Circulating heat shock protein mRNA profile in gestational hypertension, pre-eclampsia & foetal growth restriction.

BACKGROUND & OBJECTIVES: Heat shock proteins (Hsp) are ubiquitously distributed phylogenetically conserved molecules that regulate cellular homeostasis and maintain the integrity and function of cellular proteins. Increased levels of Hsp in maternal circulation have been shown to be associated with increased risk of pregnancy related complications. The objective of this study was to explore extracellular Hsp mRNA levels in maternal circulation and quantified Hsp27, Hsp60, Hsp70, Hsp90 and Hsp70 binding protein 1 (HspBP1) mRNAs in maternal plasma samples using real-time reverse-transcriptase polymerase chain reaction. METHODS: Pregnancies with gestational hypertension (GH) (n = 33), pre-eclampsia (PE) with or without foetal growth restriction (FGR) (n = 78) and FGR (n = 25) were involved in the study. Hsp gene expression was analysed in relation to the severity of the disease with respect to the degree of clinical signs, requirements for the delivery and Doppler ultrasound parameters. RESULTS: Upregulation of Hsp70 was observed in patients with mild and severe PE (P = 0.004 and P = 0.005, respectively) and in pregnancies complicated with PE delivering before and after 34 wk of gestation regardless of the degree of clinical signs (P = 0.015 and P = 0.009, respectively). No difference in the expression of other Hsp genes among the studied groups was observed. No association between Hsp gene expression and Doppler ultrasonography parameters was found. INTERPRETATION & CONCLUSIONS: These data support that maternal circulation can reflect both maternal and foetal pathologic conditions. Hsp70 represents the sole plasmatic marker, and increased Hsp70 mRNA levels reflect maternal and placental stress response to pregnancy-related complications such as GH and PE, irrespective of the severity of the disease.

Assessment of placental and maternal stress responses in patients with pregnancy related complications via monitoring of heat shock protein mRNA levels.

The study describes the stress response in the central cotyledon zone of placental tissue and in maternal whole peripheral blood to pregnancy related complications including gestational hypertension (n = 31), preeclampsia w or w/o fetal growth restriction (n = 95), and fetal growth restriction (n = 39) using real-time RT-PCR and genes encoding Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 proteins. The placental tissue does not respond to pregnancy induced hypertension, fetal growth restriction and short-term severe preeclampsia that requires immediate termination of gestation. Upregulation of Hsp27, Hsp90 and HspBP1 appears just in case of long-term deteriorated conditions (usually in mild preeclampsia, that enable further continuation of gestation, when properly treated). On the other hand, maternal circulation is able to reflect both maternal and fetal pathologic conditions. While pregnancy related complications always induce upregulation of Hsp70 and downregulation of Hsp90 in maternal whole peripheral blood, the increase of Hsp60 mRNA levels occurs entirely in patients with preeclampsia and/or fetal growth restriction. Hsp60, Hsp70 and Hsp90 are dysregulated in maternal circulation irrespective of the severity of the disease (in both mild and severe preeclampsia) and the requirements for the delivery (before and after 34th week of gestation). Nevertheless, the highest Hsp60 mRNA levels may be observed in pregnancies with signs of the centralization of the fetal circulation associated with fetal hypoxia.

First trimester screening of circulating C19MC microRNAs can predict subsequent onset of gestational hypertension.

OBJECTIVE: The objective of the study was to evaluate risk assessment for gestational hypertension based on the profile of circulating placental specific C19MC microRNAs in early pregnancy. STUDY DESIGN: The prospective longitudinal cohort study of women enrolled at first trimester screening at 10 to 13 weeks was carried out (n = 267). Relative quantification of placental specific C19MC microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 28 normal pregnancies and 18 pregnancies which developed gestational hypertension using real-time PCR and a comparative Ct method relative to synthetic C. elegans microRNA (cel-miR-39). RESULTS: Increased extracellular C19MC microRNA plasmatic levels (miR-516-5p, p<0.001; miR-517*, p = 0.007; miR-520h, p<0.001; miR-518b, p = 0.002) were detected in patients destined to develop gestational hypertension. MiR-520h had the best predictive performance with a PPV of 84.6% at a 7.1% false positive rate. The combination of miR-520h and miR-518b was able to predict 82.6% of women at the same false positive rate. The overall predictive capacity of single miR-518b (73.3% at 14.3% FPR), miR-516-5p (70.6% at 17.9% FPR) and miR-517* (57.9% at 28.6% FPR) biomarkers was lower. CONCLUSION: The study brought interesting finding that the up-regulation of miR-516-5p, miR-517*, miR-520h and miR-518b is associated with a risk of later development of gestational hypertension. First trimester screening of extracellular miR-520h alone or in combination with miR-518b identified a significant proportion of women with subsequent gestational hypertension.

Circulating C19MC microRNAs in preeclampsia, gestational hypertension, and fetal growth restriction.

The objective of the study was to identify the profile of circulating C19MC microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525, and miR-526a) in patients with established preeclampsia (n = 63), fetal growth restriction (n = 27), and gestational hypertension (n = 23). We examined the correlation between plasmatic concentrations and expression levels of microRNAs and the severity of the disease with respect to clinical signs, requirements for the delivery, and Doppler ultrasound parameters. Using absolute and relative quantification approaches, increased extracellular C19MC microRNA levels (miR-516-5p, P = 0.037, P = 0.009; miR-517*, P = 0.033, P = 0.043; miR-520a*, P = 0.001, P = 0.009; miR-525, P = 0.026, P = 0.01; miR-526a, P = 0.03, P = 0.035) were detected in patients with preeclampsia. The association analysis pointed to no relationship between C19MC microRNA plasmatic concentrations and expression profile and identified risk factors for a poorer perinatal outcome. However, the dependence between the levels of plasmatic C19MC microRNAs and the pulsatility index in the middle cerebral artery and the values of cerebroplacental ratio was demonstrated. The study brought the interesting finding that the upregulation of miR-516-5p, miR-517*, miR-520a*, miR-525, and miR-526a is a characteristic phenomenon of established preeclampsia.

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.

Placental-specific microRNA in maternal circulation--identification of appropriate pregnancy-associated microRNAs with diagnostic potential.

The goal of this study was to identify placental specific microRNAs present in maternal plasma that differentiate between women with normal pregnancies and nonpregnant individuals. The selection of appropriate pregnancy-associated microRNAs with diagnostic potential was based on the following criteria: (1) detection rate of 100% in full-term placentas, (2) detection rate of ≥ 67% in maternal plasma throughout gestation (at least four positive wells out of six tested wells) and (3) not detectable in whole peripheral blood and plasma samples of nonpregnant individuals. Initially, we tested microRNAs (miR-34c, miR-372, miR-135b and miR-518b), which had been previously identified as pregnancy-associated microRNAs. Additionally, we selected 16 other highly specific placental microRNAs (miR-512-5p, miR-515-5p, miR-224, miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d, miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526a, and miR-526b) from the miRNAMap database. Seven microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) were identified as new pregnancy associated microRNAs with diagnostic potential. Their levels in maternal plasma during the 36th week of gestation corresponded to 45.0-427.0 pg of total RNA (enriched for small RNAs) per milliliter of maternal plasma.

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.

Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma.

OBJECTIVE: In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene. METHODS: We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery. RESULTS: Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhD-negative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D-C-E-c+e+ C(w)- and the infant D+C-E-c+e+ C(w)+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DC(w)- paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (C(T)) for RHD exon 10 or 7 amplicons reached nearly the same value like C(T) for control beta-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined. CONCLUSION: Prediction offoetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products.

Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies.

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies.

BACKGROUND: We investigated fetal and total DNA levels in maternal plasma in patients bearing fetuses affected with Down syndrome in comparison to controls carrying fetuses with normal karyotype. METHODS: DNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and beta-globin genes as markers. Twenty-one pregnant women with a singleton fetus at a gestational age ranging from 15 to 19 weeks recruited before amniocentesis (carried out for reasons including material serum screening and advanced material age), and 16 pregnant women bearing fetuses affected with Down syndrome between 17 to 22 weeks of gestation were involved in the study. RESULTS: The specificity of the system reaches 100% (no Y signal was detected in 14 women pregnant with female fetuses) and the sensitivity 91.7% (SRY amplification in 22 of 24 examined samples). The median fetal DNA levels in women carrying Down syndrome (n=11) and the controls (n=13) were 23.3 (range 0-58.5) genome-equivalents/ml and 24.5 (range 0-47.5) genome-equivalents/ml of maternal plasma, respectively (P = 0.62). The total median DNA levels in pregnancies with Down syndrome and the controls were 10165 (range 615-65000) genome-equivalents/ml and 7330 (range 1300-36750) genome-equivalents/ml, respectively (P = 0.32). The fetal DNA proportion in maternal plasma was 0%-6 % (mean 0.8%) in women carrying Down syndrome and 0%-2.6 % (mean 0.7 %) in the controls, respectively (P=0.86). CONCLUSIONS: Our study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.