210923例孕妇外周血胎儿游离DNA无创产前筛查研究

目的 评价孕妇外周血胎儿游离DNA无创产前筛查(non-invasive prenatal testing, NIPT)对胎儿染色体异常产前筛查与产前诊断的临床应用价值。方法 收集2017年1月至2020年12月上海市所有产前诊断中心孕妇外周血胎儿游离DNA产前筛查210 923例,进行研究分析。结果 NIPT筛查出染色体高风险异常孕妇2225例,检出率1.05%;其中850例行羊膜腔穿刺染色体核型产前诊断,确诊506例染色体异常,确诊率59.53%,其中21三体综合征284例、18三体综合征65例、13三体综合征17例、性染色体异常104例、其他染色体异常36例。NIPT筛查中自愿要求检测占51.56%;行羊膜腔穿刺孕妇中,高龄妊娠(≥35岁)占39.85%。经统计分析,高龄妊娠在不同分型染色体异常中有差异(P<0.05)。NIPT实验室检测失败占0.15%,其中主要原因为质控标准未达标。结论 对于无明确筛查指征的孕妇,NIPT是优选有效筛查方法;NIPT染色体疾病筛查中,21三体综合征检出率高;NIPT实验室检测失败原因亦值得关注。     

无创DNA检测在诊断高龄孕妇胎儿非整倍体中的应用

目的:探讨高龄妊娠胎儿染色体异常的风险以及无创DNA产前检测(NIPT)在诊断高龄孕妇胎儿非整倍体染色体病中的应用价值。方法:选择≥35岁的高龄孕妇2714例,按年龄分为35~39岁,≥40岁两组,采用NIPT高通量测序检测孕妇血浆游离DNA,并对检测结果提示21-三体、18-三体、13-三体及性染色体高风险者行羊膜腔穿刺术及胎儿染色体核型分析,对检测结果阴性者通过电话随访进行验证。计算NIPT检测的敏感度、特异度、阳性预测值、阴性预测值及Youden指数。结果:2714例高龄孕妇NIPT检测结果提示胎儿非整倍体染色体异常高风险47例,6例高风险孕妇拒绝侵入性产前诊断,余41例高风险孕妇行羊膜腔穿刺术及羊水细胞染色体核型分析,结果显示21-三体19例,18-三体1例,13-三体2例,性染色体异常7例。与现有的金标准羊膜腔穿刺术核型分析相比较,NIPT对高龄孕妇除外性染色体异常的胎儿非整倍体染色体异常检出敏感度为100.00%,特异度为99.93%,阳性预测值为90.91%,阴性预测值为100.00%,Youden指数为0.99。进一步通过年龄分组发现,40岁及以上年龄组异常率显著高于35~39岁年龄组(P=0.011)。结论:高龄孕妇可通过NIPT快速、安全地筛查出胎儿非整倍体染色体异常,减少侵入性产前检测比例,降低胎儿出生缺陷率。     

无创产前筛查在双胎妊娠染色体非整倍体筛查中的应用及胎儿游离DNA浓度分析

目的 评估无创产前筛查(non-invasive prenatal testing, NIPT)在双胎妊娠染色体非整倍体的应用价值,并对胎儿游离DNA浓度进行分析。方法 收集2018年1月至2023年5月在广州市花都区妇幼保健院接受NIPT检测的双胎样本617例(辅助生殖双胎284例,自然妊娠双胎333例)为研究对象,同期12688例单胎妊娠样本作为对照,采用高通量测序方法进行检测,对染色体非整倍体高风险样本行核型分析,根据Y染色体唯一比对条数对男胎进行胎儿游离DNA浓度计算,使用基于浅深度的母亲血浆DNA测序方法(SeqFF)对女胎进行胎儿游离DNA浓度计算。结果 双胎妊娠检出2例T21和1例T18,未检出T13,T21、T18的阳性预测值分别为100%、0,阴性预测值均为100%;单胎妊娠T21、T18、T13的阳性预测值分别为88.89%、45.45%、25%,阴性预测值均为100%。双胎妊娠胎儿游离DNA浓度为10.48%,较单胎妊娠(11.35%)稍低;双胎妊娠首次建库成功率为99.03%,较单胎妊娠(99.99%)低,差异有统计学意义(χ²=104.1...     

孕妇血浆游离DNA无创产前筛查检测失败及相关妊娠结局

孕妇血浆游离DNA(cell-free DNA, cfDNA)无创产前筛查(noninvasive prenatal screening, NIPS)是胎儿常见染色体非整倍体最有效的一种产前筛查方法,国内外已广泛开展。值得注意的是,该技术存在无法避免的检测失败,且近几年的一些研究报道发现,这种检测失败可能与胎儿染色体非整倍体和妊娠相关并发症发生存在一定相关性。目前,这方面的报道文献还相对有限,临床意义尚未完全明确。本文将回顾研究报道,探讨无创产前筛查检测失败原因及与胎儿染色体非整倍体、妊娠并发症相关性,探索在检测失败后对孕妇进行临床咨询和妊娠管理指导的理论依据。     

无创产前检测在胎儿染色体非整倍体筛查中的应用

<正>胎儿染色体非整倍体异常可导致严重的不良妊娠结局及出生缺陷,其产前筛查及诊断是出生缺陷二级预防的重要内容之一。常见严重或致死的胎儿染色体非整倍体有21-三体(唐氏综合征,Down′s Syndrome)、18-三体(爱德华氏综合征,Edwards Syndrome)和13-三体(帕陶氏综合征,Patau Syndrome),其中以21-三体最常见,发病率为0.7‰～1.5‰^[1]。     

游离胎儿DNA高通量基因测序技术在产前筛查的临床应用

目的:探讨应用孕妇血浆中游离胎儿DNA高通量基因测序技术在无创性产前诊断中的可行性.方法:采用化学发光法对22977例孕中期妇女进行唐氏综合征筛查,对筛查结果为高风险的1173例孕妇采用高通量基因测序技术检测母体血浆胎儿游离DNA,分析胎儿染色体拷贝数,检测结果为高危的孕妇再做羊膜腔穿刺及脐血穿刺术进行胎儿染色体核型分析.结果:1173例孕妇中游离胎儿DNA高通量基因测序技术检测出11例染色体高风险胎儿,羊水/脐血核型分析证实其中10例为染色体异常,分别为7例21-三体和2例18-三体,1例13-三体.游离胎儿DNA高通量基因测序技术的敏感度为100.00％(10/10),特异度为99.91％(1162/1163),假阴性率为0(0/1173),假阳性率为0.09％(1/1173).结论:母体血浆游离DNA高通量基因测序技术可用于胎儿染色体拷贝数异常的检测,有准确性高、假阴性和假阳性率低、无创取样的优点,但由于费用较高,可结合血清学唐氏综合征筛查在有条件的地区进行推广应用.     

产前筛查——从血清学筛查到无创产前检测

        产前筛查一般指的是对胎儿常见染色体非整倍体的筛查,即21-三体综合征、18-三体综合征、13-三体综合征,不包括其他染色体非整倍体。 浏览更多请关注本刊微信公众号及当期杂志。     

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。     

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。     

染色体微阵列分析在无创产前筛查非整倍体高风险中的应用价值

目的:探讨染色体微阵列分析(CMA)技术在无创产前筛查(NIPT)提示非整倍体高风险胎儿的产前诊断中的应用价值。方法:241例NIPT提示胎儿染色体非整倍体高风险的孕妇,经羊膜腔或绒毛穿刺获取胎儿细胞,进行CMA分析。对于NIPT提示21-三体和性染色体非整倍体高风险的孕妇根据筛查原因(高龄筛查组、唐筛异常或超声软指标异常组、非高龄筛查组)进行组间NIPT阳性预测值的比较,并对CMA检测提示有染色体拷贝数变异(CNV)的病例进行致病性分析。结果:NIPT对于染色体非整倍体总的阳性预测值为76.3%。NIPT对于21-三体、18-三体、13-三体和性染色体非整倍体的阳性预测值分别为95.0%、88.8%、75.0%和53.3%。经CMA分析确认,在101例NIPT提示21-三体高风险病例中,以高龄筛查组阳性预测值最高(100.0%),3组间比较,差异有统计学意义(P=0.001)。在90例NIPT提示性染色体非整倍体高风险病例中,仍以高龄筛查组阳性预测值最高(61.9%),3组间比较,差异有统计学意义(P=0.002)。12例NIPT提示染色体非整倍体高风险经CMA检测存在CNV。结论:...     

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     

Fetal origin of single nucleated erythroblasts and free DNA in the peripheral blood of pregnant women.

OBJECTIVES: To investigate the feasibility of using single fetal nucleated erythroblasts (FNRBCs) and free DNA in maternal blood for non-invasive prenatal diagnosis. METHODS: Single FNRBCs were isolated from 51 of 116 samples of maternal blood analyzed by micromanipulation after density gradient centrifugation. Furthermore, the nested polymerase chain reaction (PCR) method was used to amplify the SRY gene of single FNRBCs. Primer extension pre-amplification and nested PCR were used to amplify the SRY gene of the plasma DNA extracted from 65 samples of maternal blood. RESULTS: The detection rate of single FNRBCs was 90.20% (46/51). The concordance rates between real fetal sex and sex determined by amplification of the SRY gene from single cells and from free DNA analysis were 82.61% (38/46) and 90.77% (59/65), respectively. CONCLUSIONS: Single nucleated erythroblasts and free DNA in maternal blood are of fetal origin and can be valuable fetal material sources for non-invasive prenatal diagnosis.     

Non-invasive prenatal diagnosis by isolation of both trophoblasts and fetal nucleated red blood cells from the peripheral blood of pregnant women

Objective To isolate fetal trophoblasts and nucleated red blood cells from the peripheral blood of pregnant women.
Design Trophoblasts were isolated from whole blood of women in the first trimester of pregnancy by a specific monoclonal antibody, 340. Nucleated red blood cells were isolated by separating whole blood on a triple gradient, staining with ferromagnetic particles coated with an antitransferrin monoclonal antibody and separated on a mini magnetic activated cell sorting (MACS) column. Sorted cells were sexed using a nested polymerase chain reaction for a specific sequence on the Y chromosome and the sex was confirmed by karyotyping of chorionic villus samples.
Participants Patients between 10 and 14 weeks of pregnancy who were undergoing elective chorionic villus sampling for the detection of fetal aneuploidies.
Main outcome measure Fetal sex determined by polymerase chain reaction on fetal cells sorted from maternal blood.
Results When both trophoblasts and nucleated red blood cells were sorted, fetal sex was correctly predicted in 12/13 cases (92%), which included correct diagnosis of five of six male pregnancies. More importantly the two techniques were complementary, with only one male pregnancy being diagnosed on both trophoblasts and nucleated red blood cells, two being detected only with trophoblasts and two on nucleated red blood cells alone. No false positives (male signal from a female pregnancy) were diagnosed with either trophoblasts or nucleated red blood cells even with the highly sensitive nested polymerase chain reaction technique, which is very prone to contamination. This study also shows that it is possible to isolate both trophoblasts and nucleated red blood cells from the same sample of maternal blood.
Conclusion Fetal cells can be isolated from maternal blood at around 10 weeks of pregnancy.     

Simple non-invasive prenatal detection of Hb Bart's disease by analysis of fetal erythrocytes in maternal blood

OBJECTIVE: To investigate a simple non-invasive technique for early detection of Hemoglobin (Hb) Bart's disease. METHOD: Maternal blood smears from 8 known Hb Bart's pregnancies and 40 at-risk pregnancies were investigated. Maternal peripheral blood smears were stained with fluorescence-labeled monoclonal antibodies against alpha- and embryonic zeta-globin chains. RESULTS: Fetal nonnucleated red blood cells, stained with anti-zeta but not with anti-alpha globin antibodies were found in 15 out of 16 affected pregnancies but were not detected in 23 out of 24 unaffected pregnancies. CONCLUSION: Results showed that non-invasive immunofluorescence staining of maternal blood is a feasible approach for screening Hb Bart's disease before ultrasound manifestation in affected pregnancies.     

Feasibility study of using fetal DNA in maternal plasma for non-invasive prenatal diagnosis

BACKGROUND: The discovery of the presence of fetal genetic material in maternal blood has opened up a new approach to prenatal diagnosis. One approach that has been extensively investigated over the past few decades is the isolation of fetal cells from maternal blood (Herzenberg et al. Proc Natl Acad Sci USA. 1979;76:1453-5; Bianchi et al. Proc Natl Acad Sci USA. 1990;87:3279-83; Cha et al. Prenat Diagn. 2005;25:586-91). As the fetal cells are scarce and the enrichment is of low efficiency, the technique could not be implemented in clinics. In 1997, Lo et al. (Lancet 1997;350(9076):485-7) discovered that cell-free fetal DNA is present in the plasma and serum of pregnant women. This discovery suggests that maternal plasma/serum DNA may be a useful source of material for non-invasive prenatal diagnosis. The objective of our study was to investigate the feasibility of using fetal DNA in maternal plasma for prenatal diagnosis. METHODS: Plasma DNA in 277 blood samples of 40 pregnant women at the gestational period from 5 to 40 weeks and 24 h after delivery were extracted by column separation. FQ-PCR was used to amplify the SRY sequence in 237 plasma samples of 30 pregnant women. Fluorescent PCR was used to amplify 9 short tandem repeat loci simultaneously in 40 plasma samples of 10 pregnant women, and genomic DNA samples from their husbands were amplified by the same method. RESULTS: The fetal SRY sequence could be detected from the 7th week of gestation, with a concentration that increased with progressing gestational age, attaining its highest peak before delivery. Twenty-four hours after delivery, fetal SRY sequence could not be detected in the maternal plasma. The concordance rate of the SRY sequence amplification results of plasma-free DNA, with real fetal gender was 100%. Analysis of maternal plasma samples collected during pregnancy revealed the presence of paternally inherited fetal-specific alleles. Among the 30 collected plasma samples, fetal-specific alleles were detected in 23 plasma DNA samples. The rate of positive results was 76% (23/30), and the frequency of positive results was 6/10 in early pregnancy, 8/10 in middle pregnancy, and 9/10 in late pregnancy. Short tandem repeats could not be detected from the maternal plasma 24 h after delivery. CONCLUSION: Fluorescent PCR can be used for amplification of fetal SRY sequence and STRs in maternal plasma to obtain fetal genetic information, which may have implications for non-invasive prenatal diagnosis of certain hereditary diseases.     

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood

Prenatal diagnosis of aneuploidy and single-gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non-invasive prenatal diagnostic procedures. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD-negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non-invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin-based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non-invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non-invasive prenatal diagnosis.