OBJECTIVE: To analyze methodological influences and characterize the concentrations of cell-free fetal DNA (cffDNA) circulating in maternal plasma at different gestational ages in physiological pregnancies. METHODS: We investigated 238 independent samples from single male-bearing pregnancies of different gestation age. In the other 50 pregnancies, the samples were collected three times during pregnancy (at all trimesters) to evaluate the kinetics of cffDNA. The manual and automated DNA extraction methods (Roche) were compared. cffDNA was amplified using real-time PCR method and Y-specific sequences SRY and DYS14. Total cell-free DNA circulating in maternal plasma was determined by the use of the GADPH sequence. RESULTS: The elevation in the concentration of cffDNA during pregnancy with the highest value in the third trimester was observed independently on the DNA extraction method and on the Y-specific amplified sequence. The same is documented for the percentage of fetal DNA in total cell-free DNA in maternal plasma. It increases also in successive trimesters (8.3, 10.7 and 23.2%). CONCLUSIONS: We discuss methodological problems and describe statistical parameters of cffDNA concentrations in maternal plasma during pregnancy as the basic information for comparison with pregnancies having a pathological outcome. Copyright 2008 S. Karger AG, Basel

3rd Medical Department Charles University Pilsen Czech Republic

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