Maternal urine for prenatal diagnosis--an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester.

OBJECTIVES: The aim of the study was the detection, quantification and correlation of cell-free fetal (cff) DNA in maternal urine and plasma in normal and complicated pregnancies during the third trimester. METHODS: One hundred and fifty-one urine and plasma samples obtained from 96 women carrying male fetuses, and 55 carrying female fetuses were collected and analyzed for cff-DNA using fluorescent PCR and quantitative real-time PCR. DNA was extracted from 1 mL maternal urine and analyzed with two different primer sets (SRY and DYS-14). The concentrations of cff and total DNA in maternal plasma were correlated with maternal and obstetric parameters using appropriate correlation analyses. RESULTS: Y-chromosome-specific sequences were detected in 31 of 96 (32.3%) urine samples collected from women pregnant with male fetuses using DYS-14 and in 6 of 96 (6.3%) urine samples using SRY as primers using real-time PCR. All 96 plasma samples obtained from women carrying male fetuses were positive for cff-DNA using real-time PCR. Cff-DNA exhibited a correlation with gestational age (R = 0.244; P = 0.018) and an inverse correlation with the latency between blood collection and birth (R = - 0.218; P = 0.036). Total DNA showed a correlation with placental weight (R = 0.182; P = 0.034) and pregnancy-associated complications (R = 0.280; P < 0.001). CONCLUSION: Our data confirm that cff-DNA is cleared by the kidneys in detectable amounts, but due to its low concentration or problematic detection in maternal urine this source seems inappropriate for noninvasive prenatal diagnosis.

A prospective analysis of cell-free fetal DNA concentration in maternal plasma as an indicator for adverse pregnancy outcome.

OBJECTIVES: To evaluate whether cell-free fetal (cff) DNA in maternal plasma during the second trimester is a marker for developing pregnancy-associated complications. Two PCR techniques for the detection and quantitation of fetal DNA were compared. METHODS: Plasma samples were prospectively collected from 84 pregnant women carrying male fetuses before amniocentesis (14-29 weeks). We later recorded 26 pregnancies with complicated outcomes, including five cases of fetal chromosomal abnormalities. For statistical analysis, two overlapping subgroups A and B were made. Each group was separately compared for total and fetal DNA with a corresponding group considered normal using Wilcoxon rank sum test. Male fetal DNA concentration in maternal plasma was quantified using real-time quantitative polymerase chain reaction (PCR) of SRY sequences. The samples were also analyzed by quantitative fluorescent PCR (QF-PCR) using highly polymorphic short tandem repeat DNA sequences (STRs), and the percentage of relative fetal allele concentration in maternal alleles was calculated and compared to the fetal/total DNA ratio obtained by real-time PCR. RESULTS: Quantities of total and fetal circulating DNA were significantly correlated (r(2) = 0.44, P < 0.0001) with a median total DNA concentration of 522 GE/mL (range 51-3047) and a median fetal DNA concentration of 8 GE/mL (range 0-879). Neither level was correlated with gestational age in pregnancies with normal (r(2) = -0.05; P = 0.66, and r(2) = 0.02; P = 0.88, respectively) and abnormal (r(2) = 0.45; P = 0.17, and r(2) = 0.11; P = 0.76, respectively) outcomes. Although both total and fetal DNA levels were always higher in women carrying pregnancies with chromosomal aberrations or having other pregnancy complications (P-values range from 0.028 to 0.267), these differences reached statistical significance only for total DNA levels between the group A and corresponding normal pregnancies (P = 0.028). The correlation between the fetal/total DNA ratio obtained by real-time PCR and the percentage of relative fetal allele concentration in maternal alleles obtained by QF-PCR was not found to be statistically significant (r(2) = 0.04; P = 0.76). CONCLUSION: Our results confirm the clinical value of fetal DNA measurement in maternal plasma during the second trimester as a supplement for the diagnosis of aneuploidies. Its use as a screening instrument for complications that develop later in pregnancy seems to be limited but needs further investigation. Although the QF-PCR assay has the advantage of being applicable to both female and male fetuses, this approach cannot be used for quantitation of cff DNA in maternal plasma samples.