎盘类型无关,差异无统计学意义(P>0.05)。结论:孕妇外周血中胎儿DNA量与胎盘粘连与否有关,即与胎盘组织的功能状态有关。因此测定孕妇外周血中胎儿DNA量可能有助于预测前置胎盘合并胎盘粘连。     

孕激素对重度子痫前期TLR4信号通路的调控作用

目的:研究孕激素在重度子痫前期孕妇外周血血浆中的水平及其在重度子痫前期发病机制Toll样受体4(TLR4)信号通路中的作用。方法:选择重度子痫前期孕妇30例(重度子痫前期组)和正常孕妇30例(正常对照组),采用稀释定量法分别测定其血浆孕激素水平;原代培养重度子痫前期组孕妇外周血单个核细胞(PBMC),以不同浓度(0、10-8mol/L、10-6mol/L、10-4mol/L)孕激素(孕酮)刺激,实时定量聚合酶联反应检测TLR4、髓样分化因子88(My D88)、核转录因子-κB(NF-κB)mRNA的表达;酶联免疫吸附试验检测肿瘤坏死因子-α(TNF-α)及白介素-6(IL-6)蛋白的表达。结果:重度子痫前期组孕妇血浆孕激素水平明显低于正常对照组(P0.05)。随着孕激素浓度的增加,重度子痫前期组孕妇TLR4、My D88、NF-κB mRNA的相对表达量逐渐减少,4组组间两两比较,差异均有统计学意义(P0.01);TNF-α及IL-6蛋白的表达均减少,4组组间两两比较,差异均有统计学意义(P0.01)。结论:孕激素水平在重度子痫前期孕妇外周血浆中明显降低,孕激素可在重度子痫前期发病机制TLR4信号通路中发挥抑制作用,从而对机体产生保护作用。     

Increased fetal DNA in the maternal circulation in early pregnancy is associated with an increased risk of preeclampsia

OBJECTIVE: The aim of our study was to determine if fetal DNA is present in the maternal circulation in early pregnancy before the clinical manifestation of preeclampsia, and if this could be predictive of the development of preeclampsia. STUDY DESIGN: Blood were obtained from patients attending for a first antenatal visit. Cases were asymptomatic women who subsequently developed preeclampsia matched to control women for parity and gestational age. Real-time polymerase chain reaction (PCR) using TaqMan primers and probes directed against SRY gene sequences quantified fetal DNA in the maternal circulation. RESULTS: There were 88 cases of women with preeclampsia and 176 control women, both sampled at a mean gestation (+/-SD) of 15.7 +/- 3.6 weeks. The presence of fetal DNA in the maternal circulation in early pregnancy is associated with an 8-fold increased risk of developing preeclampsia. CONCLUSION: Increased fetal DNA is present in the maternal circulation in early pregnancy in women who subsequently develop pre-eclampsia and there appears to be a graded response between the quantity of fetal DNA and the risk of developing pre-eclampsia.     

Increased fetal RhD gene in the maternal circulation in early pregnancy is associated with an increased risk of pre-eclampsia

OBJECTIVE: To determine whether the fetal RhD gene is present in the maternal circulation in early pregnancy prior to the clinical manifestation of pre-eclampsia. DESIGN: This is a nested case-control study. SETTING: Blood samples were obtained from patients attending for a first antenatal visit. SAMPLE: Cases were asymptomatic RhD negative women (n= 23) who subsequently developed pre-eclampsia matched to RhD negative controls (n= 23) for parity and gestational age. METHODS: Real time PCR using TaqMan primers and probes directed against the RhD gene quantified fetal DNA in the maternal circulation. MAIN OUTCOME MEASURES: Quantity of RhD gene detected. RESULTS: As the copy number of RhD gene per millilitre of whole blood at 15 weeks of gestation increased, there was a significantly increased risk of developing pre-eclampsia. There was a graded association between copy number of RhD gene in early pregnancy and severity of disease with controls having 6942, mild pre-eclamptics 83,273 and severe pre-eclamptics 285,793 copies/mL (logscale 3.6, 4.0 and 4.5, respectively). CONCLUSION: Increased fetal RhD gene is present in the maternal circulation in early pregnancy in women who subsequently develop pre-eclampsia and there appears to be a graded response between the quantity of fetal DNA and severity of pre-eclampsia.     

Cell-free fetal DNA in the plasma of pregnant women with severe fetal growth restriction

OBJECTIVE: Although there have been reports of increased fetal nucleated erythrocytes in the blood of pregnant women who are carrying growth-restricted fetuses, there have been no reports of quantification of fetal DNA concentration in the plasma of women with fetal growth restriction. We quantified fetal DNA concentration in the plasma of pregnant women with preeclampsia and/or fetal growth restriction. STUDY DESIGN: We examined maternal plasma from 9 pregnant women with fetal growth restriction and 9 with preeclampsia and from 20 women who were gestational age-matched normal control subjects. All women carried a male fetus. DNA was extracted from 1.5-mL plasma samples, and the DYS14 and beta-globin gene were analyzed by real-time quantitative polymerase chain reaction. RESULTS: The concentration of fetal DNA was significantly higher in subjects with preeclampsia than in fetal growth restriction subjects and normal control subjects. Fetal DNA concentrations in fetal growth restriction subjects were similar to those of normal control subjects. The concentration of total DNA (beta-globin) was significantly higher in subjects with preeclampsia when compared with healthy control subjects. CONCLUSION: We demonstrated that there was no increase in fetal DNA in the plasma of pregnant women with fetal growth restriction and that most fetal DNA in maternal plasma originates from trophoblasts.     

孕妇及胎儿乙型肝炎病毒DNA的检测及意义

目的定量检测乙型肝炎病毒(HBV)DNA阳性孕妇及其胎儿血清、PBMCs、胎盘、胎肝组织中的HBVDNA,探讨HBV宫内传播与宫内感染的可能途径以及孕妇HBV含量与宫内传播发生的量化关系。方法用PCR荧光定量分析法筛选血清HBVDNA阳性的不同孕龄孕妇,定量检测孕妇及其流产胎儿血清、PBMCs、胎盘、胎肝组织中的HBVDNA;对阳性胎肝组织进行HBVDNA原位杂交;对阳性PBMCs检测HBVcccDNA。结果21例血清HBVDNA阳性的孕妇中,11例PBMCsHBVDNA检测阳性,其中9例HBVcccDNA检测为阴性,而胎儿PBMCsHBVDNA检测均为阴性。母血HBVDNA含量不同数量级之间胎盘、胎肝HBVDNA阳性率差异有显著性(P=0.0001,P=0.001)。HBVDNA原位杂交检测显示,2例胎肝细胞内见阳性染色,相应的胎肝组织、胎儿脐血、胎盘组织PCR检测均为HBVDNA阳性。结论HBV宫内传播经胎盘发生,孕妇PBMCs内的HBV不会发生宫内传播;母血HBVDNA含量在106拷贝/ml及以上时,胎盘感染HBV的危险性显著增加,母血HBVDNA含量在107拷贝/ml及以下时,胎肝感染HBV的危险性显著降低。     

Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia

OBJECTIVE: Elevated amounts of circulating fetal deoxyribonucleic acid in maternal plasma have recently been detected in pregnancies complicated by preeclampsia. We attempted to confirm this finding and simultaneously examined the quantity of maternal circulating deoxyribonucleic acid. STUDY DESIGN: Circulating deoxyribonucleic acid was measured by realtime quantitative polymerase chain reaction in plasma samples obtained from 44 women with preeclampsia and a matched cohort of 53 normotensive pregnant women. RESULTS: We confirmed that circulating fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (3194.6 vs 332.8 copies/mL; P < .001). We also showed for the first time that circulating maternal deoxyribonucleic acid levels are also elevated (219,023.9 vs 20,235.8 copies/mL; P < .001). The increases in these deoxyribonucleic acid levels corresponded to the severity of the disorder, and values were correlated with each other in pregnancies complicated by preeclampsia (r = 0.556; P < .001) but not normotensive pregnancies (r = 0.046; P = .747). CONCLUSION: The releases of both free fetal and maternal deoxyribonucleic acid were found to be affected in preeclampsia.     

Correlation of fetal DNA levels in maternal plasma with Doppler status in pathological pregnancies

OBJECTIVES: To evaluate whether intrauterine growth restriction (IUGR) as seen in preeclampsia is associated with high levels of fetal DNA in maternal circulation, and whether fetal DNA is related to altered uterine and/or umbilical artery Doppler velocimetry. METHODS: Fetal DNA quantification was performed by real-time PCR on SRY sequences in 64 male-bearing pregnant women with IUGR and/or preeclampsia and 89 controls. RESULTS: Fetal DNA content was significantly elevated in IUGR pregnancies similar to preeclampsia and correlated with altered umbilical Doppler velocimetry, while no correlation was found with uterine Doppler status. CONCLUSION: Increased fetal DNA levels in maternal plasma may be a sign of placental or fetal pathology even in the presence of normal uterine Doppler velocimetry, allowing a more precise diagnostic evaluation. The finding that elevated fetal DNA in IUGR pregnancies correlates with abnormal umbilical Doppler velocimetry suggests that fetal DNA release is associated more with fetal chronic hypoxia than with fetal size.     

Cell-free foetal DNA in maternal plasma does not appear to be derived from the rich pool of cell-free foetal DNA in amniotic fluid

Background: Large quantities of cell-free foetal DNA have been detected in amniotic fluid, and it has been proposed that this material may contribute to the pool of cell-free foetal DNA in maternal plasma. Methods: Twelve maternal blood samples were obtained from pregnant women about to undergo an amniocentesis. Cell-free DNA was extracted from the maternal plasma samples and the matched amniotic fluid samples. The amount of cell-free foetal DNA was quantified by real-time PCR assays for the SRY and RHD genes. Results: Amniotic fluid was found to contain vast quantities of cell-free DNA (median concentration = 3,978 copies/ml amniotic fluid). The concentration of cell-free foetal DNA in maternal plasma was much lower (median concentration = 96.6 copies/ml maternal plasma). No significant correlation could, however, be determined between these two pools of cell-free foetal DNA. Conclusions: Our data confirm that amniotic fluid contains prodigious quantities of cell-free foetal DNA, but as no relationship exists between this material and that in the maternal circulation, it is unlikely that the amnion contributes to the presence of cell-free foetal DNA in maternal plasma.     

Comparison of activin A and cell-free fetal DNA levels in maternal plasma from patients at high risk for preeclampsia

OBJECTIVES: We examined the concentration of activin A in a prospective manner before the clinical manifestation of preeclampsia and compared the data with those of cell-free fetal DNA in the maternal plasma. METHODS: The levels of activin A were analysed by enzyme-linked immunosorbent assay (ELISA) for pregnant women: (1) with preeclampsia (n = 34) in the third-trimester and normal controls (n = 44); and (2) at-risk of preeclampsia in the second-trimester (n = 15) as indicated by uterine artery Doppler and normal controls (n = 68). Correlation between activin A level and cell-free fetal DNA level was examined using the Spearman rank test. RESULTS: The level of plasma activin A was significantly higher in the preeclamptic samples (12.056 vs 7.068 ng/mL, p = 0.000). The increase in the activin A concentration was observed prior to the onset of preeclampsia (3.483 vs 1.324 ng/mL, p = 0.000). This increase in activin A correlated significantly with the increased level of cell-free fetal DNA, in the maternal circulation prior to the onset of preeclampsia (r = 0.977, p = 0.000). CONCLUSION: Our data suggest that circulatory activin A could be an independent biomarker for the early identification and monitoring of preeclampsia.