Katerina Kotlabova

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.

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.

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.

Cardiovascular and Cerebrovascular Disease Associated microRNAs Are Dysregulated in Placental Tissues Affected with Gestational Hypertension, Preeclampsia and Intrauterine Growth Restriction.

Aims To demonstrate that pregnancy-related complications are associated with alterations in cardiovascular and cerebrovascular microRNA expression. Gene expression of 32 microRNAs (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-33a-5p, miR-92a-3p, miR-100-5p, miR-103a-3p, miR-122-5p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-208a-3p, miR-210-3p, miR-221-3p, miR-342-3p, miR-499a-5p, and miR-574-3p) was assessed in placental tissues, compared between groups (35 gestational hypertension, 80 preeclampsia, 35 intrauterine growth restriction and 20 normal pregnancies) and correlated with the severity of the disease with respect to clinical signs, delivery date, and Doppler ultrasound parameters. Initially, selection and validation of endogenous controls for microRNA expression studies in placental tissues affected by pregnancy-related complications have been carried out. Results The expression profile of microRNAs was different between pregnancy-related complications and controls. The up-regulation of miR-499a-5p was a common phenomenon shared between gestational hypertension, preeclampsia, and intrauterine growth restriction. Preeclamptic pregnancies delivering after 34 weeks of gestation and IUGR with abnormal values of flow rate in the umbilical artery demonstrated up-regulation of miR-1-3b. Preeclampsia and IUGR requiring termination of gestation before 34 weeks of gestation were associated with down-regulation of miR-26a-5p, miR-103a-3p and miR-145-5p. On the other hand, some of microRNAs (miR-16-5p, miR-100-5p, miR-122-5p, miR-125b-5p, miR-126-3p, miR-143-3p, miR-195-5p, miR-199a-5p, miR-221-3p, miR-342-3p, and miR-574-3p) were only down-regulated or showed a trend to down-regulation just in intrauterine growth restriction pregnancies requiring the delivery before 34 weeks of gestation. Conclusion Epigenetic changes induced by pregnancy-related complications in placental tissue may cause later onset of cardiovascular and cerebrovascular diseases in offspring.

Gestational hypertension, preeclampsia and intrauterine growth restriction induce dysregulation of cardiovascular and cerebrovascular disease associated microRNAs in maternal whole peripheral blood.

AIMS To demonstrate that pregnancy-related complications are associated with alterations in cardiovascular and cerebrovascular microRNA expression. Gene expression of 29 microRNAs (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-92a-3p, miR-100-5p, miR-103a-3p, miR-122-5p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-210-3p, miR-221-3p, miR-342-3p, miR-499a-5p, and miR-574-3p) was assessed in maternal whole peripheral blood, compared between groups (39 gestational hypertension, 68 preeclampsia, 33 intrauterine growth restriction and 20 normal pregnancies) and correlated with the severity of the disease with respect to clinical signs, delivery date, and Doppler ultrasound parameters. Initially, selection and validation of endogenous controls for microRNA expression studies in patients affected by pregnancy-related complications have been carried out. RESULTS The expression profile of microRNAs was different between pregnancy-related complications and controls. The down-regulation of miR-100-5p, miR-125b-5p and miR-199a-5p was a common phenomenon shared between gestational hypertension, preeclampsia, and intrauterine growth restriction. Moreover, IUGR pregnancies induced down-regulation of miR-17-5p, miR-146a-5p, miR-221-3p and miR-574-3p in maternal circulation. Irrespective of the severity of the disease, preeclampsia was associated with the dysregulation of miR-100-5p and miR-125b-5p and IUGR with dysregulation of miR-199a-5p. Preeclampsia requiring termination of gestation before 34 weeks was associated with down-regulation of miR-146a-5p, miR-199a-5p and miR-221-3p. Weak negative correlation between miR-146a-5p and miR-221-3p expression and the pulsatility index in the umbilical artery was found. Additional microRNAs (miR-103a-3p, miR-126-3p, miR-195-5p and miR-499a-5p) showed a trend to down-regulation in appropriate pregnancy-related complications. CONCLUSION Epigenetic changes are induced by pregnancy-related complications in maternal whole peripheral blood.

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.

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.

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma--next step toward reliable non-invasive prenatal diagnostics.

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n=51) or female (n=19) fetus sampled throughout gestation and absent in non-pregnant individuals (n=29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ=0.66, P<0.001), total RASSF1A and GLO (ρ=0.65,P<0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ=0.62, P<0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.

First trimester screening of circulating C19MC microRNAs and the evaluation of their potential to predict the onset of preeclampsia and IUGR

Objectives A nested case control study of a longitudinal cohort comparing pregnant women enrolled at 10 to 13 gestational weeks was carried out to evaluate risk assessment for preeclampsia and IUGR based on circulating placental specific C19MC microRNAs in early pregnancy. Methods The expression of placental specific C19MC microRNAs (miR-516b-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, and miR-525-5p) was determined in plasma samples from pregnancies that subsequently developed preeclampsia (n = 21), IUGR (n = 18), and 58 normal pregnancies using real-time PCR and comparative Ct method relative to synthetic Caenorhabditis elegans microRNA (cel-miR-39). Results Circulating C19MC microRNAs were up-regulated (miR-517-5p, p = 0.005; miR-518b, p = 0.013; miR-520h, p = 0.021) or showed a trend toward up-regulation in patients destined to develop preeclampsia (miR-520a-5p, p = 0.067; miR-525-5p, p = 0.073). MiR-517-5p had the best predictive performance for preeclampsia with a sensitivity of 42.9%, a specificity of 86.2%, a PPV of 52.9% and a NPV of 80.6%. The combination of all examined circulating C19MC microRNAs had no advantage over using only the miR-517-5p biomarker to predict the occurrence of preeclampsia (a sensitivity of 20.6%, a specificity of 90.8%, a PPV of 44.8%, and a NPV of 76.0%). Conclusions Up-regulation of miR-517-5p, miR-518b and miR-520h was associated with a risk of later development of preeclampsia. First trimester screening of extracellular miR-517-5p identified a proportion of women with subsequent preeclampsia. No circulating C19MC microRNA biomarkers were identified that could predict later occurrence of IUGR.

The Occurrence of Fetal Microchimeric Cells in Endometrial Tissues Is a Very Common Phenomenon in Benign Uterine Disorders, and the Lower Prevalence of Fetal Microchimerism Is Associated with Better Uterine Cancer Prognoses

This is the first study carried out to describe the role of fetal microchimerism (FM) in the pathogenesis of uterine cancer. The prevalence and concentration of male fetal microchimeric cells (FMCs) were examined in endometrial tissues in relation to subtypes of uterine cancer, and the histological grade and stage of the tumor. FM occurrence was analyzed in relation to risk factors, including hypertension, obesity, type 2 diabetes, dyslipidemia, age at cancer diagnosis, and patient pregnancy history. The prevalence and concentration of FMCs were examined in endometrial tissues using real-time polymerase chain reaction, SRY and β-globin sequences as markers for male fetal FMCs and total DNA. The studied group involved 47 type 1 endometrial cancers, 28 type 2 endometrial cancers, and 41 benign uterine diseases. While the prevalence of FM was decreased only in type 1 endometrial cancer, compared with benign uterine disorders (38.3% vs.70.7%; odds ratio [OR]=0.257, 95% confidence interval [CI]: 0.105 to 0.628, p=0.003), FMC concentrations did not differ within examined groups. The lower FM prevalence was detected in low-grade (grade 1 and grade 2) endometrioid cancer (38.3% vs. 70.7%, OR=0.256, 95% CI: 0.105 to 0.627, p=0.003) and in FIGO 1 tumors (40.7% vs. 70.7%, OR=0.285, 95% CI: 0.120 to 0.675, p=0.004). No correlation between FM prevalence or FMC concentrations and risk factors was demonstrated. A lower prevalence of male FM seemed to be associated with better prognoses in uterine cancer based on tumor subtype, histological grade, and stage of the tumor.