Liu Fu-Min, Wang Xiu-Ying, Feng Xia, Wang Wen, Ye Yue-Xian, Chen Hong
Department of Obstetrics and Gynecology, Affiliated Hospital of Xuzhou Medical College, Jiangsu Province, Xuzhou, P.R. China.
Acta Obstet Gynecol Scand. 2007;86(5):535-41. doi: 10.1080/00016340601159124.
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.
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.
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.
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.
母体血液中胎儿遗传物质的发现为产前诊断开辟了一条新途径。在过去几十年中广泛研究的一种方法是从母体血液中分离胎儿细胞（赫岑伯格等人。《美国国家科学院院刊》。1979年；76:1453 - 5；比安奇等人。《美国国家科学院院刊》。1990年；87:3279 - 83；查等人。《产前诊断》。2005年；25:586 - 91）。由于胎儿细胞稀少且富集效率低，该技术无法在临床中应用。1997年，罗等人（《柳叶刀》1997年；350(9076):485 - 7）发现孕妇血浆和血清中存在游离胎儿DNA。这一发现表明母体血浆/血清DNA可能是无创产前诊断的有用材料来源。我们研究的目的是探讨利用母体血浆中的胎儿DNA进行产前诊断的可行性。
采用柱分离法提取40名孕周为5至40周的孕妇及产后24小时的277份血样中的血浆DNA。采用荧光定量聚合酶链反应（FQ-PCR）对30名孕妇的237份血浆样本中的SRY序列进行扩增。采用荧光聚合酶链反应同时扩增10名孕妇的40份血浆样本中的9个短串联重复序列位点，并采用相同方法扩增其丈夫的基因组DNA样本。
妊娠第7周可检测到胎儿SRY序列，其浓度随孕周增加而升高，在分娩前达到最高峰。产后24小时，母体血浆中未检测到胎儿SRY序列。游离DNA的SRY序列扩增结果与实际胎儿性别一致率为100%。对孕期采集的母体血浆样本分析发现存在父系遗传的胎儿特异性等位基因。在采集的30份血浆样本中，23份血浆DNA样本检测到胎儿特异性等位基因。阳性结果率为76%（23/30），早孕时阳性结果频率为6/10，中孕时为8/10，晚孕时为9/10。产后24小时母体血浆中未检测到短串联重复序列。
荧光聚合酶链反应可用于扩增母体血浆中的胎儿SRY序列和短串联重复序列以获取胎儿遗传信息,这可能对某些遗传性疾病的无创产前诊断有意义。
