胎儿幼稚红细胞产前遗传学诊断的研究

目的：探讨利用早期孕妇血中胎儿幼稚红细胞，进行无创伤性胎儿染色体异常产前诊断的可行性。方法：对１３例孕早期（８～１４周）已知孕男性胎儿孕妇外周血中糖蛋白Ａ（ＧＰＡ）表达阳性的细胞，进行荧光激活细胞分离（ＦＡＣＳ）及Ｙ染色体特异的引物介导原位标记（ＰＲＩＮＳ）检测，同时在测定４１例正常孕妇孕早期（８～１４周）血清妊娠相关血浆蛋白Ａ（ＰＡＰＰ┐Ａ）作为正常参考值的基础上，对５例可疑孕Ｄｏｗｎ′ｓ综合征胚胎的高龄孕妇进行ＰＡＰＰ┐Ａ检测、ＧＰＡ阳性细胞的ＦＡＣＳ及２１号染色体ＰＲＩＮＳ检测。结果：１３例孕男性胎儿的孕妇血ＧＰＡ阳性细胞中，胎儿幼稚红细胞的平均含量达到１４．５％；５例高龄孕妇血清ＰＡＰＰ┐Ａ均在正常范围内，ＧＰＡ阳性细胞中胎儿幼稚红细胞的２１号染色体ＰＲＩＮＳ检测未见异常。结论：早期孕妇血中存在胎儿幼稚红细胞，并可利用这些细胞对胎儿染色体非整倍性异常进行产前分子┐细胞遗传学诊断。     

人类ZFY基因巢式PCR技术的建立及产前诊断的初步研究

目的：建立一种对性连锁遗传病胎儿进行产前性别诊断的方法及探讨利用孕妇外周血中胎儿细胞检测ＺＦＹ基因的可行性。方法：用计算机辅助设计的两对寡核苷酸引物位于染色体ＺＦＹ基因保守区内，并建立了巢式聚合酶链反应（ｎｅｓｔｅｄ ＰＣＲ）技术扩增男性特异性的ＺＦＹ基因。结果：扩增产物分别为３５４ｂｐ和３０７ｂｐ，对不同标本的扩增结果表明，该引物具有高度的敏感性和特异性，２０份男性标本中均发现特异扩增片段，但在２０份女性标本中均未发现。应用该引物对３１份孕妇外周血连续监测，胎儿ＺＦＹ基因检出率随孕周增加而增高，总准确率达９６８％（３０／３１），４５份绒毛标本的阴性／阳性之比为１：１１４３，接近实际的胎儿性别比。结论：用巢式ＰＣＲ技术检测ＺＦＹ基因具有特异、敏感、快速、准确的优点，提示该法检测胎儿细胞ＤＮＡ是可行的，在临床上可用于性连锁遗传病的产前性别诊断。     

从孕妇外周血中分离胎儿有核红细胞方法学初探

从孕妇外周血中分离胎儿细胞进行无创性产前诊断成为新的研究热点,胎儿有核红细胞被认为是最理想的研究对象［１］。本研究采用简单的不连续密度梯度离心法分离富集孕妇外周血中有核红细胞,进行Ｙ染色体特异性ＤＹＺ１基因的聚合酶链反应（ＰＣＲ）扩增以预测胎儿性别,探讨胎儿有核红细胞用于无创性产前诊断的一些问题一、资料和方法　　１．研究对象孕龄６～１４周,年龄２１～３０岁的孕妇共４５例,分为细胞制片组１５例,包括早孕８例,中孕７例;胎儿性别预测组３０例,包括早孕１８例,中孕１２例。此外,以未婚、无妊娠史的２例女…     

利用母血胎儿游离DNA检测胎儿遗传信息的应用进展

目前临床广泛开展的产前诊断技术主要是侵入性产前诊断技术,包括妊娠早期(7～11周)绒毛活检技术、妊娠早中期胚胎(胎儿)镜检查技术、妊娠中期羊膜腔穿刺取样技术、妊娠中晚期脐静脉穿刺胎血取样技术等,因其创伤性、有一定的流产风险使得有创产前诊断的普及率不高.目前,利用孕妇外周血中胎儿细胞或胎儿DNA开展产前诊断已成为非侵入性产前诊断临床探索的新方向.然而,由于参与母体血循环的胎儿细胞数量极少、母血胎儿有核红细胞含量随孕周变化差异巨大,且分离、提纯、富集胎儿有核红细胞技术复杂、价格昂贵,导致利用母血胎儿细胞检测胎儿遗传信息发展缓慢[1].     

细胞游离胎儿DNA产前诊断胎儿非整倍体异常

细胞游离胎儿DNA（cffDNA）已被成功在妊娠妇女血循环中检测到。与母体血循环中胎儿细胞不同,cffDNA最早可在妊娠4周检测出,并能在产后迅速消失。cffDNA可能源于胎盘部位的滋养细胞等并通过凋亡机制释放进入母体血循环中。通过检测cffDNA可产前诊断胎儿非整倍体异常,但因其在妊娠妇女血中含量较少,检测具有挑战性。目前已有多种新检测方法用于非整倍体异常的产前诊断,尚需不断完善。     

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。     

运用流式细胞技术分析孕妇外周血中胎儿有核红细胞的比例与孕周的关系

目的 :探讨母亲外周血中胎儿有核红细胞 (NRBCs)的比例与孕周的关系。方法 :对 6 1名孕龄为 11～ 34周、年龄为 2 1～ 35岁的妇女的外周血中的胎儿细胞进行分离纯化 ,采用荧光激活细胞分离技术 (FACS)分选表达转铁蛋白受体的阳性细胞 (CD71+细胞 )。将孕妇组、未孕组及新生儿脐血组进行对照。结果 :孕 11～ 34周的孕妇外周血中 CD71+细胞的比例为 (0 .35± 0 .2 5 )× 10 - 2 ,与未孕组及新生儿脐血组均有极显著性差异。CD71+细胞在外周血中的比例在孕 17～18周达峰值 ,分别为 (0 .6 4± 0 .2 5 )× 10 - 2和 (0 .5 4± 0 .35 )× 10 - 2。结论 :早、中、晚期孕妇外周血中存在胎儿有核红细胞。利用母血循环中的胎儿细胞进行产前诊断的最佳时间应在妊娠 17～ 18周     

胎儿有核红细胞与无创性产前诊断

自妊娠妇女外周血中发现并分离到XY核型的胎儿细胞以来，发现运用母体外周血中胎儿细胞可以进行无创性产前诊断，目前发现妊娠妇女外周血中的胎儿细胞有：胎儿有核红细胞（nucleated red blood cells，NRBCs）、滋养细胞、胎儿淋巴细胞和粒细胞。越来越多的研究发现，胎儿有核细胞，尤其是NRBCs以其独特的细胞学特点倍受关注，就近十年来无创性产前诊断的最佳候选细胞——胎儿NRBCs的细胞学特点、富集、分离和鉴定方法、在临床产前诊断中的应用等综述。     

细胞游离胎儿DNA产前诊断胎儿非整倍体异常

细胞游离胎儿DNA(cffDNA)已被成功在妊娠妇女血循环中检测到。与母体血循环中胎儿细胞不同,cffDNA最早可在妊娠4周检测出,并能在产后迅速消失。cffDNA可能源于胎盘部位的滋养细胞等并通过凋亡机制释放进入母体血循环中。通过检测cffDNA可产前诊断胎儿非整倍体异常,但因其在妊娠妇女血中含量较少,检测具有挑战性。目前已有多种新检测方法用于非整倍体异常的产前诊断,尚需不断完善。     

孕妇外周血中胎儿有核红细胞的出现频率及其与孕周的关系

目的 探讨用检测孕妇外周血中的胎儿有核红细胞（ＮＲＢＣ）进行无创性产前诊断的最佳时间。方法 对４４名孕龄６～４０周的孕妇外周血进行不连续密度梯度离心,将分离后的细胞进行制片,显微镜下行有核红细胞计数,然后用显微操作法一一获取,进行Ｙ染色体特异性ＤＹＺ１基因的聚合酶链反应（ＰＣＲ）,以确定其胎儿来源。结果 ４４名孕妇中有１７例其外周血中检出有ＮＲＢＣ,分布于妊娠第９～２６周,其中以妊娠第１１～２０周     

Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies

BACKGROUND: We investigated fetal and total DNA levels in maternal plasma in patients bearing fetuses affected with Down syndrome in comparison to controls carrying fetuses with normal karyotype. METHODS: DNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and beta-globin genes as markers. Twenty-one pregnant women with a singleton fetus at a gestational age ranging from 15 to 19 weeks recruited before amniocentesis (carried out for reasons including material serum screening and advanced material age), and 16 pregnant women bearing fetuses affected with Down syndrome between 17 to 22 weeks of gestation were involved in the study. RESULTS: The specificity of the system reaches 100% (no Y signal was detected in 14 women pregnant with female fetuses) and the sensitivity 91.7% (SRY amplification in 22 of 24 examined samples). The median fetal DNA levels in women carrying Down syndrome (n=11) and the controls (n=13) were 23.3 (range 0-58.5) genome-equivalents/ml and 24.5 (range 0-47.5) genome-equivalents/ml of maternal plasma, respectively (P = 0.62). The total median DNA levels in pregnancies with Down syndrome and the controls were 10165 (range 615-65000) genome-equivalents/ml and 7330 (range 1300-36750) genome-equivalents/ml, respectively (P = 0.32). The fetal DNA proportion in maternal plasma was 0%-6 % (mean 0.8%) in women carrying Down syndrome and 0%-2.6 % (mean 0.7 %) in the controls, respectively (P=0.86). CONCLUSIONS: Our study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.     

Earliest gestational age for fetal sexing in cell-free maternal plasma

OBJECTIVES: To evaluate at what gestational age fetal DNA can reliably be detected at the earliest in maternal plasma. METHODS: We performed consecutive blood sampling in the first trimester of pregnancy in 17 women who were pregnant after in vitro fertilization (IVF) or intrauterine insemination (IUI). DNA was isolated and the Y-chromosome specific SRY was amplified by real-time polymerase chain reaction (PCR).We likewise studied 31 women prior to invasive prenatal diagnosis procedures for test validation purposes. All test results were compared to cytogenetic sex or sex at birth. RESULTS: The earliest SRY detection was at a gestational age of 5 weeks and 2 days. In none of 4 pregnancies ending in a miscarriage was SRY detected. We detected SRY in maternal plasma in 1 of 2 patients (50%) carrying a male fetus at a gestational age of 5 weeks, in 4 of 5 (80%) at a gestational age of 7 weeks, in 4 of 4 (100%) at a gestational age of 9 weeks. In all 7 women pregnant with a male fetus, the correct fetal sex was detected by 10 weeks. In none of the 6 patients who delivered a girl was SRY detected. In the validation group, SRY was detected in 13 of the 13 male, and none of the 18 female fetuses. CONCLUSIONS: We conclude that real-time PCR of the SRY gene promises to be a reliable technique for early fetal sexing in maternal plasma.     

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。     

Maternal serum free beta-hCG levels in uncomplicated pregnancies at the 10th-15th week of gestation and the development of obstetric complications

OBJECTIVE: To determine if free beta-human chorionic gonadotropin (hCG) serum levels at the 10th-14th week of gestation were different in groups of women who had experienced pregnancy complications. STUDY DESIGN: The obstetric records of women who had uncomplicated pregnancies when they consented to donate blood for biochemical research purposes early in pregnancy were reviewed. Two hundred thirteen of these women had donated blood at the 10th-14th week of gestation. Of these, 135 had uneventful pregnancies and delivered at term, 19 delivered before 37 weeks'gestation, 10 had fetuses small for gestational age, 4 developed pregnancy-induced hypertension, 7 developed gestational diabetes, 10 aborted spontaneously, 4 had an intrauterine fetal death after 20 weeks' gestation, and 24 were lost to follow-up. After the clinical groups had been identified, the 213 maternal serum stored samples were thawed and free beta-hCG measured by enzyme-linked immunosorbent assay. After normalization of the data, ANOVA was used to compare mean gestational age and mean free beta-hCG levels within groups. RESULTS: The overall mean gestational age at maternal blood sampling was 12.5 weeks. All groups had similar gestational ages at blood sampling (P = .18). The overall mean free beta-hCG serum level was 18.05 mIU/mL. Only the group of women who went on to experience spontaneous abortions had significantly lower free beta-hCG lev- els (mean, 10.45 mIU/mL; P < .03) CONCLUSION: Our data suggest that of the groups with obstetric complications evaluated, only the group of women who experienced spontaneous abortions had significantly different serum levels of free beta-hCG at the 10th-14th week of gestation.     

Analysis of peripheral blood of pregnant women for the presence of fetal Y chromosome-specific ZFY gene deoxyribonucleic acid sequences.

Deoxyribonucleic acid sequences of human ZFY (zinc-finger-Y) gene, a Y-chromosome-specific gene and candidate for the testis-determining factor, has been identified by an in vitro enzymatic deoxyribonucleic acid amplification method in peripheral blood specimens of women pregnant with male fetuses. This technique permits detection of ZFY gene deoxyribonucleic acid sequences in as few as a single male cell among 1,000,000 female cells. Maternal blood results were confirmed by amplification of ZFY gene deoxyribonucleic acid sequences in chorionic villus cells and by karyotyping in 33 of 36 pregnant women. There was no false-positive male result, and two of the three blood specimens with false-negative results were obtained from pregnant women at a very early gestational age. With properly designed guidelines, this deoxyribonucleic acid amplification method may be an alternative to determine the fetal sex for those pregnancies at risk for X-linked genetic disorders.     

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.     

Culture of endothelial cells isolated from maternal blood using anti-CD105 and CD133

OBJECTIVES: We hypothesized that fetal cells in maternal blood that do not respond to hematopoietic culture conditions represent endothelial cells. We investigated whether endothelial progenitor cells of fetal origin may be selected from maternal blood on the basis of their expression of CD133 or CD105 and expanded in culture. METHODS: Peripheral blood mononuclear cells from 16 pregnant women (gestational age: 11 to 24 weeks) were labeled with magnetic beads coupled to anti-CD133 or anti-CD105. Selection of labeled cells was performed using MACS. Resulting CD133+, CD105+, and CD133-/CD105- cell fractions were placed in culture in conditions favoring endothelial cells for 7 days (positive fractions) to 30 days (depleted fractions). Cells from women carrying male fetuses were analyzed by conventional PCR (SRY primers) for detection of male cells. RESULTS: Expansion of cells isolated from all subjects occurred in each of the cell fractions. No PCR products consistent with the presence of male cells were detected in women carrying male fetuses. CONCLUSION: CD133+ and CD105+ cells isolated from maternal blood can be expanded in vitro under endothelial conditions. These cells appear to be of maternal, rather than fetal, origin.     

First-trimester fetal sex determination in maternal serum using real-time PCR.

Fetal sex prediction can be achieved using PCR targeted at the SRY gene by analysing cell-free fetal DNA in maternal serum. Unfortunately, the results reported to date show a lack of sensitivity, especially during the first trimester of pregnancy. Therefore, determination of fetal sex by maternal serum analysis could not replace karyotype analysis following chorionic villus sampling. A new highly sensitive real-time PCR was developed to detect an SRY gene sequence in maternal serum. Analysis was performed on 121 pregnant women during the first trimester of pregnancy (mean gestational age: 11.8 weeks). Among them, 51 had at least one previous male-bearing pregnancy. Results were compared with fetal sex. SRY PCR analysis of maternal serum was in complete concordance with fetal sex. Among the 121 pregnant women, 61 were bearing a male fetus and 60 a female fetus. No false-negative results were observed. Furthermore, no false-positive results occurred, even though 27 women carrying a female fetus during the current pregnancy had at least one previous male-bearing pregnancy. This study demonstrates that a reliable, non-invasive sex determination can be achieved by PCR analysis of maternal serum during the first trimester of pregnancy. This non-invasive approach for fetal sex prediction should have great implications in the management of pregnant women who are carriers of an X-linked genetic disorder. Prenatal diagnosis might thus be performed for male fetuses only, avoiding invasive procedures and the risk of the loss of female fetuses.     

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination

BACKGROUND: We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. METHODS: Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. RESULTS: The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). CONCLUSIONS: Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.     

Elevated amniotic fluid leptin levels in pregnant women who are destined to develop preeclampsia

OBJECTIVE: To analyze whether leptin levels of the amniotic fluid elevate during early pregnancy in women destined to develop preeclampsia and to evaluate the relationship between amniotic fluid leptin levels and gestational age, maternal body mass index, and fetal sex. STUDY DESIGN: Leptin levels of the amniotic fluid were compared in two groups of women, preeclamptic (n = 20) and normotensive pregnant (n = 40), matched for fetal sex, maternal body mass index at sampling, gravidity and fetal gestational age at sampling. Furthermore, amniotic leptin levels in 400 normotensive pregnant women were analyzed for their correlation with gestational age, maternal body mass index, and fetal sex. RESULTS: Median leptin concentrations were significantly higher (p < 0.001) in the women with preeclampsia (7.3+/-0.7 ng/ml) than in the normotensive pregnant women (4.1 +/- 0.3 ng/ml), independent of fetal sex. The leptin levels in the amniotic fluid decreased with advanced gestational age (r = 0.24, p < 0.001). Amniotic fluid leptin levels in the pregnant women carrying a female fetus (5.6+/-0.3ng/ml) were significantly higher than those carrying a male fetus (4.7+/-0.2 ng/ml) (p = 0.004). CONCLUSION: Higher amniotic fluid leptin levels were observed in the preeclamptic pregnant women, and they decreased as gestational age advanced. Furthermore, the women with a female fetus were noted to have higher amniotic fluid leptin levels.