Postnatal Expression Profile of MicroRNAs Associated with Cardiovascular Diseases in 3- to 11-Year-Old Preterm-Born Children

(1) Background: Preterm-born children have an increased cardiovascular risk with the first clinical manifestation during childhood and/or adolescence. (2) Methods: The occurrence of overweight/obesity, prehypertension/hypertension, valve problems or heart defects, and postnatal microRNA expression profiles were examined in preterm-born children at the age of 3 to 11 years descending from preterm prelabor rupture of membranes (PPROM) and spontaneous preterm birth (PTB) pregnancies. The whole peripheral blood gene expression of 29 selected microRNAs associated with cardiovascular diseases was the subject of our interest. (3) Results: Nearly one-third of preterm-born children (32.43%) had valve problems and/or heart defects. The occurrence of systolic and diastolic prehypertension/hypertension was also inconsiderable in a group of preterm-born children (27.03% and 18.92%). The vast majority of children descending from either PPROM (85.45%) or PTB pregnancies (85.71%) had also significantly altered microRNA expression profiles at 90.0% specificity. (4) Conclusions: Postnatal microRNA expression profiles were significantly influenced by antenatal and early postnatal factors (gestational age at delivery, birth weight of newborns, and condition of newborns at the moment of birth). These findings may contribute to the explanation of increased cardiovascular risk in preterm-born children. These findings strongly support the belief that preterm-born children should be dispensarized for a long time to have access to specialized medical care.


Introduction
Preterm-born children have an increased cardiovascular risk, with the first clinical manifestation during childhood and/or adolescence. Usually, clinical symptomatology appears during early adulthood at the very latest.
A strong association between preterm birth before 32 weeks of gestation and the risk of incident heart failure [53] and cerebrovascular disease [54] has been recently reported in children and/or young adults. In parallel, low gestational age at birth has been observed to be associated with increased risk of venous thromboembolism in young adulthood [55]. In addition, an association between low birth weight and the risk of ischemic heart disease and myocardial infarction in young adulthood has been demonstrated, but independently of gestational age at delivery [56].
Besides, children and young adults born extremely preterm had significantly altered neuropsychological outcomes (lower general cognitive abilities, visuomotor skills, prospective memory, and aspects of executive functions and language) [32,57,58]. Gestational age of birth, including preterm birth, has also been observed to be associated with the risk of autism spectrum disorders [59].
Recently, it has been shown that children descending from pregnancies affected with hypertensive disorders (gestational hypertension (GH) or preeclampsia (PE)), metabolic disorders (gestational diabetes mellitus (GDM)), and/or fetal growth restriction (FGR) have altered postnatal expression profiles of microRNAs, playing a role in the pathogenesis of diabetes mellitus and cardiovascular diseases. Altered expression of these microRNAs present in peripheral blood leukocytes of children at the age of 3-11 years may contribute, besides other factors, to the development of diabetes mellitus and cardiovascular diseases later in life [60][61][62].
In addition, the microRNA expression profiles of groups of preterm-born children (descending from either PPROM or PTB pregnancies) and term-born children with re-gard to the presence of abnormal clinical findings (overweight/obesity, prehypertension/hypertension, valve problems and heart defects) were compared. In parallel, the impact of multiple antenatal and early postnatal factors on postnatal microRNA expression profiles of children was studied. These factors involved therapies applied to mothers to postpone the preterm delivery with the aim to accelerate fetal lung maturation (corticosteroid therapy, antibiotic therapy, and tocolytic therapy), gestational age at delivery, mode of delivery, and clinical parameters of newborns (the birth weight, and the condition of infants at the moment of birth determined using the Apgar scores and pH of cord arterial blood).
To the best of our knowledge, there are currently no data on postnatal expression profiles of microRNAs associated with cardiovascular diseases in preterm-born children if other pregnancy-related complications (GH, PE, FGR, and GDM) are not present. In addition, no data on the long-term impact of corticosteroid, tocolytic, and antibiotic therapies administered to pregnant women at risk of preterm delivery on postnatal microRNA expression profile of preterm-born children are available.

Participants
A total of 355 preterm-born children fulfilling the inclusion criteria of the study were identified in the medical databases of the Institute for the Care of the Mother and Child and were invited to participate in our study. Finally, 111 preterm-born children arrived at examination, which indicates a 31.27% success rate.
The inclusion criteria involved preterm-born children at the age of 3-11 years descending from singleton pregnancies affected with PPROM and PTB. PTB was defined as preterm delivery prior to the 37th gestational week with the occurrence of regular uterine contractions (at least 2 contractions within 10 min), together with cervical changes. In case of PPROM, leakage of amniotic fluid appeared at a minimum of 2 h before the onset of labor, once again prior to the 37th gestational week [66][67][68]. Term-born children were defined as healthy infants weighing more than 2500 g, who were born after 37 completed weeks of gestation to women with a normal course of gestation (no presence of medical, obstetrical, or surgical complications). Term-born children were selected randomly from medical databases on the basis of equal age.
The exclusion criteria involved children descending from preterm birth pregnancies complicated, in addition, with hypertensive disorders, gestational diabetes mellitus, and fetal growth restriction. Concurrently, children affected with inborn defects and chromosomal aneuploidy disorders were not involved in the study. In addition, preterm-born children descending from pregnancies affected with placenta previa, placental abruption, and significant vaginal bleeding were also not included in the study.
The prospective study finally included various ethnic populations of Caucasian children living in the Czech Republic descending from pregnancies with a normal course of gestation (NP, n = 92), and pregnancies complicated with PPROM (n = 55) and PTB (n = 56). The national composition of the inhabitants of the Czech Republic is the following: the Bohemians, the Moravians, the Silesians, the Poles, the Slovaks, the Germans, the Ukrainians, the Hungarians, and the Russians, all belonging to Caucasian population. In the Czech Republic ethnic minorities are represented by the Vietnamese people (Mongoloid race, 0.3%) and Romani people (Gypsies, various ethnic subgroups, 0.1%) (demographic data collected in 2011 during the census). Only a few preterm-born children from these ethnic minority groups were delivered in the Institute for the Care of the Mother and Child, Prague, and none of them were interested in participating in our study. The clinical characteristics of preterm-and term-born children are summarized in Table 1. Maternal prenatal corticosteroid administration to accelerate fetal lung maturation was applied in 82 out of 111 women at risk of preterm delivery [69][70][71]. Prophylactic antibiotic therapy was administered to 86 out of 111 women at risk of preterm delivery [72,73]. The tocolytic therapy was applied, only if non-contraindicated, to 64 out of 111 women at risk of preterm delivery [72,74,75].
The case group involved 19 extremely preterm children (delivered before the 28th gestational week), 33 very preterm children (delivered within the 28th and 32nd gestational weeks) and 59 moderate to late preterm children (delivered within the 32nd and 37th gestational weeks) [72,75,76]. Forty-six preterm-born children were delivered via cesarean section and 65 children were delivered via vaginal birth.
The Apgar scores were determined after the birth of newborns at 1 min, 5 min, and 10 min [77][78][79][80]. A reassuring Apgar score after five minutes (7-10) was detected in 101 preterm-born infants. A moderately abnormal Apgar score after five minutes (4-6) was detected in six preterm-born infants, and a concerning Apgar score after five minutes (0-3) was detected in three preterm-born infants. Significant metabolic acidosis (umbilical cord arterial blood pH < 7.0) was detected in 7 out of 111 preterm-born infants.
Informed consents were signed by legal representatives of the children included in the study. The Ethics Committees (the Institute for the Care of the Mother and Child and the Third Faculty of Medicine, Charles University, Prague, Czech Republic) approved the study (grant no. AZV 16-27761A, "Long-term monitoring of complex cardiovascular profile in the mother, fetus and offspring descending from pregnancy-related complications", dates of approval: 27 March 2014 and 28 May 2015). All procedures were in compliance with the Helsinki declaration of 1975, as revised in 2000.

Clinical Examination of Children
Standardized BMI assessment, BP measurement and an ultrasound of the heart (echocardiography) were performed as described previously [60][61][62]. At least three BP measurements were performed on different occasions. Children with normal BP had average SBP and DBP values below the 90th percentile for age, sex, and height. Children with prehypertension had repeatedly average systolic or diastolic BP within the range of ≥90th percentile and <95th percentile. Children with hypertension had during three independent measurements average systolic or diastolic BP ≥ 95th percentile [81].
The BMI in children was assessed using the age-specific and sex-specific BMI percentile calculator [82]. Children with normal BMI values ranged within >5th percentile and <85th percentile. Overweight children had BMI within the range of >85th percentile and <95th percentile. Obese children were those with BMI ≥95th percentile.
A complete two-dimensional echocardiography was performed by a pediatrician experienced in echocardiography using a Philips HD15 ultrasound machine (Philips Ultrasound, Bothell, WA, USA) and a sector array transducer (3)(4)(5)(6)(7)(8) incorporating color flow, pulse-wave, and continuous-wave Doppler measurements with adaptive technology. Preterm-and term-born children with abnormal clinical findings were referred to the Department of Pediatric Cardiology.

Processing of Samples and Relative MicroRNA Quantification via Real-Time RT-PCR
Processing of samples, reverse transcription, and relative quantification of microR-NAs were done as previously described [60][61][62][63][64][65]. In brief, homogenized cell lysates were prepared from samples of unclotted whole peripheral venous blood (200 µL) as soon as possible after blood collection using a QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany, no. 52304).
Reverse transcription was performed using microRNA-specific stem-loop RT primers, the components of TaqMan MicroRNA Assays, and a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Branchburg, NJ, USA, no. 4366597) in a total reaction volume of 10 µL on a 7500 Real-Time PCR system (Applied Biosystems, Branchburg, NJ, USA) under the following conditions: 30 min at 16 • C, 30 min at 42 • C, 5 min at 85 • C, and then held at 4 • C.
Relative quantification of microRNAs was performed using real-time PCR in a total reaction volume of 15 µL, in which case cDNA (3 µL) was mixed with specific TaqMan MGB primers and probes (TaqMan MicroRNA Assay; Applied Biosystems, Branchburg, NJ, USA), and the constituents of TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ, USA, no. 4318157). The samples were tested in duplicate and regarded as positive if the amplification occurred at Ct <40 (Ct, threshold cycle). The expression of the studied microRNAs was determined using the comparative Ct method [83], and normalized to endogenous controls with the lowest expression variability between the studied samples (geometric mean of RNU58A and RNU38B) [84]. A reference sample, the fetal part of one randomly selected placenta of normally ongoing pregnancy, was used throughout the study for relative microRNA quantification.

Processing of Experimental Data
Logistic regression was used to compare the presence of abnormal clinical findings (the incidence of overweight/obesity, systolic and diastolic prehypertension/hypertension, valve problems or heart defects) among the groups of preterm and term-born children. Since microRNA gene expression data did not show a normal distribution using the Shapiro-Wilk test [85], microRNA levels were compared among the appropriate groups using non-parametric tests (Mann-Whitney test and Kruskal-Wallis test).
Using receiver operating characteristic (ROC) curves, the areas under the curves (AUC), the optimal cut-off points, and the respective sensitivities at 10.0% false positive rate (FPR) for the studied microRNAs were assessed (MedCalc Software bvba, Ostend, Belgium). The p-values of p < 0.05 were considered statistically significant. Logistic regression combined with ROC curve analysis was used to assess the optimal combinations of microRNA biomarkers (MedCalc Software bvba, Ostend, Belgium). In brief, in this setting, the power of the model's predicted values to discriminate between positive and negative cases is quantified by the area under the ROC curve. To perform a full ROC curve analysis, the predicted probabilities are first saved and then used as a new variable in ROC curve analysis. The dependent variable used in logistic regression then acts as the classification variable in the ROC curve analysis dialog box.
The box plots representing log-normalized expression values [quantitative reverse transcription polymerase chain reaction (RT-qPCR) expression, log 10 2 −∆∆Ct ] for selected individual microRNAs with the best sensitivity at 10.0% FPR are presented.
Correlation between variables was calculated using the Spearman rank correlation coefficient (ρ). If the correlation coefficient values ranged within <−1.0; −0.5>, there was a strong negative correlation. If it was within the interval <−0.5; 0>, there was a weak negative correlation.

Information on MicroRNA-Gene-Biological Pathways/Disease Interactions
The MiRWalk database and the Predicted Target module were used to provide information on predicted targets of microRNAs dysregulated in the whole peripheral blood of preterm-born children [86].
MiRWalk is a comprehensive database that provides information on microRNAs from humans, mice, and rats on their predicted and/or validated target genes. miRWalk2.0 not only documents miRNA binding sites within the complete sequence of a gene, but also combines this information with a comparison of binding sites from 12 existing miRNA-target prediction programs (DIANA-microTv4.0, DIANA-microT-CDS, miRanda, mirBridge, miRDB4.0, miRmap, miRNAMap, DoRiNA, PicTar2, PITA, RNA22v2, RNAhybrid2.1, and Targetscan6.2) to build novel, comparative platforms of binding sites for the promoter (4 prediction datasets), cds (5 prediction datasets), 5 -UTR (5 prediction datasets), and 3 -UTR (13 prediction datasets) regions. Information on the miRNA-target interactions of 2035 disease ontologies (DO), 6727 human phenotype ontologies (HPO), and 4980 OMIM disorders is available. This information provides possible interactions between the microR-NAs and genes associated with the 597 KEGG, 456 Panther, and 522 Wiki pathways.
Normal clinical findings (normal anamnesis, normal BP, normal BMI, and normal reference values of echocardiographic measurements) were found in 45 out 111 pretermborn children and in 53 out of 92 term-born children.
Logistic regression revealed a higher incidence of valve problems and/or heart defects and a higher incidence of systolic prehypertension/hypertension in a group of pretermborn children (PPROM and PTB). There was also a trend toward a higher incidence of diastolic prehypertension/hypertension in a group of preterm-born children (PPROM and PTB). However, no difference in the incidence of overweight/obesity between the groups of preterm and term-born children was observed ( Table 2). Logistic regression is used to compare the incidence of abnormal clinical findings between the groups of preterm-and term-born children.
The significance level is established at a p-value of < 0.05. A higher incidence of valve problems and/or heart defects is observed in a group of preterm-born children (PPROM and PTB). In addition, a higher incidence of systolic prehypertension/hypertension is observed in a group of preterm-born children (PPROM and PTB). Diastolic prehypertension/hypertension shows a trend toward statistical significance in a group of preterm-born children (PPROM and PTB). No difference in the incidence of overweight/obesity is observed between the groups of preterm and term-born children.
MicroRNA gene expression profiles differentiated between children descending from PPROM pregnancies and term-born children with a sensitivity (range 16.36-78.18%) at 10.0% FPR (Supplementary Table S1). Likewise, microRNAs were able to differentiate between children descending from PTB pregnancies and term-born children with a sensitivity (range 17.86-73.21%) at 10.0% FPR (Supplementary Table S1).

Postnatal MicroRNA Expression Profiles in Preterm-Born Children (PPROM and PTB) with Regard to the Absence or Presence of Abnormal Clinical Findings (Overweight/Obesity, Prehypertension/Hypertension, Valve Problems and Heart Defects)
Furthermore, microRNA expression profiles between groups of preterm-born children (descending from either PPROM or PTB pregnancies) and term-born children with regard to the presence and the absence of abnormal clinical findings (overweight/obesity, prehypertension/hypertension, valve problems and heart defects) were compared.
In addition, an increased expression of miR-23a-3p and miR-145-5p was observed in both groups of preterm-born children with the presence of abnormal clinical findings (PPROM (Supplementary Table S2

Postnatal MicroRNA Expression Profiles in Preterm-Born Children (PPROM and PTB) with Regard to Mode of Delivery
No association between the mode of delivery (vaginal birth and cesarean birth) and postnatal microRNA gene expression was observed in preterm-born children (PPROM and PTB).

Postnatal MicroRNA Expression Profiles in Preterm-Born Children (PPROM and PTB) with Regard to Condition of Newborns at Moment of Birth
No association between the Apgar scores (AS) determined at 5 min and 10 min after the birth of newborns and postnatal microRNA gene expression was observed in preterm-born children with the exception of miR-133a-3p, which showed a weak negative correlation with the Apgar scores (AS) determined at 5 min (ρ = −0.227; p = 0.017) and 10 min (ρ = −0.192; p = 0.045) after the birth of newborns.

Postnatal MicroRNA Expression Profiles in Preterm-Born Children (PPROM and PTB) with Regard to Antenatal Application of Corticosteroids, Antibiotics, and Tocolytics to Mothers
The antenatal administration of corticosteroids to mothers with the aim to accelerate lung maturation in the fetus had no impact on the postnatal microRNA gene expression profile in preterm-born children (PPROM and PTB). In parallel, the treatment with tocolytics to postpone premature labor also had no impact on the postnatal microRNA gene expression profile in preterm-born children (PPROM and PTB). Similarly, no association between the antibiotic administration to women in preterm delivery and postnatal microRNA gene expression was observed in preterm-born children (PPROM and PTB).

Information on MicroRNA-Gene-Biological Pathways/Disease Interactions
Information on microRNA-gene-biological pathways/disease interactions was provided on microRNAs dysregulated in the whole peripheral blood of preterm-born children. Predicted targets of microRNAs involved in key human biological pathways, with a role in the pathogenesis of preterm delivery, were reported. These biological pathways involve apoptosis, inflammatory response, senescence, and autophagy. In addition, predicted targets of microRNAs associated with cardiovascular risk factors (hypertension, congenital heart defects, patent foramen ovale, patent ductus arteriosus, and heart valve disease) that appeared more frequently in our group of preterm-born children were also reported.

Discussion
Initially, a comprehensive physical examination of preterm-and term-born children delivered in our hospital to obtain baseline medical information was performed. Standardized BMI assessment, BP measurement, and an ultrasound of the heart (echocardiography) were performed in children at the age of 3 to 11 years. From the clinical examination, it was evident that nearly one-third of preterm-born children descending from PPROM or PTB pregnancies (32.43%) had valve problems and/or heart defects that should be checked at least by pediatric cardiologists and if needed the children should be dispensarized to have access to specialized medical care.
In addition, the occurrence of systolic prehypertension/hypertension (27.03%) and diastolic prehypertension/hypertension (18.92%) was also inconsiderable in a group of preterm-born children (PPROM or PTB) when compared with term-born children. No child had a confirmed diagnosis of chronic hypertension or was on blood pressure treatment, when physical examination was performed. Definitely, children indicated to have prehypertension/hypertension during several visits should be checked from a long-term perspective to exclude an accidental BP increase resulting from the visit to the medical department or to confirm the diagnosis of chronic hypertension.
A certain concern arises also from the fact that the incidence of overweight/obesity was nearly the same within the groups of age-matched preterm and term-born children in spite of the substantially lower weight of preterm-born newborns.
Nevertheless, the findings resulting from this study were consistent with the findings of other researchers who identified increased peripheral SBP, increased peripheral DBP and/or a trend toward increased peripheral DBP in preterm-born children [4,[6][7][8][10][11][12]. The data were also in compliance with the observation of another scientific group [40], which found no differences in BMI and waist-hip ratio between full term, healthy preterm and sick preterm young adults. Nevertheless, this study was not held during childhood, but at early adulthood, at the age of 23 years [40].
With the exception of a study reporting a higher prevalence of congenital heart disease inclusive of atrial and ventricular septal defects and patent ductus arteriosus in pretermborn school children [87] and a study demonstrating a higher prevalence of interarterial septal aneurysm in young adults born preterm [88], no data on the incidence of valve problems (tricuspid, mitral, pulmonary, and bicuspid aortic valve regurgitations) or other heart defects (foramen ovale apertum, and arrhythmia) are available in preterm-born children older than 3 years.
Furthermore, postnatal microRNA expression profiles were compared between preterm and term-born children. Preterm-born children descending from pregnancies with other parallel complications were not involved in the study by intent, since postnatal microRNA gene expression in children prenatally affected with fetal growth restriction, maternal hypertensive and metabolic disorders had already been investigated separately [60][61][62]. Concurrently, preterm-born children, whose mothers had labor complicated with placenta previa, placental abruption, and significant vaginal bleeding, were also not included in the study.
The majority of the tested microRNAs (25 out of 29 microRNAs) showed higher expression levels in the whole peripheral blood of preterm-born children, irrespective of the condition which led to preterm delivery (PPROM or PTB). The postnatal screening based on only one microRNA, miR-1-3p, reached a very high accuracy to discriminate between preterm and term-born children. In total, 78.18% children descending from PPROM pregnancies and 73.21% children descending from PTB pregnancies showed increased postnatal miR-1-3p expression at 10.0% FPR (Supplementary Table S1, Supplementary  Figures S1 and S2).
So far, this finding is comparable with the finding in children descending from GDM pregnancies, where our group identified, through combination screening, altered postnatal microRNA expression profiles in 88.14% of the children [61]. With respect to this fact, preterm-born children descending from either PPROM or PTB pregnancies have, next to children descending from GDM pregnancies, the most serious alterations in postnatal microRNA expression profiles out of all groups of children affected with pregnancy-related complications [60][61][62].
On the grounds of these clinical and experimental findings, we strongly support the efforts of other investigators suggesting the long-term dispensarization of pretermborn children as a highly risky group for a later development of cardiovascular diseases deserving regular monitoring, implementation of preventive measures and therapeutic interventions if needed.
In addition, we studied the relation between postnatal microRNA gene expression and multiple antenatal and early postnatal factors, including gestational age at delivery, birth weight of newborns, the condition of newborns at the moment of birth, mode of delivery, and antenatal application of corticosteroids, antibiotics, and tocolytics to mothers. It was apparent that both gestational age at delivery and birth weight of newborns were significantly associated with postnatal levels of microRNAs in preterm-and term-born children. The highest postnatal microRNA expression levels were observed in children born prematurely at lower gestational ages and in children born with lower birth weight. These findings may also contribute to the explanation of why preterm-born children with lower birth weight have an increased cardiovascular risk and to the explanation of the increase in cardiovascular risk with the decrease in gestational age at delivery [2,5,[9][10][11]15,19,20,22,23,26,27,29,31,35,39,43,44,51,58,59].
Similarly, the condition of newborns at the moment of birth also had an impact on the postnatal microRNA expression profiles of preterm-born children, even though this impact was not as significant as the impact of gestational age at delivery and birth weight of newborns. miR-133a-3p showed a weak negative correlation with the Apgar scores determined at 5 min and 10 min and the pH of cord arterial blood after the birth of newborns. In addition, a weak negative correlation between the pH of cord arterial blood and the postnatal gene expression of miR-199a-5p and miR-221-3p was observed in preterm-born children. These findings indicate that preterm-born children with the worst values of parameters assessed immediately after the delivery may have higher postnatal levels of some cardiovascular disease-associated microRNAs. These findings may also contribute to the explanation of why preterm-born children have an increased cardiovascular risk.
On the other hand, the administration of tocolytic drugs, the administration of antenatal corticosteroids, and the administration of antibiotics to mothers at the time of preterm delivery had no impact on the postnatal microRNA expression profiles of preterm-born children. Likewise, the mode of delivery (vaginal or cesarean birth) had no influence on the postnatal microRNA expression profiles of preterm-born children.
On the grounds of these data, we suppose that altered microRNA expression profiles in the whole peripheral blood of preterm-born children might be associated with increased cardiovascular risk. We believe that the dysregulation of cardiovascular disease-associated microRNAs in preterm-born children might be induced by in utero exposure to a gestation with a pathological course. The pathogenesis of preterm delivery is multifactorial, but some principal pathogenic mechanisms such as cervical insufficiency, uterine malformations, acute inflammation of the membranes and chorion of the placenta followed by an exaggeration of inflammatory processes have already been discovered [68,[89][90][91][92][93][94]. Moreover, premature aging of the fetal membranes encompassing telomere shortening, senescence, apoptosis, and proteolysis has also been demonstrated to play a key role in the pathogenesis of preterm delivery [95][96][97]. Interactions between cardiovascular diseaseassociated microRNAs with altered expression profiles in the whole peripheral blood of preterm-born children and specific genes involved in key biological processes related to pathogenesis of preterm delivery, such as the apoptosis pathway, inflammatory response pathway, senescence, and autophagy pathways, were demonstrated in this study.
Although the association between the presence of prehypertension/hypertension (combined systolic and diastolic prehypertension/hypertension and/or isolated systolic or diastolic prehypertension/hypertension) and the expression of cardiovascular diseaseassociated microRNAs has not yet been identified in the association analysis previously performed in children born from normal and complicated pregnancies [62], we assume that the presence of chronic hypertension might exacerbate current alterations and induce de-novo alterations in microRNA expression profiles in preterm-born children similarly as in young and middle-aged mothers with confirmed diagnosis of systolic hypertension and on blood pressure treatment [65]. Previously, it was demonstrated that 7 out of 29 tested microRNAs (miR-17-5p, miR-24-3p, miR-92a-3p, miR-126-3p, miR-130b-3p, miR-181a-5p, and miR-210-3p) showed increased expression in mothers with systolic hypertension when the comparison to mothers with normal SBP values was performed regardless of the course of previous gestation (women with a history of normal and complicated pregnancies). The expression of microRNAs (miR-24-3p, miR-210-3p, miR-221-3p, and miR-342-3p) also differed between mothers on blood pressure treatment and without medication [65]. In parallel, an association between the presence of hypertension and altered expression of cardiovascular disease-associated microRNAs (miR-21-5p, miR-100-5p, miR-103a-3p, miR-145-5p, miR-181a-5p, and miR-199a-5p) was observed by other investigators [98][99][100][101][102][103][104]. However, large cohort studies involving a sufficient number of preterm-born children with the confirmed diagnosis of chronic prehypertension/hypertension are needed to prove the association between the presence of chronic prehypertension/hypertension and altered microRNA expression profiles.
Interactions between microRNAs dysregulated in the whole peripheral blood of preterm-born children and specific genes related to human disease ontologies, such as hypertension, congenital heart defects, patent foramen ovale, patent ductus arteriosus, and heart valve disease, were also demonstrated in this study. Nevertheless, further studies are definitely needed to evaluate the possible role of postnatal microRNA expression as a predictor for a later development of cardiovascular diseases in preterm-born children.
This study has some limitations. A set of 29 microRNAs, known to be involved in the pathogenesis of cardiovascular diseases, was selected for our previous and currently ongoing studies [60][61][62][63][64][65]. However, the list of selected microRNAs did not include all the microRNAs known to be involved in the pathogenesis of cardiovascular diseases. In addition, the list of selected microRNAs for our previous and currently ongoing studies [60][61][62][63][64][65] was not based on genome-wide expression studies since these analyses had not been performed in the cohorts of our patients. In addition, another possible limitation of this study is the fact that, while dysregulated microRNAs play a role in the functioning of the cardiovascular system, there are certainly other risk factors that can contribute to the development of cardiovascular diseases.

Conclusions
In conclusion, nearly one-third of preterm-born children descending from either PPROM or PTB pregnancies had valve problems and/or heart defects. The occurrence of prehypertension/hypertension was also inconsiderable in a group of preterm-born children. A vast majority of preterm-born children also had severe postnatal alterations in microRNA expression profiles at the age of 3 to 11 years. These postnatal microRNA expression profiles were significantly influenced by many antenatal and early postnatal factors. Among these factors are conditions leading to preterm birth, gestational age at delivery, birth weight of newborns, and the condition of newborns at the moment of birth. These findings may contribute to the explanation of the increased cardiovascular risk in preterm-born children. These findings strongly support the belief that preterm-born children should be dispensarized for a long time to have access to specialized medical care.

Patents
National patent granted-Industrial Property Office, Czech Republic (Patent No. 308102).

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9070727/s1: Table S1: Postnatal microRNA expression profiles in preterm-born children; Table S2: Postnatal microRNA expression profiles in children descending from PPROM pregnancies with regard to clinical findings; Table S3: Postnatal microRNA expression profiles in children descending from PTB pregnancies with regard to clinical findings; Table S4: Postnatal microRNA expression profiles in children descending from preterm pregnancies with regard to gestational age at delivery; Figure