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.


Introduction
Normal pregnancy is associated with a systemic inflammatory response. Many of the physiologic changes of normal pregnancy are part of an acute-phase reaction, which is generated by an inflammatory response. The placenta is the proximal cause of these problems [1]. Since the placenta is being continuously remodelled during normal placental development, extracellular nucleic acids of both fetal and placental origin, packed into either trophoblast-derived apoptotic bodies or shedding syncytiotrophoblast microparticles, may be detected in maternal circulation during the course of normal gestation [2][3][4][5][6].
Circulating syncytiotrophoblast debris has been hypothesized to contribute to maternal inflammation and some of the features of the maternal syndrome [7]. Signs of maternal inflammation which appear to be present in normal pregnancies at term are exaggerated in preeclampsia (PE) and fetal growth restriction (FGR) and can account for their clinical features [1,8].
Preeclampsia and fetal growth restriction (FGR) are major complications affecting about 2-10% of pregnancies responsible for maternal and perinatal morbidity and mortality [9,10]. Preeclampsia usually develops after 20 weeks of gestation and is characterized by chronic or gestational hypertension combined with proteinuria, which results from defective placentation eliciting inadequate uteroplacental blood perfusion and ischemia [8,11,12]. The causes of preeclampsia and FGR remain unknown. Trophoblastic debris and the microparticles shed during normal pregnancy are proinflammatory and this process is amplified in preeclampsia [13]. A hypoxic environment induces excessive trophoblast cell death and increased shedding of placenta debris into the maternal circulation; as a result, placental insufficiency related pregnancy complications are associated with abnormal levels of extracellular fetal DNA and mRNA transcripts [5,14].
Recent evidence suggests that preeclampsia can be further subdivided into early PE (before 34 weeks of gestation), intermediate PE (between 34 and 37 weeks of gestation), and late PE (after 37 weeks of gestation) [15,16]. The concept of early and late PE is modern, and it is widely accepted that these two entities have different etiologies and should be regarded as different forms of the disease, where early onsets of PE and IUGR are considered to be placenta-mediated diseases [17][18][19].
MicroRNAs belong to a family of small noncoding RNAs that regulate gene expression at the posttranscriptional level by degrading or blocking translation of messenger RNA (mRNA) targets [40,41].
The results of our pilot study indicated no differentiation between normal and complicated pregnancies, but could not come to definitive conclusions due to the low number of studied subjects involved [51,54]. The current study is a followup of our previous studies [51,54] and describes comprehensively for the first time the expression profile of circulating C19MC microRNAs (miR-516-5p, miR-517 * , miR-518b, miR-520a * , miR-520h, miR-525, and miR-526a) in the entirely new sample set of patients with clinically established preeclampsia and/or fetal growth restriction. To our knowledge, no study describing the profile of circulating C19MC microRNAs in gestational hypertension has been carried out.

Patients.
The studied cohort consisted of Caucasian women involving 63 preeclampsia (PE) w or w/o fetal growth restriction (FGR), 27 FGR, 23 gestational hypertension (GH), and 55 controls. Twenty-four women had signs of mild preeclampsia, 39 women were diagnosed with severe preeclampsia, 24 preeclamptic patients required the delivery before 34 weeks of gestation and 39 patients delivered after 34 weeks of gestation. In 18 cases, preeclampsia superposed on previous hypertension; otherwise, it occurred in normotensive patients (45 cases). Eight growth-retarded foetuses were delivered before 34 weeks of gestation and 19 those after 34 weeks of gestation. Oligohydramnios or anhydramnios were present in 7 growth restricted foetuses.
Doppler studies showed an abnormal pulsatility index (PI) in the umbilical artery (14 preeclampsia ± FGR and 14 FGR) and/or in the middle cerebral artery (10 preeclampsia ± FGR and 11 FGR). Cerebroplacental ratio (CPR), expressed as a ratio between umbilical artery and middle cerebral artery pulsatility index, was below the fifth percentile in 21 cases (9 preeclampsia ± FGR and 12 FGR). Absent or reversed enddiastolic velocity waveforms in the umbilical artery occurred in 8 cases (2 preeclampsia + FGR and 6 FGR).
Normal pregnancies were defined as those without complications who delivered full term, singleton, healthy infants The association between the plasmatic concentration (absolute quantification) and gene expression levels (relative quantification) of C19MC microRNAs and Doppler ultrasonography parameters such as arteria umbilicalis pulsatility index, middle cerebral artery pulsatility index, and cerebroplacental ratio. PI: pulsatility index; : Spearman's correlation coefficient; P: level of significance; bold font: statistically significant results.
weighting >2500 g after 37 completed weeks of gestation. Preeclampsia was defined as blood pressure >140/90 mmHg in two determinations 4 hours apart that was associated with proteinuria >300 mg/24 h after 20 weeks of gestation. Severe preeclampsia was diagnosed by the presence of one or more of the findings according to the guidelines of ACOG Committee [11]. Fetal growth restriction was diagnosed when the estimated fetal weight (EFW), calculated using the Hadlock formula (Astraia Software GmbH), was below the tenth percentile for the evaluated gestational age.
Gestational hypertension was defined as high blood pressure that developed after the twentieth week of pregnancy.
All patients provided written informed consent. The study was approved by the Ethics Committee of the Third Faculty of Medicine, Charles University in Prague. The samples for the study were chosen on the basis of equal times in storage and gestation age. Gestational age was assessed using ultrasonography.

Processing of Samples.
Nine millilitres of peripheral blood were collected into EDTA tubes and centrifuged twice at 1200 g for 10 min at room temperature. Plasma samples were stored at −80 ∘ C until subsequent processing.
Total RNA was extracted from 1 mL of plasma and 25 mg of normal placental tissue preserved in RNAlater (Ambion, Austin, USA) followed by an enrichment procedure for small RNAs using a mirVana microRNA Isolation kit (Ambion, Austin, USA). Trizol LS reagent was used in plasma samples for total RNA extraction from biological fluids (Invitrogen, Carlsbad, USA) and preceded the small RNAs enrichment procedure. To minimize DNA contamination, we treated the eluted RNA with 5 L of DNase I (Fermentas International, ON, Canada) for 30 min at 37 ∘ C.

Reverse Transcriptase Reaction.
Each microRNA was reverse-transcribed into complementary DNA using TaqMan MicroRNA Assay, containing microRNA-specific stem-loop RT primers (Table 1), and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Branchburg, USA) in a total reaction volume of 50 L on a 7500 real-time PCR system (Applied Biosystems, Branchburg, USA) with the following thermal cycling parameters: 30 minutes at 16 ∘ C, 30 minutes at 42 ∘ C, 5 minutes at 85 ∘ C, and then held at 4 ∘ C.

Quantification of MicroRNAs.
15 L of cDNA corresponding to each microRNA was mixed with components of TaqMan MicroRNA Assay and the ingredients of the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, USA) in a total reaction volume of 35 L. TaqMan PCR conditions were set as described in the TaqMan guidelines. The analysis was performed using a 7500 realtime PCR system. All PCRs were performed in duplicates. A sample was considered positive if the amplification signal occurred before the 40th threshold cycle. Concentrations of individual microRNAs were expressed as pg of total RNA enriched for small RNAs per millilitre of plasma.
The expression of particular microRNA in maternal plasma was determined using the comparative Ct method [55] relative to the expression of the same microRNA in the reference sample, randomly selected placenta derived from gestation with normal course.
RNA fraction highly enriched for small RNA isolated from the fetal part of the placenta (the part of the placenta derived from the chorionic sac that encloses the embryo, consisting of the chorionic plate and villi) was used to buildup the standard curves and as a reference sample for relative quantification throughout the study.
Synthetic C. elegans microRNA (cel-miR-39, Qiagen, Hilden, Germany) was used as an internal control for variations during the preparation of RNA, cDNA synthesis, and real-time PCR. Due to a lack of generally accepted standards, all experimental real-time qRT-PCR data were normalized to cel-miR-39, as it shows no sequence homology to any human microRNA. 1 l of 0.1 nM cel-miR-39 was spiked in after incubation with Trizol LS reagent to the human plasma samples.

Statistical Analysis.
MicroRNA levels were compared between groups by nonparametric tests (the Mann-Whitney U test for the comparison between two groups and the Kruskal-Wallis test for the comparison between three or more groups) using Statistica software (StatSoft Inc., USA). Correlation between variables including absolute and/or relative microRNA quantification and Doppler ultrasonography parameters (the pulsatility index in the umbilical artery, the pulsatility index in the middle cerebral artery, and the cerebroplacental ratio) was calculated using the Spearman's rank correlation coefficient ( ). If it varies from −0.5 to 0, there is a weak negative correlation. The significance level was established at a value of < 0.05.

The Association Study of Circulating C19MC Micrornas and the Severity of the Disease with respect to Doppler
Ultrasonography Monitoring. The association between the plasmatic concentration and gene expression levels of C19MC microRNAs and Doppler ultrasonography parameters (the pulsatility index in the umbilical artery the pulsatility index in the middle cerebral artery, and the cerebroplacental ratio) was studied in the cohort of pregnancies complicated with preeclampsia and/or fetal growth restriction.
No difference within the group of complicated pregnancies with normal and abnormal values of flow rate in the umbilical artery was found out with the exception of miR-526a, which was upregulated in the group of patients with abnormal blood flow velocity waveforms (absolute quantification: = 0.038; relative quantification: = 0.05).
Further, the statistical analysis showed no effect of the pulsatility index in the middle cerebral artery and the cere- 195) of all microRNAs (miR-516-5p, miR-517 * , miR-520a * , miR-525, and miR-526a) that were identified to be upregulated in plasma samples derived from preeclampsia with or without fetal growth restriction.
The correlation between variables including absolute and/or relative quantification of particular microRNA in maternal plasma and the values of flow rate in the umbilical artery and the fetal blood vessel was calculated using Spearman's rank correlation coefficient. The pulsatility index in the umbilical artery did not show any correlation with microRNA plasmatic concentrations and/or microRNA gene expression. However, a weak negative correlation between the   Figure 2(b)) was found (Table 1).

Function and Functional Relationship Analysis of Target Genes of Differentially Expressed Extracellular C19MC
MicroRNAs in Preeclampsia. The function and functional relationship analysis of predicted targets of the five elevated extracellular C19MC microRNAs in patients with established preeclampsia indicated that a large group of genes was connected to the regulation of the immune system and inflammatory response ( Table 2). The data were collected from miRDB database (http://mirdb.org/miRDB/). All the targets were predicted by a bioinformatics tool MirTarget2, which was developed by analyzing thousands of genes impacted by miRNAs with an SVM learning machine.

Discussion
The results of our previous pilot study strongly supported the need for a more detailed exploration of extracellular microRNAs in maternal circulation with the view toward their recognition as potential biomarkers for placental insufficiency related complications [51,54]. Initially, some extracellular placental specific microRNAs (miR-516-5p, miR-520 * , miR-518b, and miR-526a) trended just to a higher level in the small cohort of patients with placental insufficiency related complications (16 preeclampsia, 5 preeclampsia with IUGR and 11 IUGR), however, did not reach statistical significance when compared to gestationalage-matched controls. Although we have previously demonstrated that normal pregnancies and the manifestation of placental-insufficiency-related complications did not influence the levels of miR-16 and let-7d in maternal plasma [51,54], it is now no doubt that ubiquitously expressed miR-16 and let-7d should not be further used to normalize expression profiles of various extracellular microRNAs as was done before in our pilot study. Our latest research revealed that the expression levels of miR-16 were significantly decreased in placental tissues derived from patients with preeclampsia (data submitted for publication). Similarly, Maccani et al. [56] also reported that reduced expression of miR-16 in placental tissue may be relevant to the low birth weight in term infants born small for gestational age. In contrast, miR-16 was previously observed to be overexpressed in placental tissues affected with severe preeclampsia, respectively [38]. The latest study of Wu et al. [44] brought the evidence of circulating mir-16 down-regulation in patients with severe preeclampsia. Furthermore, Yang et al. [43] showed decreased expression of circulating let-7d in preeclamptic patients applying more sophisticated approach such as next generation sequencing technology.
For that reason, alternative endogenous control candidates to normalize extracellular microRNA gene expression data should be used.
In the current study, the cohort of pregnancy related complications was expanded to achieve adequate power of the study. This time, the normalization of circulating microRNA expression was done against synthetic C. elegans microRNA (cel-miR-39). Using both absolute and relative quantification approaches, the ability of extracellular C19MC microRNAs (miR-516-5p, miR-517 * , miR-520a * , miR-525, and miR-526a) to differentiate between normal and complicated pregnancies  during the onset of preeclampsia w or w/o fetal growth restriction was confirmed. Unfortunately, limited data comparing extracellular C19MC microRNA levels between the groups of normal and complicated pregnancies are available. Our data are inconsistent with Yang et al. [43], who observed the upregulation of extracellular miR-520h in four patients with preeclampsia.
Our findings may be supported by Mouillet et al. [45], who have recently also observed no significant difference in relative placental specific microRNA levels (miR-518b) in plasma samples from those with normally progressing and fetal growth restriction pregnancies.
On the basis of the results of our study, we further studied the association between circulating C19MC microRNAs and the severity of the disease with respect to the degree of clinical signs, requirements for the delivery (before and after 34 weeks of gestation), and Doppler ultrasound examination.
The association analysis pointed to no relationship between C19MC microRNA plasmatic concentrations and/or gene expression and identified risk factors for a poorer perinatal outcome. There was no difference in microRNA plasmatic levels and/or gene expression between pregnancies with mild and severe preeclampsia, pregnancy-related complication with the need for the delivery before 34 weeks of gestation, and those who delivered after this critical period and pregnancies with abnormal and normal blood flow velocity waveforms. Nevertheless, the levels of miR-526a were significantly increased in the group of patients with abnormal values of flow rate in the umbilical artery.
On the other hand, 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 relation between the increased levels of plasmatic C19MC microRNAs (miR-516-5p, miR-517 * , miR-520a * , miR-525, and miR-526a) and decreased values of flow rate in the middle cerebral artery reached a statistical significance in complicated pregnancies.
Similarly, the relationship between increased levels of plasmatic C19MC microRNAs (miR-520a * and miR-526a) and decreased values of cerebroplacental ratio was revealed.
In conclusion, microRNAs play a fundamental role in a variety of physiological and pathological processes involving  TOX  ACVR2B  CD109  IL6ST  CD24  AHSA2  CD1A  IL9R  CD302  ATRN  DNAJC25  IRAK3  CFLAR  CD2  FLT1  LILRA2  DNAJC21  CD300LB  IL17RE  MTDH  HSP90AA1  CD46  IRAK1  PAPPA  IGFBP1  CD93  LILRB5  TLR2  HSF5  PDCD6IP  TNFRSF19  IGF1R  SOCS2  TNFSF15  IL10RA  TRAF6 MMD2 PPARA TLR7 VSIG4 All the targets were predicted by a bioinformatics tool MirTarget2 using miRDB online database. A negative regulator of T-cell responses structurally related to the B7 family of immune regulatory proteins pregnancy-related complications. Current study demonstrated for the first time that circulating C19MC microRNAs might play a role in the pathogenesis of preeclampsia, but not in the pathogenesis of gestational hypertension and fetal growth restriction. The study brought interesting finding that the upregulation of circulating C19MC microRNAs (miR-516-5p, miR-517 * , miR-520a * , miR-525, and miR-526a) is a characteristic phenomenon of established preeclampsia.