Cell-free fetal DNA in amniotic fluid: unique fragmentation signatures in euploid and aneuploid fetuses

Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are novel biomarkers with many clinical applications. Amniotic fluid (AF) is a rich source of cffDNA. We investigated the biophysical characteristics of cffDNA in AF, hypothesizing that they would differ from cffDNA in maternal plasma.

We obtained 10 mL of fresh AF supernatant from women carrying euploid fetuses (n = 39) and aneuploid fetuses (n = 4). To test the effects of storage and karyotype, samples from euploid fetuses (n = 19) and aneuploid fetuses with trisomies 21 (n = 16), 18 (n = 9), or 13 (n = 3); triploidy (n = 4); or monosomy X (n = 2) were frozen at -80 degrees C. AF cffDNA was characterized by real-time quantitative PCR amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and analysis of the DNA fragmentation signature.

We observed a significant correlation of concentration with gestational age for fresh AF cffDNA from euploid fetuses (R(2) = 0.77, P <0.0001) but not for frozen cffDNA (P = 0.63). The median amount of cffDNA in frozen euploid samples was significantly lower than in fresh samples (P <0.0001). After adjustment for gestational age, there was a statistically significant decrease in the median amount of cffDNA in frozen aneuploidy samples compared with frozen euploid samples (P = 0.0005). Analysis of the cffDNA size distribution showed different and qualitatively unique patterns for each karyotype.

Gestational age, karyotype, and sample storage time affect concentrations and fragment size of AF cff DNA. These effects may be attributable to fundamental differences in tissue sources, excretion modes, or kinetic pathways. Characteristic signature patterns for each common aneuploidy offer the possibility of using DNA fragmentation analysis as a means of triaging AF samples.