Extracellular chromosome 21-derived microRNAs in euploid & aneuploid pregnancies.

BACKGROUND & OBJECTIVES: Trisomy 21 is the most common chromosomal aneuploidy in live born infants. Recently, the over expression of chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802) in human fetal hippocampus and heart samples from individuals with Down syndrome was observed. Therefore, concentrations and expression profile of extracellular chromosome 21-derived microRNAs were studied to verify their ability to distinguish noninvasively between pregnancies bearing euploid fetuses and those affected with Down syndrome. METHODS: RNA enriched for small RNAs was isolated from plasma samples of 12 pregnant women with high risk of bearing Down syndrome foetuses (median gestation 18.5 wk), 12 women with normal course of gestation and 10 non-pregnant women. MicroRNA transcribed into cDNA using specific stem-loop primer was detected using real-time PCR assay. Simulation experiments using RNA pools of healthy non-pregnant individuals and aneuploid amniotic fluid samples in descending dilution ratio ranging from 1:1 to 1000:1 were used to test the detection limit of the technique for overexpressed chromosome 21-derived microRNAs specific for Down syndrome. The expression profile of the gene encoding microRNA was studied through the relative gene expression using the comparative Ct (threshold cycle) method. Concentrations of individual microRNAs were subtracted from the calibration curves in the course of analyses and expressed as pg of total RNA per milliliter of plasma. RESULTS: Four of the five extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) were reliably detected in plasma samples. Simulation experiments revealed the detection limit of aneuploidy at a ratio 100:1 for let-7c, miR-125b-2 and miR-155, and a ratio of 1000:1 for miR-99a. Overexpression of extracellular miR-99a, miR-125b-2 and miR-155 was observed in pregnant women compared to non-pregnant women. Similarly, increased concentrations of extracellular miR-99a and miR-125b-2 were detected in pregnant women than in non-pregnant women. The concentrations and relative gene expression of extracellular chromosome 21-derived microRNAs did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. INTERPRETATION & CONCLUSIONS: Analysis of extracellular chromosome 21-derived microRNAs has no benefit for screening programmes and non-invasive diagnosis of Down syndrome.

Absolute and relative quantification of placenta-specific micrornas in maternal circulation with placental insufficiency-related complications.

Placental insufficiency-related complications are one of the leading causes of maternal and perinatal morbidity and mortality. This study investigated the quantification of placenta-specific microRNAs (miRNAs) in the maternal circulation during gestation in a cohort of women with normally progressing pregnancies, the differentiation between placental insufficiency-related complications and normally progressing pregnancies, and the differentiation between placental insufficiency and normally progressing pregnancies during the early stages of gestation. Both absolute and relative quantification of placenta-specific miRNAs (ie, miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525, and miR-526a) was determined in 50 women with normally progressing pregnancies, 32 with complicated pregnancies [21 with preeclampsia with or without intrauterine growth retardation (IUGR) and 11 with IUGR], and 7 women with pregnancies at various gestational stages who later developed preeclampsia and/or IUGR using real-time PCR and a comparative C(T) method relative to normalization factor (ie, geometric mean of ubiquitous miR-16 and let-7d). Both quantification approaches revealed significant increases in extracellular placenta-specific miRNA levels over time in women with normally progressing pregnancies; however, they were not able to differentiate between normally progressing and complicated pregnancies at the time of preeclampsia and/or IUGR onset. Nevertheless, significant elevation of extracellular miRNA levels was observed during early gestation (ie, within the 12th to 16th weeks) in pregnancies with later onset of preeclampsia and/or IUGR. Early gestation extracellular miRNA screening can differentiate between women with normally progressing pregnancies and those who may later develop placental insufficiency-related complications.

Quantification of extracellular DNA using hypermethylated RASSF1A, SRY, and GLO sequences--evaluation of diagnostic possibilities for predicting placental insufficiency.

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.

The effect of DYS-14 copy number variations on extracellular fetal DNA quantification in maternal circulation.

The aims of our research involved to investigate DYS-14 copy number variations in healthy males, to quantify extracellular DNA in maternal circulation in normal versus complicated pregnancies, and to study variations in the DYS-14 copy number in extracellular male fetal DNA. Fifty-five healthy males, 43 uncomplicated male singleton pregnancies (23 sampled at the 16th week and 20 sampled at the 36th week), and 15 pregnancies with placental insufficiency (PI)-related complications (mean 34.1 weeks) were analyzed using real-time PCR with DYS-14 sequence, sex determining region Y (SRY), and beta-globin (GLO) genes used as markers. Increased levels of extracellular DNA were detected in PI-related complications relative to gestational age-matched controls (SRY, p < 0.001; DYS-14, p = 0.007; GLO, p < 0.001). When the mean + 2SD (standard deviation) of controls was used as a cutoff, SRY, DYS-14, and GLO achieved 91.7%, 68.8%, and 94.4% accuracy, respectively, for differentiation between normal and complicated pregnancies. Considerable variations in the DYS-14 copy number in healthy males (mean 52.6) and extracellular DNA were found. A lower DYS-14 copy number was observed in PI-related complications (mean 83.5) compared to uncomplicated pregnancies (16th week: mean 114.2, p = 0.02; 36th week: mean 142.8, p = 0.04). The DYS-14 copy number was higher in extracellular DNA throughout gestation relative to healthy males. We concluded that, regarding interindividual copy number variations, the DYS-14 sequence is not an optimal marker for extracellular fetal DNA quantification for differentiation between normal and complicated pregnancies.