Tse N. Leung

Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

Rapid Clearance of Fetal DNA from Maternal Plasma

Fetal DNA has been detected in maternal plasma during pregnancy. We investigated the clearance of circulating fetal DNA after delivery, using quantitative PCR analysis of the sex-determining region Y gene as a marker for male fetuses. We analyzed plasma samples from 12 women 1-42 d after delivery of male babies and found that circulating fetal DNA was undetectable by day 1 after delivery. To obtain a higher time-resolution picture of fetal DNA clearance, we performed serial sampling of eight women, which indicated that most women (seven) had undetectable levels of circulating fetal DNA by 2 h postpartum. The mean half-life for circulating fetal DNA was 16.3 min (range 4-30 min). Plasma nucleases were found to account for only part of the clearance of plasma fetal DNA. The rapid turnover of circulating DNA suggests that plasma DNA analysis may be less susceptible to false-positive results, which result from carryover from previous pregnancies, than is the detection of fetal cells in maternal blood; also, rapid turnover may be useful for the monitoring of feto-maternal events with rapid dynamics. These results also may have implications for the study of other types of nonhost DNA in plasma, such as circulating tumor-derived and graft-derived DNA in oncology and transplant patients, respectively.

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

mRNA of placental origin is readily detectable in maternal plasma

The discovery of circulating fetal nucleic acid in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Thus far, a gender- and polymorphism-independent fetal-specific target that can be used for prenatal screening and monitoring in all pregnant women has not been reported. In addition, the origin of such circulating nucleic acid has remained unclear. Here we provide direct evidence that the placenta is an important source of fetal nucleic acid release into maternal plasma by demonstrating that mRNA transcripts from placenta-expressed genes are readily detectable in maternal plasma. The surprising stability of such placental mRNA species in maternal plasma and their rapid clearance after delivery demonstrate that such circulating mRNA molecules are practical markers for clinical use. The measurement of such plasma mRNA markers has provided a gender-independent approach for noninvasive prenatal gene expression profiling and has opened up numerous research and diagnostic possibilities.

Prenatal exclusion of β thalassaemia major by examination of maternal plasma

Summary The discovery of the presence of fetal DMA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of p thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (–CTTT) mutation in the β globin gene from maternal plasma by real-time PCR. The specificity and sensitivity of the allele-specific assay was confirmed by subjecting plasma, buffy coat, and amniotic fluid samples from 100 pregnancies to screening for the mutation. Subsequently, the assay was applied to the prenatal testing of eight fetuses at risk of β thalassaemia major, the aim being to exclude fetal inheritance of paternally transmitted COdon 41/42 mutation. The fetal genotype was completely concordant with conventional analysis and β thalassaemia major was excluded in two of the pregnancies non-invasively.

The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia

BACKGROUND Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia. METHODS We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay. RESULTS CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann-Whitney test, P <0.001). CONCLUSION Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.

Prenatal detection of fetal Down's syndrome from maternal plasma

Fetal DNA is present in maternal plasma, and a proportion of such DNA is seen in intact fetal cells. We investigated the use of fluorescence in-situ hybridisation (FISH) techniques on maternal plasma samples. In plasma samples obtained from three women carrying fetuses affected by trisomy 21 (Downs syndrome), we identified fetal cells with three chromosome-21 signals. These results show the feasibility of non-invasive detection of fetal chromosomal aneuploidy by maternal plasma analysis.

Serial Analysis of Fetal DNA Concentrations in Maternal Plasma in Late Pregnancy

The discovery of fetal DNA in maternal plasma/serum has suggested new possibilities for noninvasive prenatal diagnosis (1) with applications in investigations of sex-linked diseases (2), rhesus D status (3), and β-thalassemia (4). Biologically, fetal DNA concentrations have been found to increase with gestational age, with a sharp increase toward the end of pregnancy (5). This latter phenomenon has been postulated to be related to the imminence of delivery (6). Consistent with the latter hypothesis, women with preterm labor have been found to have a higher concentration of fetal DNA in their plasma (7)(8). These results suggest that circulating fetal DNA concentrations are undergoing a time of flux during the late third trimester, but detailed information on the dynamics of maternal plasma fetal DNA is not available for this stage of gestation. In this study, we performed a serial analysis of fetal DNA concentrations in maternal plasma during the late third trimester. We recruited 33 healthy pregnant women from the Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong. All had ultrasonic evidence of male fetuses. Fetal sex was confirmed at delivery. Ethics approval was obtained from the Clinical Research Ethical Committee of the Chinese University of Hong Kong. Informed consent was obtained in all …

Circulating Fetal RNA in Maternal Plasma

Abstract: The discovery of cell‐free nucleic acids in plasma has opened up new possibilities for noninvasive clinical diagnosis. We demonstrate the presence of cell‐free fetal RNA in maternal plasma, indicating that plasma fetal RNA might be used as a marker for noninvasive prenatal diagnosis.

Prenatal diagnosis of intra-abdominal mature testicular teratoma.

Ovarian teratoma is a common neoplasm among women in their reproductive age. On the contrary, testicular teratoma is a rare tumor. 1,2 It usually appears during infancy as a palpable scrotal mass. We report a case of mature teratoma of the right undescended testis diagnosed on the basis of prenatal ultrasonography. The ultrasonographic diagnostic features and clinical importance will be discussed.