Quantification of Extracellular DNA Using HypermethylatedRASSF1A,SRY, andGLOSequences—Evaluation of Diagnostic Possibilities for Predicting Placental Insufficiency

Abstract

This study evaluated quantification of fetal extracellular DNA in maternal plasma for differentiation between cases at risk of onset of placental-insufficiency-related complications and normal pregnancies. Using real-time polymerase chain reaction, fetal (sex-determining region Y [SRY] and hypermethylated RASSF1A sequence) and total (beta-globin [GLO] gene) extracellular DNA was examined in 70 normal pregnancies, 18 at risk of placental-insufficiency-related pregnancy complications, 24 preeclampsia with or without (w or w/o) intrauterine growth retardation (IUGR) (median 34.0 week), and 11 IUGR (median 28.5 week). IUGR was diagnosed when estimated fetal weight was below the 10th percentile for evaluated gestational age. Although increased levels of extracellular DNA were detected in pregnancies with preeclampsia w or w/o IUGR relative to controls (RASSF1A, p < 0.001; SRY, p = 0.009; GLO, p < 0.001), quantities of fetal extracellular DNA in IUGR were not statistically significant (RASSF1A, p = 0.21; SRY, p = 0.2). RASSF1A, SRY, and GLO achieved 93.1%, 93.6%, and 92.1% accuracy for differentiation between normal pregnancy and preeclampsia w or w/o IUGR. Lower sensitivity was observed for pregnancies with onset of IUGR (RASSF1A, 60.0%; SRY, 80.0%; GLO, 72.7%), but did not influence final accuracy (RASSF1A, 91.6%; SRY, 92.5%; GLO, 89.5%). Among 18 patients at risk, 8 pregnancies involving 3 female and 5 male fetuses developed preeclampsia (n = 4), IUGR (n = 3), and chronic placentopathy causing hypoxia (n = 1). Elevation of extracellular DNA was demonstrated in 3/5 (SRY), 1/8 (hypermethylated RASSF1A), and 4/8 (GLO) patients at the earliest 26 weeks and at the latest 2 weeks before the onset of symptoms. These data indicate that fetal and total extracellular DNA concentrations can be significantly elevated in plasma of patients who later developed placental-insufficiency-related pregnancy complications. However, this is strongly individualized, and not a rule for all cases, and probably depends on the actual occurrence of excessive placental trophoblast apoptosis.

Extracellular Nucleic Acids in Maternal Circulation as Potential Biomarkers for Placental Insufficiency

“…Similarly, our previous studies revealed significantly increased levels of extracellular fetal and total DNA in pregnancies with PE with or without IUGR (median 34 weeks, range 26-40 weeks), relative to controls (using SRY, hypermethylated RASSF1A sequence, and b-globin as markers and real-time PCR) (Hromadnikova et al, 2009(Hromadnikova et al, , 2010b. While increased levels of extracellular DNA were detected in pregnancies with PE w/or w/o IUGR relative to controls (RASSF1A p < 0.001; SRY p = 0.009; and GLO p < 0.001), quantities of fetal extracellular DNA in IUGR (median 28.5 weeks) were not statistically significant (RASSF1A p = 0.21; SRY p = 0.2).…”

“…Lo et al (1999) showed a fivefold increase in the median concentration of extracellular fetal DNA, using realtime quantitative PCR assays for the SRY gene on the Y chromosome, in pre-eclamptic pregnancies (mean 32 weeks, range 27-41 weeks) compared with gestation age-matched controls). These data were later confirmed by other investigators using real-time quantitative PCR and either the SRY gene or the DYS-14 sequence as markers to differentiate between normal and complicated pregnancies (Smid et al, 2001;Zhong et al, 2001;Lau et al, 2002;Farina et al, 2004b;Engel et al, 2007;Alberry et al, 2009;Hromadnikova et al, 2009Hromadnikova et al, , 2010b. It was suggested that a rise in fetal DNA represented a valuable marker of placenta-related pregnancy complications, which could predict PE several weeks before clinical manifestation; however, with regard to IUGR, a rise in fetal DNA was less well correlated (Caramelli et al, 2003;Sekizawa et al, 2003;Farina et al, 2004c;Zhong et al, 2007;Hromadnikova et al, 2010b).…”

“…93.6%, and 92.1% accuracy for differentiation between normal pregnancy and those with PE w/or w/o IUGR, respectively (Hromadnikova et al, 2010b). Lower sensitivity was observed for all examined markers in pregnancies with the onset of IUGR (RASSF1A: 60.0%; SRY: 80.0%; and GLO: 72.7%), but it did not influence the final accuracy (RASSF1A: 91.6%; SRY: 92.5%; and GLO: 89.5%).…”

“…While our data indicated an elevation of extracellular fetal and total DNA in patients at a risk of placental-insufficiency-associated pregnancy complications before the onset, this phenomenon was strongly individualized, could not be generalized to all cases, and was probably dependent on excessive placental trophoblast apoptosis. Interestingly, SRY and GLO quantification provided superior results compared with the hypermethylated RASSF1A sequence (Hromadnikova et al, 2009(Hromadnikova et al, , 2010b.…”

“…Our study evaluated the quantification of fetal extracellular DNA in maternal plasma for the differentiation between cases at a risk of onset of placental-insufficiency-related complications and normal pregnancies (Hromadnikova et al, 2010b). Using real-time PCR, fetal (SRY and hypermethylated RASSF1A) and total (GLO gene) extracellular DNA were examined in 18 pregnancies at a risk of placental-insufficiency-related pregnancy complications.…”