WO2020088703A1

WO2020088703A1 - A method of predicting a risk of cardiovascular disease for a child born from a complicated pregnancy

Info

Publication number: WO2020088703A1
Application number: PCT/CZ2019/050050
Authority: WO
Inventors: Ilona HROMADNIKOVA
Original assignee: Univerzita Karlova, 3.Lekarska Fakulta
Priority date: 2018-10-31
Filing date: 2019-10-30
Publication date: 2020-05-07
Also published as: CZ308102B6; CZ2018595A3

Abstract

The present invention provides a method of predicting a risk of onset and development of cardiovascular disease for a child born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction, or in a child born from a physiological pregnancy having an abnormal postnatal clinical finding, said method comprising the steps of: - determining the level of expression of at least one microRNA marker selected from the group consisting of miR-1-3p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-133a-3p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-210-3p, miR-342-3p and combinations thereof, in a sample of blood taken from the child; - comparing the determined level(s) of expression of the said at least one microRNA marker with reference cut-off value, wherein said reference cut-off value is obtained by statistical analysis of samples from children from physiological pregnancies and samples from children from complicated pregnancies; - wherein the aberrant regulation of the said at least one microRNA marker exceeding the reference cut-off value, or below the reference cut-off value, respectively, indicates the risk of onset and development of cardiovascular disease for the child.

Description

A method of predicting a risk of cardiovascular disease for a child born from a complicated pregnancy

Field of Art

The present invention relates generally to the determination of cardiovascular risk in children born from a complicated pregnancy. In particular, the invention relates to molecular markers for the stratification of patients at risk of onset and development of cardiovascular diseases. The presence of an aberrant cardiovascular microRNA expression profile in the (whole peripheral venous) blood of children born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction) selects a risk population that may benefit from preventative measures to reduce or prevent onset and development of cardiovascular diseases.

Background Art

Gestational hypertension (GH), preeclampsia (PE) and fetal growth restriction (FGR) are major complications that affect 2-10% of pregnant women and contribute significantly to maternal and perinatal morbidity and mortality (WFlO, 1988; Bamfo et al., 2011). Gestational hypertension (GF1) usually develops after the 20th gestational week and is characterized by elevated blood pressure > 140/90 mmHg found in 2 independent measurements over a 4 hour period. Preeclampsia also occurs in pregnant women after the 20th gestational week and is characterized by the presence of chronic and/or gestational hypertension in combination with proteinuria (ACOG, 2002). Fetal growth restriction (FGR) is diagnosed if the fetus lags in growth with respect to the relevant gestational age (estimated fetal weight is below the lOth percentile for the respective gestational period) and at the same time pathological flows are present in the umbilical artery, uterine artery, or signs of centralization of fetal circulation are present (ACOG, 2013).

Early preeclampsia (onset before the 34th gestational week) and early FGR (onset before the 32nd gestational week) are commonly due to placental insufficiency with subsequent maladaptation of the maternal cardiovascular system (Brosens et al., 2011; Thilaganathan, 2017; Nardozza et al., 2017 Figueras et al., 2014; Baschat 2011). On the other hand, late preeclampsia (onset after 34 gestational week) and late FGR (onset after 32 gestational week) may occur as a result of secondary placental insufficiency due to dysfunction of the maternal cardiovascular system (Thilaganathan, 2016 and 2017; Nardozza et al., 2017; Figueras et al., 2014; Baschat 2011). Epidemiological studies suggest that children born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction have an increased cardiovascular risk during their whole life, including the early years of their life (Davis et al., 2012; Alsnes et al., 2017). Children whose prenatal condition has been complicated by gestational hypertension or maternal preeclampsia generally have a higher BMI, waist circumference, systolic and diastolic blood pressure, both in early childhood and at age of 18 (Davis et al., 2012; Alsnes et al., 2017; Tenhola et al., 2003; 0glaend et al., 2009; Fraser et al., 2013). Children in the age group of 6-13 years which were born prematurely due to early maternal preeclampsia (before the 34th gestational week) usually have an even higher systolic blood pressure and an even higher nocturnal systolic and diastolic pressure than children of the same age who were born from a pregnancy complicated by late preeclampsia (occurrence after the 34th gestational week) (Lazdam et a , 2012). There is even a positive correlation between maternal blood pressure during pregnancy and blood pressure in children of 3 years of age (Lim et al., 2015). Moreover, the incidence of preeclampsia in the mother during pregnancy induces the development of defective pulmonary circulation in children (about 30% higher pulmonary arterial pressure and 30% lower dilatation), predisposing these children to develop a hypoxic pulmonary hypertension and to a premature occurrence of cardiovascular diseases affecting systemic circulation (Jayet et al., 2010). Even worse is the situation in children who were diagnosed as growth retarded fetuses (FGR) during pregnancy. Cardiac remodeling is more common in children born from FGR-related pregnancies (smaller and more spherical hearts, smaller left ventricular length, smaller ascendent aorta, smaller left ventricular discharge diameter), which results in a decreased cardiac performance (decreased longitudinal contraction and impaired relaxation) (Sarvari et al., 2017; Yiallourou et al., 2017). In addition to cardiac remodeling, fetal malnutrition due to placental insufficiency during pregnancy is also associated with more frequent hypertension, obesity and diabetes mellitus (Fibby et al., 2007; Tapia et al., 2006). For these reasons, children born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction represent a population group that would benefit significantly from early implementation of preventative regimen measures such as diet adjustment, appropriate physical activity, or pharmacological interventions if regimen fails.

Disclosure of the Invention

The present invention is based on the identification of postnatal expression profile of microRNAs which play a role in pathogenesis of cardiovascular diseases in children born from pregnancies complicated by gestational hypertension, preeclampsia and/or fetal growth restriction. The tested set of microRNAs consisted of: miR-l-3p, miR-l6-5p, miR-l7-5p, miR-20a-5p, miR-20b-5p, miR-2l-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-92a-3p, miR-l00-5p, miR-l03a-3p, miR- l25b-5p, miR-l26-3p, miR-l30b-3p, miR-l33a-3p, miR-l43-3p, miR-l45-5p, miR-l46a-5p, miR-l55- 5p, miR-l8la-5p, miR-l95-5p, miR-l99a-5p, miR-2lO-3p, miR-22l-3p, miR-342-3p, miR-499a-5p and miR-574-3p.

From the herein listed microRNAs, the following microRNAs show aberrant expression profile in children born from complicated pregnancies and are useful as markers of the increased risk of development of cardiovascular disease in such children: miR-l-3p, miR-l7-5p, miR-20a-5p, miR-20b- 5p, miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-l03a-3p, miR-l25b-5p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p, miR-l8la-5p, miR-l95-5p, miR-2lO-3p, miR-342-3p, and combinations thereof.

The present invention thus provides a method of predicting a risk of onset and development of cardiovascular disease for a child born from a pregnancy complicated by either gestational hypertension, preeclampsia, or fetal growth restriction, or in a child born from a physiological pregnancy having currently an abnormal postnatal clinical finding, said method comprising the steps of:

- determining the level of expression of at least one microRNA marker selected from the group consisting of miR-l-3p, miR-l7-5p, miR-20a-5p, miR-20b-5p, miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-l03a-3p, miR-l25b-5p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p, miR-l8la-5p, miR-l95-5p, miR-2lO-3p, miR-342-3p and combinations thereof, in a sample of blood taken from the child;

- comparing the determined level(s) of expression of the said at least one microRNA marker with reference cut-off value, wherein said reference cut-off value is obtained by statistical analysis of samples from children from physiological pregnancies and samples from children from complicated pregnancies;

- wherein aberrant regulation of the said at least one microRNA marker exceeding the reference cut-off value, or below the reference cut-off value, respectively, indicates the risk of onset and development of cardiovascular disease for the child.

Preferably, the aberrant regulation means a downregulation (below a reference cut-off value) in case of miR-342-3p in children born from a pregnancy complicated by early preeclampsia, and an upregulation (exceeding a reference cut-off value) for all other microRNA markers and combinations thereof.

Preferably, the children in risk of onset and development of cardiovascular disease are subjected to preventative measures such as diet adjustment, appropriate physical activity, and/or pharmacological intervention (administration of cardiovascular preventative medication).

Preferably, the method according to the invention is carried out in childred at an age of at least 3 years, more preferably between 3 and 11 years.

The risk of onset and development of cardiovascular disease (or increased cardiovascular risk, or increased risk of onset and development of cardiovascular disease) means a higher risk of onset and development of cardiovascular disease for the tested child than the risk of onset and development for a healthy child from a physiological pregnancy. Healthy child from a physiological pregnancy means a child born from a physiological pregnancy and having no abnormal postnatal clinical finding. The risk of cardiovascular disease represents a risk of onset and development of cardiovascular disease in the child. The cardiovascular diseases include, in particular, myocardial infarction, ischemia/reperfusion-induced cardiac injury, ischemia, ischemic stroke, coronary arterial disease, hypertension, pulmonary hypertension, heart failure, hypertension-induced heart failure, cerebral ischemia, cardiac hypertrophy, hypertrophic cardiomyopathy, dyslipidemia, vascular inflammation, insulin resistance and diabetes, atherosclerosis, angiogenesis, intracranial aneurysm, peripartal cardiomyopathy.

The sample of blood is preferably a sample of whole peripheral venous blood.

Herein, the term„abnormal clinical findings" or„abnormal postnatal findings" or„abnormal postnatal clinical findings" means abnormal heart echocardiography, high blood pressure, overweight/obesity (or high BMI).

„Abnormal heart echocardiography" or„abnormal echocardiogram findings" include, in particular, tricuspid valve regurgitation, mitral valve regurgitation, pulmonary valve regurgitation, bicuspid aortic valve regurgitation, ventricular septum defect, atrial septum defect, hypertrophic ventricular septum, foramen ovale apertum, arrhythmia.

„High blood pressure (BP)“,„prehypertension/hypertension“ means systolic and/or diastolic blood pressure equal to or greater than the 90th percentile, preferably equal to or greater than the 95th percentile, as measured by the Age -based Pediatric Blood Pressure Reference Charts calculator (https://www.bcm.edu/bodvcomplah/Flashapps/BPVAgeChartpage.html. USDA/ARS Children Nutrition Research Center, Baylor College of Medicine, Houston, Texas).

„0verweight/0besity“ or„high BMI" means BMI above the 85th percentile, preferably above the 95th percentile, evaluated by age- and sex-specific BMI Percentile Calculator for Child and Teens (https://www.cdc.gov/healthvweight/hmi/caiculator.html. Centers for Disease Control and Prevention, USA).

„Normal postnatal findings" or„normal clinical findings" or„normal postnatal clinical finding" means that none of the above symptoms comprised in the term„abnormal clinical findings" was found in the child.

MicroRNA marker is an individual microRNA. Combination of microRNAs is a combination of individual microRNAs. The statistical analysis assesses each microRNA separately. In case of combination of microRNAs, individual microRNA markers are assessed in one combined statistical test. In the combination of microRNAs, the expressions of individual microRNAs contained in the combination are determined, and the combination of their expressions is assessed in the statistical analysis with one cut-off value. To select the optimal combinations of microRNA biomarkers, logistic regression was used (MedCalc Software bvba, Ostend, Belgium) by the inventors. The logistic regression procedure allows to analyse the relationship between one dichotomous dependent variable and one or more independent variables.

Determination of expression of microRNA markers can be performed using commercially available kits from various manufacturers, the procedure is described in manufacturer's instructions. In this invention, the expression of microRNA markers is preferably normalized using geometric mean of 2 endogenous controls, such as RNU58A and RNU38B.

MicroRNAs belong to a family of small non-coding RNA molecules of 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by degradation and / or blocking of the target rnRNA. A number of tissues affected by the pathological process have a characteristic microRNA expression profile that distinguishes the physiological tissue from the tissue affected by the pathology, which is successfully used in diagnostics. Recent studies have documented the aberrant expression profile of microRNAs in placental tissue, cord blood and maternal circulation in gestational hypertension, preeclampsia and fetal growth restriction.

It is an object of the invention to describe the expression profile of microRNAs in whole peripheral venous blood in children born of pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction. We focused especially on those microRNAs that play a role in the pathogenesis of dyslipidemia, hypertension, vascular inflammation, insulin resistance and diabetes, atherosclerosis, angiogenesis, coronary arterial disease, myocardial infarction, heart failure, stroke, intracranial aneurysm, pulmonary arterial hypertension and peripartal cardiomyopathy.

This is the first study of this type. To date, no study has been conducted documenting the expression profile of microRNAs associated with cardiovascular and cerebrovascular diseases in whole peripheral venous blood in children born from such complicated pregnancy (gestational hypertension, preeclampsia and/or fetal growth restriction).

It is an object of the invention to describe which microRNAs and optionally combinations thereof could be used in screening for stratification of children born of complicated pregnancy at increased cardiovascular risk. A newly identified risk group of children based on microRNA screening, using miroRNAs associated with cardiovascular diseases, may benefit from follow-up of general practitians and/or specialized physicians and the implementation of targeted preventive regimen measures to reduce or prevent the onset and development of cardiovascular and cerebrovascular diseases.

The method of the present invention can also be used in sports medicine, for determining the risk of cardiovascular disease (most preferably - stroke, heart failure, aneurysm and myocardial infarction) in sports physical activity.

Pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction) induce epigenetic changes that are usually associated with the occurrence of cardiovascular and cerebrovascular diseases. These epigenetic changes can be detected in whole peripheral venous blood in children in the long term (3-11 years after birth). Postnatal screening of individual microRNAs associated with the occurrence of cardiovascular and cerebrovascular diseases (and/or optimal combinations thereof) identifies children at increased cardiovascular risk. The group of children with an aberrant cardiovascular microRNA expression profile in whole peripheral venous blood represents a newly identified risk group of patients who may benefit from preventive measures to reduce or prevent the onset or development of cardiovascular and cerebrovascular diseases.

The expression profile of miR-2l-5p differs between the control group of healthy children born of physiological pregnancy with normal postnatal blood pressure, BMI, normal cardiac morphology and function, and the group of children born of physiological pregnancy with abnormal postnatal findings (overweight/obesity, and/or prehypertension/hypertension and/or abnormal cardiac morphology and function). In 28.95% of children with abnormal postnatal findings, upregulation of miR-2l-5p was found. The group of children born from normal pregnancy who have overexpressed miR-2l-5p represents a risk group of children that should be followed up at a specialized cardiology center and monitored regularly.

Furthermore, children born from pregnancy that were complicated by gestational hypertension (miR-l- 3p, miR-l7-5p, miR-20a-5p, miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-l03a-3p, miR- l25b-5p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p, miR-l8la-5p, miR-l95-5p, and miR-342-3p), preeclampsia (miR-l-3p, miR-20a-5p, miR-20b-5p, miR-l03a-3p, miR-l33a-3p, miR-342-3p) and fetal growth restriction (miR-l7-5p, miR-l26-3p, miR-l33a-3p) show an aberrant epigenetic profile characterized by aberrant expression (downregulation in case of miR-342-3p in early preeclampsia, overexpression in all other cases) of the above microRNAs, which are associated with the occurrence of cardiovascular and cerebrovascular diseases. Furthemore, the epigenetic profile varies between groups of children born of pathological pregnancy with respect to actual postnatal findings (overweight/obesity, and/or prehypertension/hypertension and/or abnormal cardiac morphology and function). Children born of pathological pregnancy (preeclampsia and/or FGR) with abnormal postnatal findings more frequently show an aberrant microRNA expression profile. Postnatal microRNA biomarker combined screening reveals a proportion of children at increased cardiovascular risk in children born from pregnancy with severe preeclampsia (miR-l03a-3p and miR-l33a-3p, 21.28% of children), late form preeclampsia (miR-l-3p, miR-20a-5p, miR-l03a-3p a miR-l33a-3p, 32.43% of children), and in children born from FGR pregnancy (miR-l7-5p, miR-l26-3p and miR- l33a-3p, 40.91 % of children). MiR-20b-5p is dysregulated in the proportion of children (36.36%) born from pregnancy complicated by mild preeclampsia and in whom normal postnatal findings are present.

Postnatal miR-2lO-3p levels detected in whole peripheral venous blood of children also correlate with the severity of preeclampsia and/or FGR as assessed by Doppler ultrasound examination during pregnancy-related complications. Pathological flow in the ductus venosus during pregnancy-related complications (preeclampsia and/or fetal growth restriction) induces upregulation of miR-2lO-3p in the circulation of children.

In children born of pregnancy affected by gestational hypertension, the epigenetic profile differs between children with normal and abnormal postnatal findings. For children with normal postnatal findings, the postnatal combined screening of two biomarkers (miR-26a-5p and miR-l95-5p, 34.78% of children) appears to be optimal. In children with abnormal postnatal findings, only miR-20a-5p is upregulated (25.81% of children). Furthermore, aberrant expression profile of a number of microRNAs occurs in children born of pregnancy affected by gestational hypertension regardless of postnatal findings, the optimal combination of which leads to the identification of a risk group of children. The combination of 5 microRNA biomarkers (miR-l-3p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, and miR-l8la-5p) identifies a group of children born from GH pregnancy with an increased cardiovascular risk (47.83% children) who still have normal postnatal findings. The combination of all 7 microRNA biomarkers (miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p, and miR- l8la-5p) then identifies a group of children born from GH pregnancy who already have an abnormal postnatal finding (38.71% of children).

Some epigenetic changes (particularly upregulation of miR-l-3p and miR-l33a-3p) that have been induced by previous pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction) are of a long-term and/or even permanent nature. Other epigenetic changes have not been observed by us during pregnancy-related complications in placental tissue, umbilical cord blood, or peripheral venous blood of mothers, and have likely formed de-novo during the life of children. However, both may predispose these children to development of cardiovascular disease. Therefore, postnatal screening of the above microRNAs can significantly contribute to the stratification of risk groups of children born from complicated pregnancy. These children should be followed up at specialized cardiology centers and regularly monitored.

Isolation of RNA enriched for small RNAs and RNA quality control

Peripheral venous blood was collected into EDTA tubes. Homogenized cell lysates were prepared immediately after collection of 200 pl whole peripheral blood samples, using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany, no: 52304) according to manufacturer's instructions, and stored at -80 °C until further processing. Total RNA was extracted from homogenized cell lysates using a mirVana microRNA Isolation kit (Ambion, Austin, USA, no: AM1560) and followed by an enrichment procedure for small RNAs (siRNA, microRNA) according to manufacturer's instructions. To minimize DNA contamination, the eluted RNA was treated for 30 min at 37 °C with 5 pL of DNase I (Thermo Fisher Scientific, California, USA, no: EN0521). A RNA fraction highly enriched for short RNAs (<200nt) was obtained. The concentration and quality of RNA was assessed spectrophotometrically (NanoDrop ND-1000 spectrophotometer, NanoDrop Technologies, USA). The A(260/280) absorbance ratio of isolated RNA is 1.8 - 2.0 (RNA is free of DNA) and the A(260/230) absorbance ratio greater than 1.6 (RNA is free of polysaccharides) indicate that the RNA fraction is pure and can be used for further analysis.

Reverse transcriptase reaction Individual microRNAs were reverse transcribed into complementary DNA (cDNA) in a total reaction volume of 10 pL using TaqMan MicroRNA Assay which contains a microRNA-specific stem-loop RT primer for the relevant microRNA, and a reverse transcription kit (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems, Branchburg, USA, no: 4366597) according to manufacturer's instructions. Reverse transcriptase reactions were performed using a cycler (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.

Relative quantification of microRNAs by real-time PCR

3 pL of cDNA were mixed with specific TaqMan MGB probes and primers (TaqMan MicroRNA Assay, Applied Biosystems, Branchburg, USA), and the ingredients of a master mix (TaqMan Universal PCR Master Mix, Applied Biosystems, Branchburg, USA, no: 4318157) in a total reaction volume of 15 pL. TaqMan PCR standard conditions were set up as described in the TaqMan guidelines for the cycler (7500 Real-Time PCR system, Applied Biosystems, Branchburg, USA). All PCRs were performed in duplicates with the involvement of multiple negative controls: NTC (water for PCR applications instead of cDNA sample), NAC (non-transcribed RNA samples), and genomic DNA (DNA isolated from equal biological samples), which did not generate any signal during PCR reactions. The samples were considered positive if the amplification signal occurred at Ct < 40 (threshold value before the 40^th cycle).

The expression of a particular microRNA was determined using a comparative Ct method relative to normalization factor (geometric mean of two endogenous controls). Two non-coding small nucleolar RNAs, RNU58A and RNU38B, were optimal for qPCR data normalization in this setting. They demonstrated equal expression in all tested groups. RNU58A and RNU38B also served as positive controls for successful extraction of RNA from all samples and were used as internal controls for variations during the isolation of RNA, cDNA synthesis, and real-time PCR.

A reference sample, RNA fraction highly enriched for small RNAs isolated from the fetal part of one randomly selected placenta derived from physiological pregnancy, was used throughout the study for relative quantification.

Statistical analysis

Data normality was assessed using the Shapiro-Wilk test. Since the experimental data did not follow a normal distribution, nonparametric tests were used. Gene expression of microRNAs was compared between groups using the Kruskai -Wallis one-way analysis of variance with post-hoc test for the comparison among multiple groups. The significance level was established at a - value of p < 0.05. Receivers operating characteristic (ROC) curves were constructed for individual microRNAs associated with cardiovascular and cerebrovascular diseases within the framework of postnatal screening in childen born from complicated pregnancies. The area under the curve (AUC) and the optimum cut-off value were calculated for the microRNA, as well as sensitivity and specificity. Furthermore, the sensitivity of a given microRNA biomarker at 90.0% specificity was determined, i.e., the information which % of the children has an increased/decreased expression of the given microRNA at 10.0% false positivity (MedCalc Software bvba, Ostend, Belgium). For every possible threshold or cut-off value, the MedCalc program (MedCalc Software bvba, Ostend, Belgium) reports the sensitivity, specificity, likelihood ratio positive (LR+), likelihood ratio negative (LR-).

To select the optimal combinations of microRNA biomarkers for a given situation, combined statistical analysis (logistic regression and ROC analysis) was used (MedCalc Software bvba, Ostend, Belgium). This procedure provides the following parameters: area under curve, sensitivity, specificity, optimum cut-off value, sensitivity of the given microRNA biomarker combination at 90.0% specificity.

This analysis identifies children having a higher risk of a subsequent occurence of cardiovascular and cerebrovascular diseases as a result of mother's complicated pregnancy. Based on individual ROC curve analyses and combined ROC curve analyses, the children born from complicated pregnancies were differentiated into a group/groups with no risk of subsequent development of cardiovascular and cerebrovascular diseases and a group/groups with a risk of subsequent development of cardiovascular and cerebrovascular diseases.

Correlation between variables (postnatal relative microRNA quantification in whole peripheral venous blood of children vs. Doppler ultrasound parameters during pregnancy: pulsatility index in the umbilical artery, pulsatility index in the arteria cerebri media, pulsatility index in the uterine artery, pulsatility index in the ductus venosus, and cerebroplacental index) was calculated using the Spearman’s rank correlation coefficient (p). Spearman's rank correlation coefficient, a nonparametric measure of rank correlation, assesses how well the relationship between two variables can be described using a monotonic function. If the correlation coefficient value ranges within <0.5; l.0>, there is a strong positive correlation. The significance level was established at a p- value of p < 0.05.

Box plots encompassing the median (dark horizontal line) of log-normalized gene expression values for individual microRNAs were generated using Statistica software (version 9.0; StatSoft, Inc., USA). The upper and lower limits of the boxes represent the 75^th and 25^th percentiles, respectively. The upper and lower whiskers indicate the maximum and minimum values that are no more than 1.5 times the span of the interquartile range (range of the values between the 25^th and the 75^th percentiles). Outliers are marked by circles and extremes by asterisks.

Postnatal upregulation of miR-21-5p is present in the children descending from physiological pregnancies that have an abnormal postnatal finding (i.e., are overweight/obese and/or prehypertensive/hypertensive and/or have abnormal echocardiogram findings)

A total of 38 children out of 88 examined children aged 3-11 years, born from physiological pregnancy, were found to have abnormal heart echocardiography [tricuspid valve regurgitation (n = 8), mitral valve regurgitation (n = 1), pulmonary valve regurgitation (n = 2), bicuspid aortic valve regurgitation (n = 1), ventricular septum defect (n = 1), atrial septum defect (n = 1), foramen ovale apertum (n = 5), arrhythmia (n = 1)] and/or high blood pressure (n = 16) (systolic and/or diastolic blood pressure equal to or greater than the 90th percentile, as measured by the Age -based Pediatric Blood Pressure Reference Charts calculator) and/or high BMI (n = 9) (BMI above the 85th percentile, evaluated by BMI Percentile Calculator for Child and Teens). The control group consisted of 50 children born from physiological pregnancy with a normal anamnesis, normal blood pressure, normal BMI and normal reference values of echocardiographic examination of the heart.

Although the Kruskal- Wallis test did not show a significant statistical difference for miR-2l-5p (r=0.171) between the group of children born of physiological pregnancy with abnormal and normal postnatal findings, subsequent ROC analysis revealed upregulation of miR-2l-5p (AUC 0.649, r=0.013) in children with abnormal postnatal findings (sensitivity 28.95% at specificity level 90.0%). MiR-2l is encoded by a gene located on chromosome l7q23.2 and controls homeostasis of the cardiovascular system. Upregulation of miR-2l promotes the development of cardiac muscle fibrosis and heart failure while maintaining the left ventricular ejection fraction. Inhibition of miR-2l by miR- 21 antagonists leads to an improvement in cardiac muscle atrophy and fibrosis. MiR-2l is upregulated in cardiomyocytes in hypoxia by HIF-la and silencing of HIF-la and miR-21 increases apoptosis of hypoxic cardiomyocytes. Circulating miR-2l is one of the biomarkers for diagnosis and prognosis of heart failure. Serum miR-2l levels were significantly higher in patients with heart failure and correlated with ejection fraction and natriuretic peptide levels. We conclude that the group of children with the upregulated miR-2l-5p expression profile represents a group of children with increased cardiovascular risk who would benefit from implementing early prevention programs to prevent the onset and development of cardiovascular disease.

MicroRNAs associated with cardiovascular and cerebrovascular diseases are dysregulated postnatally in whole peripheral venous blood in children born from complicated pregnancy

Gene expression of microRNAs associated with cardiovascular and cerebrovascular diseases was studied postnatally in whole peripheral venous blood in children aged 3 to 11 years. MicroRNA gene expression was compared between children born of complicated pregnancy (gestational hypertension, preeclampsia and/or fetal growth restriction) and children born of physiological pregnancy. Postnatal gene expression of microRNAs was also analyzed taking into account the severity of pregnancy-related complications according to clinical symptomatology (mild vs. severe preeclampsia, absence vs. presence of anhydramnion/oligohydramnion in fetus, absence vs. presence of abnormal Doppler flow in pregnancy with preeclampsia and/or FGR) and by delivery time (before the 34th gestational week vs. after the 34th gestational week for preeclampsia, before the 32nd gestational week vs. after the 32nd gestational week for FGR). Association between postnatal gene expression of the relevant microRNA in whole peripheral venous blood in children and Doppler US parameters during pregnancy-related complications (pulsatility index in the umbilical artery, pulsatility index in the arteria cerebri media, pulsatility index in the uterine artery, pulsatility index in the ductus venosus and cerebroplacental index) was analyzed in children born from pregnancy complicated by preeclampsia and/or FGR.

Postnatal upregulation of miR-l-3p, miR-17-5p, miR-20a-5p, miR-21-5p, miR-23a-3p, miR-26a- 5p, miR-29a-3p, miR-126-3p, miR-133a-3p, miR-146a-5p and miR-181a-5p is observed in children born from pregnancy complicated by gestational hypertension, who have an increased cardiovascular risk

Using Kruskal-Wallis test, the gene expression of miR-l-3p (p<0.00l), miR-l7-5p (p=0.006), miR- 20a-5p (p=0.054), miR-2l-5p (p=0.064), miR-23a-3p (p=0.l26), miR-26a-5p (p=0.065), miR-29a-3p (r=0.010), miR-l26-3p (p=0.052), miR-l33a-3p (r=0.014), miR-l46a-5p (p=0.097) and miR-l8la-5p (r=0.017) differed significantly or exhibited a trend towards reaching statistical significance between the control group of healthy children and the group of children born from prenancy complicated by gestational hypertension.

ROC analysis then revealed a statistically significant difference between these two groups for all the above mentioned microRNAs (miR-l-3p: AUC 0.726, p<0.00l; miR-l7-5p: AUC 0.684, p<0.00l; miR-20a-5p: AUC 0.662, p=0.003; miR-2l-5p: AUC 0.651, p=0.005; miR-23a-3p: AUC 0.635, r=0.014; miR-26a-5p: AUC 0.646, p=0.008; miR-29a-3p: AUC 0.689, p <0.001; miR-l26-3p: AUC 0.652, p=0.005; miR-l33a-3p: AUC 0.686, p <0.001; miR-l46a-5p: AUC 0.650, p=0.006 and miR- l8la-5p: AUC 0.686, p <0.001). All of these microRNAs showed increased gene expression in the whole peripheral venous blood of children born of pregnancy complicated by gestational hypertension. The percentage of children who are overexpressing a particular microRNA at 10.0% false positivity is also shown. Upregulation of individual microRNAs is observed in 16.67% - 46.3% of children born of pregnancy complicated by gestational hypertension depending on the relevant microRNA [miR-l-3p (46.3%), miR-l7-5p (29.63%), miR-20a-5p (20.37%), miR-2l-5p (29.63%), miR-23a-3p (27.78%), miR-26a-5p (16.67%), miR-29a-3p (35.19%), miR-l26-3p (29.63%), miR-l33a-3p (37.04%), miR- l46a-5p (18.52%) and miR-l8la-5p (31.48%) ].

This analysis identifies children who are at increased risk of cardiovascular and cerebrovascular disease due to maternal pregnancy-related complications.

Postnatal upregulation of miR-21-5p, miR-23a-3p, miR-26a-5p, miR-103a-3p, miR-125b-5p, miR-195-5p and miR-342-3p is observed in children born from pregnancy complicated by gestational hypertension with normal postnatal findings, who have an increased cardiovascular risk Using Kmskal -Wallis test for comparison with a control group of healthy children, the group of children with normal postnatal findings born from pregnancy complicated by gestational hypertension showed an increased expression and/or a trend towards statistically significant increased expression of miR-2l-5p (p=0.047), miR-23a-3p (p=0.082), miR-26a-5p (p=0.020), miR-l03a-3p (p=0.l32), miR- l25b-5p (p=0.l84), miR-l95-5p (p=0.l27) and miR-342-3p (p=0.09l) in the whole peripheral venous blood. Subsequent ROC analysis confirmed an increased expression of miR-2l-5p (AUC 0.683, p=0.009, sensitivity 39.13% at 10.0% false positivity (FP)), miR-23a-3p (AUC 0.673, r=0.015, sensitivity 34.78% at 10.0% FP), miR-26a-5p (AUC 0.700, p=0.003, sensitivity 21.74% at 10.0% FP), miR-l03a-3p (AUC 0.647, p=0.058, sensitivity 30.43% at 10.0% FP), miR-l25b-5p (AUC 0.653, p=0.054, sensitivity 47.83% at 10.0% FP), miR-l95-5p (AUC 0.660, p=0.032, sensitivity 34.78% at 10.0% FP) and miR-342-3p (AUC 0.676, p=0.009, sensitivity 21.74% at 10.0% FPR).

Postnatal screening based on the combination of miR-26a-5p and miR-l95-5p showed the highest accuracy in identifying children with normal postnatal findings who were exposed to gestational hypertension during prenatal life (AUC 0.717, p=0.00l, sensitivity 86.96%, specificity 52.0%, cut-off value >0.246824). Postnatal screening based on the combination of miR-26a-5p and miR-l95-5p identified a proportion of children at increased cardiovascular risk (sensitivity 34.78% at 10.0% false positivity) in a group of children born from pregnancy complicated by gestational hypertension who have normal postnatal findings.

Postnatal upregulation of miR-20a-5p is present in children born from pregnancy complicated by gestational hypertension with abnormal postnatal findings, who have an increased cardiovascular risk

A total of 31 children out of 54 examined children (57.41%) aged 3-11 years born from pregnancy complicated by gestational hypertension were found to have abnormal findings when performing cardiac echocardiography [tricuspid valve regurgitation (n = 6), mitral valve regurgitation (n = 1), pulmonary valve regurgitation (n = 3), aortic bicuspid valve regurgitation (n = 1), ventricular septal defect (n = 1), foramen ovale apertum (n = 2)], high blood pressure (n = 17) (systolic and/or diastolic blood pressure equal to or greater than the 90th percentile, as measured by the Age -based Pediatric Blood Pressure Reference Charts calculator) and/or high BMI (n = 3) (BMI above the 85th percentile, as measured by BMI Percentile Calculator for Child and Teens). Using the Kmskal -Wallis test for comparison with the control group, the group of children with abnormal postnatal findings born of pregnancy with gestational hypertension showed overexpression of miR-20a-5p (p = 0.041). ROC analysis showed that postnatal screening of miR-20a-5p (AUC 0.678, p = 0.005) identified a proportion of children at increased cardiovascular risk in the group of children born from pregnancy complicated by gestational hypertension who had abnormal postnatal findings (sensitivity 25.81% at 10.0 % false positivity). Postnatal upregulation of miR-l-3p, miR-17-5p, miR-29a-3p, miR-126-3p, miR-133a-3p, miR- 146a-5p and miR-181a-5p is present in children born from pregnancy complicated by gestational hypertension regardless of the postnatal clinical findings, who have an increased cardiovascular risk

Using the Kmskal -Wallis test, overexpression and/or a trend towards statistically significant overexpression was observed in some microRNAs in a group of children born from pregnancy complicated by gestational hypertension regardless of postnatal clinical findings (normal postnatal finding, abnormal postnatal finding) [miR-l-3p (p=0.005, p=0.008), miR-l7-5p (p=0.029, p=0.048), miR-29a-3p (p=0.055, p=0.044), miR-l26-3p (r=0.101, p=0.233), miR-l33a-3p (r=0.017, p=0.l33), miR-l46a-5p (p= 0.164, p=0.2l l) and miR-l8la-5p (r=0.161, p=0.020].

Subsequent ROC analysis revealed that in a proportion of children with prior exposure to gestational hypertension during the prenatal period, at 10.0% false positivity, the upregulated postnatal profile of the above microRNAs associated with cardiovascular and cerebrovascular diseases is present, regardless of the presence of abnormal postnatal findings [miR-l-3p (AUC 0.739, p<0.00l vs. AUC 0.716, p<0.00l; sensitivity 47.83% vs. 45.16%), miR-l7-5p (AUC 0.694, p=0.005 vs. AUC 0.677, p=0.004; sensitivity 30.43% vs. 29.03%), miR-29a-3p (AUC 0.682, p=0.020 vs. AUC 0.694, p=0.002; sensitivity 39.13% vs. 32.26%), miR-l26-3p (AUC 0.663, p=0.036 vs. AUC 0.645, p=0.022; sensitivity 39.13% vs. 22.58%), miR-l33a-3p (AUC 0.722, p<0.00l vs. AUC 0.659, r=0.014, sensitivity 43.48% vs. 32.26%), miR-l46a-5p (AUC 0.658, p=0.025 vs. AUC 0.644, p=0.020, sensitivity 26.09% vs. 12.9%) and miR-l8la-5p (AUC 0.659, p=0.036 vs. AUC 0.706, p<0.00l; sensitivity 39.13% vs. 25.81%).

Postnatal screening using the combination of miR-l-3p, miR-29a-3p, miR-l26-3p, miR-l33a-3p and miR-l8la-5p exhibited the highest accuracy in identification of children with normal postnatal clinical findings which were prenatally exposed to gestational hypertension (AUC 0.803, p<0.00l, sensitivity 82.61%, specificity 74.0%, cut-off value >0.224754). Postnatal screening using the combination of miR-l-3p, miR-29a-3p, miR-l26-3p, mir-l33a-3p and miR-l8la-5p identified in the group of children born from pregnancy complicated by gestational hypertension who have a normal postnatal clinical finding, a subgroup of children with increased cardiovascular risk (sensitivity 47.83% at 10.0% false positivity).

Furthermore, a postnatal screening based on a combination of all 7 microRNA biomarkers (miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and miR-l8la-5p) showed the highest accuracy in identifying children with abnormal postnatal clinical findings who were exposed to gestational hypertension during prenatal life (AUC 0.801, p<0.00l, sensitivity 70.97%, specificity 76.0%, cut-off value >0.353483). A postnatal screening based on the combination of miR-l-3p, miR- l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and miR-l8la-5p identified in the group of children born from pregnancy complicated by gestational hypertension having an abnormal postnatal finding, a subgroup of children with increased cardiovascular risk (sensitivity 38.71% at 10.0% false positivity).

MicroRNAs associated with cardiovascular and cerebrovascular diseases are dysregulated postnatally in children exposed to preeclampsia during prenatal life

A total of 63 children out of 133 examined children (47.37%) aged 3-11 years born from pregnancy complicated by preeclampsia were found to have abnormal heart echocardiography [tricuspid valve regurgitation (n = 7), mitral valve regurgitation (n = 2), pulmonary valve regurgitation (n = 1), ventricular septal defect (n = 1), atrial septal defect (n = 1), ventricular septal hypertrophy (n = 1), foramen ovale apertum (n = 9) and arrhythmia (n = 1)], high blood pressure (n = 36) (systolic and/or diastolic blood pressure equal to or greater than 90th percentile, as measured by the Age-based Pediatric Blood Pressure Reference Charts calculator) and/or high BMI (n = 5) (BMI above the 85th percentile, evaluated by BMI Percentile Calculator for Child and Teens). Abnormal postnatal clinical findings were found in 16 of 27 children exposed to mild preeclampsia (59.3%), 47 of 106 children exposed to severe preeclampsia (44.3%), 26 of 49 children exposed to early preeclampsia (53.1%) and 37 of 84 children exposed to late preeclampsia (44.0%).

Upregulation of miR-133a-3p in children born from pregnancy complicated by preeclampsia

Although initial statistical analysis using the Km ska 1- Wallis test did not reveal a difference in miR- l33a-3p expression (p = 0.186) between the control group and the group of children exposed to preeclampsia during prenatal life, a subsequent ROC analysis identified at 10.0% false positivity an upregulation of miR-l33a-3p (AUC 0.614, p=0.0l l) in 22.56% of children born from pregnancy complicated by preeclampsia regardless of disease severity and delivery date.

Subsequently, miR-l33a-3p (p = 0.074) showed a trend to upregulation in children born from pregnancy complicated by severe preeclampsia. MiR-l33a-3p was upregulated in children exposed to severe preeclampsia during the prenatal period (AUC 0.628, p = 0.006) with a sensitivity of 23.58% at 10.0% false positivity.

In parallel, a trend towards upregulation of miR-l33a-3p (p=0.065) was also observed in children born of pregnancy complicated by late preeclampsia. Upregulation of miR-l33a-3p (AUC 0.639, p=0.005) was observed at 10.0% false positivity in 21.43% of children born from pregnancy complicated by late preeclampsia. In addition, although the initial statistical analysis using the Kmskai -Wallis test did not reveal the upregulation of miR-l33a-3p (p=0.l07, p=0.30l) in a group of children with normal and abnormal clinical findings exposed to severe preeclampsia during prenatal life, subsequent ROC analysis demonstrated upregulation of miR-l33a-3p biomarker (AUC 0.635, p=0.0l l; AUC 0.620, p=0.035) in both groups of children, with normal and abnormal postnatal clinical findings (sensitivity 25.42% vs. 21.28% at 10.0% false positivity). Similarly, initial statistical analysis using the Kruskal -Wallis test did not identify upregulation of miR- l33a-3p (p=0.253, p=0.094) in a group of children with normal and abnormal postnatal clinical findings who were exposed to late pre-eclampsia during prenatal life. Statistically significant upregulation of miR-l33a-3p was detected in both groups of children only after ROC analysis (AUC 0.626, p=0.029; AUC 0.656, p=0.009) with a sensitivity of 19.15% and 24.32% at 10.0% false positivity.

At the same time, the use of the Kmskal -Wallis test did not reveal the upregulation of miR-l33a-3p (p=0.274) in a group of children with normal postnatal clinical findings who were exposed to early pre eclampsia during prenatal life. However, ROC analysis revealed that miR-l33a-3p (AUC 0.652, p=0.045) is upregulated at 10.0% false positivity in 39.13% of children with normal postnatal clinical findings born from pregnancy complicated by early pre-eclampsia.

Upregulation of miR-l-3p, miR-20a-5p, miR-103a-3p and downregulation of miR-342-3p in children with abnormal postnatal clinical findings born from pregnancy complicated by preeclampsia, who are at risk of cardiovascular disease

Statistical analysis by Kruskal- Wallis test did not show a difference in miR-l-3p gene expression between the control group and the group of children exposed to late pre -eclampsia during prenatal life (p = 0.120), but showed a clear trend towards upregulation of miR-l-3p (p = 0.052) in children with abnormal postnatal clinical findings. However, ROC analysis showed that at 10.0% false positivity, the sensitivity of miR-l-3p (AUC 0.617, p=0.022) in children exposed to late preeclampsia is 27.38% and is even higher in children with abnormal postnatal clinical findings (AUC 0.661, p=0.006, sensitivity 35.14%).

Furthermore, at 10.0% false positivity, upregulation of miR-l03a-3p (AUC 0.610, p=0.058; AUC 0.648, r=0.013) was demonstrated in 12.77% and 13.51% of children with abnormal postnatal clinical findings who were exposed during prenatal life to severe preeclampsia and/or late preeclampsia, although the initial Kmskal -Wallis test did not find a statistically significant difference (p=0.244, p=0.077). In addition, upregulation of miR-20a-5p (AUC 0.626, p=0.04l) was observed in 13.51% of children with abnormal postnatal clinical findings who were exposed to late preeclampsia during prenatal life, although Kmskal-Wallis test did not indicate this (p=0.255). MiR-342-3p (AUC 0.665, r=0.016) was a marker that was able to differentiate between a group of children with abnormal postnatal clinical findings who were exposed to early preeclampsia during prenatal life and a control group of children (sensitivity 26.92% at 10.0% FP), which would not be revealed in case only the Kruskal- Wallis test was used for statistical analysis (p=0.097).

Postnatal screening using the combination of miR-l03a-3p and miR-l33a-3p showed the highest accuracy in identifying children with abnormal postnatal clinical findings who were exposed to severe preeclampsia during prenatal life (AUC 0.637, r=0.015, sensitivity 70.21%, specificity 56.0%, cut-off value >0.429299). A postnatal screening based on the combination of miR-l03a-3p and miR-l33a-3p identified a proportion of children at increased cardiovascular risk (sensitivity 21.28% at 10.0% false positivity) in a group of children born from pregnancy complicated by severe preeclampsia who had abnormal postnatal clinical findings.

Furthermore, a postnatal screening using a combination of 4 microRNA biomarkers (miR-l-3p, miR- 20a-5p, miR-l03a-3p and miR-l33a-3p) showed the highest accuracy in identifying children with abnormal postnatal clinical findings who were exposed to late preeclampsia during prenatal life (AUC 0.701, p<0.00l, sensitivity 59.46%, specificity 72.0%, cut-off value >0.391116). A postnatal screening based on a combination of miR-l-3p, miR-20a-5p, miR-l03a-3p and miR-l33a-3p identified a subgroup of children with increased cardiovascular risk in a group of children born from pregnancy complicated by late preeclampsia who had abnormal postnatal findings (sensitivity 32.43% at 10.0% false positivity).

Upregulation of miR-20b-5p in children with normal postnatal clinical findings born from pregnancy complicated by mild preeclampsia

MiR-20b-5p (AUC 0.689, p = 0.022) is the only biomarker that differentiates between a control group of children and a group of children with normal postnatal clinical findings born from pregnancy complicated by mild preeclampsia (sensitivity 36.36% at 10.0% FP). Flowever, a difference in miR- 20b-5p gene expression (p=0.264) between the two groups would not be detected in case only the Kruskal-Wallis test was used for statistical analysis.

MicroRNAs associated with cardiovascular and cerebrovascular diseases are dysregulated postnatally in children who were affected by fetal growth restriction during prenatal life

A total of 22 children out of 34 examined children (64.7%) aged 3-11 years born from pregnancy complicated by fetal growth restriction were found to have abnormal clinical findings when performing echocardiography of the heart [tricuspid valve regurgitation (n = 4), pulmonary valve regurgitation (n = 3), atrial septal defect (n = 1) and foramen ovale apertum (n = 6)], high blood pressure (n = 9) (systolic and/or diastolic blood pressure equal to or greater than the 90th percentile, evaluated using an Age- based Pediatric Blood Pressure Reference Charts calculator) and/or a high BMI (n = 1) (BMI above the 85th percentile, evaluated using the BMI Percentile Calculator for Child and Teens). Abnormal postnatal clinical findings were found in 7 of 13 children with a history of early FGR (53.85%) and in 15 of 21 children with a history of late FGR (71.43%).

Upregulation of miR-17-5p, miR-126-3p and miR-133a-3p in children with abnormal postnatal clinical findings born from pregnancy complicated by fetal growth restriction, who are at risk of cardiovascular disease

Upregulation of miR-l7-5p (AUC 0.642, p=0.059, sensitivity 22.73% at 10.0% FP), miR-l26-3p (AUC 0.646, p=0.055, sensitivity 31.82% at 10.0% FP) and miR-l33a-3p (AUC 0.690, p=0.008, sensitivity 31.82% at 10.0% FP) was found in a group of children with abnormal postnatal clinical findings in whom fetal growth restriction was diagnosed during prenatal life. However, the Kruskal- Wallis test did not identify a statistically significant difference (miR-l7-5p: p = 0.323, miR-l26-3p: p = 0.278, miR-l33a-3p: p = 0.133).

The postnatal screening based on the combination of miR-l7-5p, miR-l26-3p and miR-l33a-3p showed the highest accuracy in identifying children with abnormal postnatal clinical findings diagnosed as FGR fetuses during prenatal life (AUC 0.710, p=0.002, sensitivity 63.64%, specificity 78.0%, cut-off value >0.305781). A postnatal screening using the combination of miR-l7-5p, miR- l26-3p and miR-l33a-3p identified a subgroup of children at increased cardiovascular risk in a group of children born from pregnancy complicated by FGR who had abnormal postnatal findings (sensitivity 40.91% at 10.0% false positivity).

Association between postnatal gene expression of microRNAs associated with cardiovascular and cerebrovascular diseases and the severity of preeclampsia and/or FGR with respect to Doppler US parameters during pregnancy

Pulsatility index values in the ductus venosus measured during pregnancy strongly positively correlated with postnatal miR-2lO-3p levels (p=0.580, p=0.009) in whole peripheral venous blood of children exposed to preeclampsia and/or fetal growth restriction during prenatal life. This finding means that fetuses with dilatation in the ductus venosus, which is a marker of poor prognosis in severe FGR, have postnatally elevated levels of miR-2lO-3p in whole peripheral venous blood.

The role of microRNAs in the pathogenesis of cardiovascular and cerebrovascular diseases

No studies are currently available to monitor the postnatal expression profile of microRNAs associated with cardiovascular and cerebrovascular diseases in whole peripheral venous blood in children born from complicated pregnancy (gestational hypertension, preeclampsia and/or fetal growth restriction). However, it is apparent from the available data that some microRNAs play an important role in the pathogenesis of cardiovascular and cerebrovascular diseases. We believe that the incidence of pregnancy-related complications (gestational hypertension, preeclampsia and/or fetal growth restriction), which is associated with the aberrant postnatal expression profile of some microRNAs playing a role in the pathogenesis of cardiovascular and cerebrovascular diseases, predisposes a proportion of children to subsequent development of cardiovascular diseases. This risk group of children should be recognized early in postnatal screening as it can benefit significantly from prophylactic measures to reduce or prevent onset and development of cardiovascular and cerebrovascular diseases.

MiR-l-3p is generated from the precursors for miR-l-l and miR-l-2 encoded by distinct genes located on chromosome 20ql3.3 and on chromosome 18ql 1.2. MiR-l is abundantly expressed in cardiac and skeletal muscles, especially in myocardium. Extracellular miR-l levels are significantly increased in patients with acute MI and highly correlate with circulating troponin T, a reliable marker of cardiac damage. MiR-l also represents a promising therapeutic target in treatment of cardiovascular diseases, heart ischemia and post-MI complications. Inhibition of miR-l with antisense oligonucleotides is cardioprotective, since it leads to reduction of apoptosis, increase of resistance to oxidative stress and attenuation of spontaneous arrhythmogenic oscillations.

A large proportion of children having normal or abnormal postnatal clinical findings showed an upregulated miR-l-3p profile with a prior exposure to gestational hypertension (47.83% children with normal clinical findings and 45.16% children with abnormal clinical findings) and/or late preeclampsia (35.14% children with abnormal postnatal clinical findings).

The results show that children descending from complicated pregnancies and having upregulated miR- l-3p profile are at a higher risk of onset of cardiovascular diseases, and should be carefully monitored in the long term.

MiR-17-5p, a member of miR-l7-92 cluster, located on the human chromosome l3q31.3, is overexpressed in endothelial cells and lowly expressed in vascular smooth muscle cells. MiR-l7p~92 cluster microRNAs were found to play a major role in cardiac development, since the hearts of miR- 17r~92 deficient mutant embryos presented a clear ventricular septal defect. Moreover, multiple studies have also confirmed the involvement of miR-l 7-5p in regulating ischemia/reperfusion-induced cardiac injury (I/R-I). Upregulation of miR-l 7-5p has been reported to promote apoptosis induced by oxidative stress via targeting Stat3 in in vivo I/R-I mouse model and in vitro cellular model of oxidative stress induced by H2O2. The inhibition of miR-l 7-5p by its specific inhibitors preserved cell survival and inhibited cell death in both in in vivo I/R-I mouse and in vitro cellular oxidative stress models and improved cardiac function after acute myocardial infarction via weakening of apoptosis in endothelial cells in SD rat model. MiR-l 7-5p expression was activated during kidney ischemia-reperfusion injury in mice. In humans, circulating miR-l 7 -5p represents one of potential upregulated biomarkers for diffuse myocardial fibrosis in hypertrophic cardiomyopathy, in patients with acute ischemic stroke and for the severity of coronary artery disease. Our data showed that miR-l7-5p was upregulated in a proportion of children with a prior exposure to gestational hypertension regardless of normal or abnormal clinical findings (30.43% vs. 29.03%) and in children prenatally affected with FGR, however only with abnormal clinical findings (22.73%). The results show that children with aberrant expression profile of miR-l 7-5p need to be stratified and dispensarized as soon as possible to prevent them from cardiovascular disease development.

MiR-20a-5p belongs to the miR-l 7 family and is also transcribed from the miR-l 7-92 cluster. MiR- 20a is involved with inflammatory signalling in pulmonary hypertension. Intraperitoneal injections of antagomiR-20a significantly down-regulated the expression levels of miR-20a-5p and restored functional levels of bone morphogenetic protein receptor type 2 (BMPR2) in pulmonary arteries in hypoxia-induced pulmonary hypertension mouse model. Previous studies demonstrated increased plasma levels of miR-l 7-5p and miR-20a-5p also in patients diagnosed with GDM at 16-19 weeks of gestation. Our results support the involvement of miR-20a-5p in pathogenesis of pathologies enhancing a cardiovascular risk, since overexpression of miR-20a-5p was identified only in a proportion of children with abnormal clinical findings that were previously exposed to gestational hypertension (25.81%) and/or late preeclampsia (terminated after 34 weeks of gestation) (13.51%). Early identification of this risky group of children with aberrant postnatal expression profile of miR-20a-5p and taking appropriate prophylactic measures improve their future cardiovascular health.

MiR-20b-5p also belongs to the miR-17 family, however is transcribed from miR-l06a-363 cluster. Recent study showed that increased plasmatic levels of miR-20b may serve as one of selected biomarkers in monitoring therapy efficiency and progression of hypertension-induced heart failure in animal experimental model. In humans, higher levels of miR-20b-5p were found in second trimester maternal sera of pregnancies with small-for-gestational age foetuses. In our study, mir-20b-5p represented a microRNA biomarker that was upregulated in a proportion of children with normal postnatal clinical findings that were prenatally affected with mild preeclampsia (36.36%). This group of children is at risk of cardiovascular disease and would profit from prophylactic programmes directed to decreasing of cardiovascular risk.

MiR-2l is encoded by a gene located on chromosome l7q23.2, forms two mature microRNAs (miR- 21-5p and miR-2l-3p), and mediates the homeostasis of the cardiovascular system. Upregulation of miR-2l promotes cardiac fibrosis and development of heart failure with preserved left ventricular ejection fraction. Inhibition of miR-2l by miR-2l antagonists leads to amelioration of cardiac atrophy and cardiac fibrosis. MiR-2l is upregulated by HIF-la under hypoxia in cardiomyocytes and silencing of HIF-la and inhibition of miR-2l increase the apoptosis of hypoxic cardiomyocytes. Circulating miR-2l can be used as a biomarker for the diagnosis and prognosis of heart failure. Serum levels of miR-2l were higher in patients with heart failure than in controls, and correlated with ejection fraction and brain natriuretic peptides. In our study, miR-2l-5p was upregulated in a proportion of children descending from normal pregnancies with abnormal clinical findings (28.95%) and in children descending from GH pregnancies with normal clinical findings (39.13%). This group of children would benefit from dispensarisation and implementation of primary prevention strategies, since it is at a higher risk of development of cardiovascular diseases.

MiR-23a, encoded by a gene located at chromosome 19pl 3.12, forms two mature microRNAs: miR- 23a-5p and miR-23a-3p. MiR-23a regulates cardiomyocyte apoptosis, a key pathogenesis factor of heart failure, by targeting manganese superoxide dismutase gene. MiR-23a also regulates the vasculogenesis of coronary artery disease via targeting epidermal growth factor receptor. Circulating miR-23a may be a new biomarker for coronary artery disease, since increased levels of miR-23a predict the presence and severity of coronary lesions in patients with coronary artery disease. MiR-23a- 3p also suppressed oxidative stress injury in a mouse model of focal cerebral ischemia-reperfusion. Since our study demonstrated upregulation of miR-23a-3p in a proportion of children with normal clinical findings born of gestational hypertension complicated pregnancies only (34.78%), we suppose that compensatory effect of miR-23a-3p may appear more likely in these children to normalise cardiomyocyte state and vasculogenesis.

MiR-26a-5p is produced by miR-26a-l and miR-26a-2, whose genes are located on chromosomes 3p22.2 and 12ql4.1. MiR-26a-5p was demonstrated to regulate the autophagy in cardiac fibroblasts by targeting a key component of autophagy pathway, ULK1 (unc-51 like autophagy activating kinase 1). Upregulation of miR-26a-5p reduces the expression of ULK1 and inhibits the transition of LC3-I to LC3-II (microtubule-associated protein 1 light chain) participating in the formation of autophagosome membranes during autophagy. Autophagy plays a protective role in heart failure and cardiac hypertrophy by removing damaged proteins. Nevertheless, in some cases inhibition of autophagy may also improve cardiac function. Since our study showed overexpression of miR-26a-5p in a proportion of children with normal clinical findings that were affected with gestational hypertension (21.74%), we suggest that this phenomenon may have protective effect against potential development of cardiac fibrosis.

MiR-29a-3p, a member of miR-29 family, is encoded by a gene located on chromosome 7q32.3. Antagomirs against miR-29a significantly increased Mcl-2 expression and significantly reduced myocardial infarct size and apoptosis in experimental model hearts subjected to ischaemia-reperfusion injury. Upregulation of miR-29a-3p was also observed in cardiac cachexia, a common complication of heart failure, also in experimental model. In patients with atrial fibrillation, overexpression of miR-29a- 3p found in the heart biopsies was associated with underexpression of calcium voltage-gated channel subunit alpha 1C (CACNA1C protein). Circulating miR-29a-3p represents one of potential upregulated biomarkers for diffuse myocardial fibrosis in hypertrophic cardiomyopathy. Previous studies demonstrated increased plasma levels of miR-29a-3p in GDM patients at 16-19 weeks of gestation. Serum mir-29a-3p levels were also shown to be elevated in patients with recent diagnosis of type 2 diabetes mellitus. Our study indicated the presence of upregulation of miR-29a-3p in both groups of children with normal and abnormal clinical findings descending from pregnancies complicated by gestational hypertension (39.13% vs. 32.26%). The results show that postnatal upregulation of mi-29a- 3p is a significant risk factor indicating an increased cardiovascular risk in children.

MiR-103 and miR-107 are paralogous microRNAs binding the same target sites. MiR-103 is encoded by two different genes located on chromosomes 5q34 and 20pl3. Both genes generate miR-103a-3p. MiR-103/107 regulate programmed necrosis and myocardial ischemia/reperfusion injury via targeting FADD (Fas-associated protein with death domain). Both miR-103 and miR-107 are upregulated in the ischemic zone of ischemic heart. Plasma miR-103a concentration is also upregulated in patients with hypertension and acute myocardial infarction. MiR-103/107 antagomir application leads to a reduction of FADD and induces a reduction in the myocardial necrosis and myocardial infarct sizes. MiR- 103/107 is also involved in hypoxia-induced pulmonary hypertension. In addition, miR-103/107 play the central importance in regulation of insulin sensitivity. Overexpression of miR-l03/l07 is present in obese mice and silencing of miR-l03/miR-l07 leads to the improvement of glucose homeostasis and insulin sensitivity. In our study, we identified upregulation of miR-l03a-3p in a proportion of children with abnormal postnatal clinical findings who were born from pregnancy with severe preeclampsia (12.77%) and/or late preeclampsia (developing after 34 weeks of gestation) (13.51%), thus we consider upregulation of miR-l03a-3p in peripheral venous blood of children as a highly significant risk factor of development of cardiovascular diseases.

There are two paralogs, miR-l25b-l on chromosome 11 q24.1 and miR-125h-2 on chromosome 21 q21.1, both producing miR-125b-5p. A set of upregulated circulating microRNAs including miR- l25b-5p is associated with acute ischemic stroke and acute myocardial infarction. Upregulation of miR-l25b-5p protects endothelial cells from apoptosis under oxidative stress via negative regulation of SMAD4 (SMAD family member 4). In addition, miR-l25b-5p acts as an ischemic stress-responsive protector against cardiomyocyte apoptosis caused by ischemia. Cardiomyocytes with upregulated miR- l25b-5p have increased survival and protect the heart from acute myocardial infarction by repressing pro-apoptotic bakl and klfl 3 genes. Our study has shown upregulation of miR-l25b-5p in a proportion of children with normal clinical findings that were born from pregnancy with gestational hypertension (47.83%). We suggest that a protective effect of miR-l25b-5p may be present just in children previously exposed to minor pregnancy-related complications, while in children exposed to severe pregnancy-related complications it apparently vanished. Our results indicate that postnatal upregulation of miR-l25b-5p is a marker of increased cardiovascular risk.

MiR-l26, producing miR-126-3p, is an intronic microRNA located in intron 7 of EGFL7 (epidermal growth factor-like protein 7) gene on chromosome 9q34.3. MiR-l26 regulates endothelial expression of vascular cell adhesion molecule 1 (VCAM-l) and controls vascular inflammation. Upregulation of miR-l26 decreases VCAM-l expression and, on the contrary, transfection of endothelial cells with miR-l26 antagomirs increases TNFa-stimulated VCAM-l expression. MiR-l26-3p is significantly down-regulated in sera derived from patients with acute myocardial infarction and in plasma of type 2 diabetes patients. Recent experiments also demonstrated that intercellular transfer of miR-l26-3p by endothelial microparticles reduced vascular smooth muscle cell proliferation and limited neointima formation via inhibition of FRP6 (FDF Receptor Related Protein 6). Our results show that compensatory role of miR-l26-3p also appears in a proportion of children with both normal or abnormal clinical findings with a prior exposure to gestational hypertension (39.13% vs. 22.58%) or FGR (31.82%), most likely with the aim to induce suppression of cytokine activated endothelial cells and vascular smooth muscle cell proliferation.

MiR-133a-3p belongs to the miR-l33 family and is transcribed from a gene appearing in several copies on chromosome 18ql 1.2 (miR-l33a-l) and on chromosome 20ql3.33 (miR-l33a-2). MiR-l33 is expressed in cardiomyocytes and in skeletal muscles and has an anti-apoptotic effect by suppressing the expression of caspase 9. MiR-l33 also plays an important role in the development of the heart by influencing the expression of HCN2 and HCN4 genes. MiR-l33 is downregulated in heart hypertrophy, heart failure, and downregulation of miR-133 also contributes to arrhythmogenesis in heart hypertrophy and heart failure. Induction of miR-l33 overexpression reduces cardiac hypertrophy and leads to correction of abnormalities in the transferring cardiac system. Furthermore, circulating miR-l33a-3p is one of the potential biomarkers of diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy and coronary artery calcification. Our results show upregulation of miR- l33a-3p in a proportion of children born from pregnancy complicated by gestational hypertension (37.04%) and/or preeclampsia regardless of disease severity and gestational age of pregnancy termination (22.56%) and regardless of postnatal clinical findings. However, upregulated miR-l33a-3p profile was particularly present in children who were exposed to severe preeclampsia (23.58%) and/or late preeclampsia (21.43%) during prenatal life. Upregulation of miR-l33a-3p has also been demonstrated in a proportion of children with abnormal postnatal clinical findings diagnosed with FGR during prenatal life (31.82%). Our results show that the upregulation of miR-l33a-3p present in children exposed to gestational hypertension, preeclampsia and/or FGR may be a long-term consequence of pregnancy-related complications. However, it appears that upregulation of miR-133a- 3p has a rather compensatory effect in order to normalize cardiovascular function. Nevertheless, the postnatal screening of miR-l33a-3p stratifies the at-risk group of children who are predisposed to the occurrence of cardiovascular diseases.

MiR-l46 family consists of 2 members, with nearly identical sequences, miR-146a-5p and miR-146h- 5p. MIR 146 A gene was found within a larger long noncoding RNA host gene, MIR3142HG, on chromosome 5q33.3. MiR-l46a-5p is actively involved in multiple oncological processes such as antitumor immune suppression, metastasis, and angiogenesis. MiR-l46a-5p is an anti-inflammatory microRNA, since it functions as a negative regulator of inflammation by targeting interleukin- 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor associated factor 6 (TRAF6), resulting in inhibition of NF-kB activation. Furthermore, miR-146a is one of the microRNAs that is most sensitive to hypoxia. It seems that miR-146a upregulation protects the myocardium from damage. Lenti virus expressing miR-146a transfected into mouse hearts decreased I/R-induced myocardial infarct size. Increased plasma levels of miR-146a also correlate with the severity of coronary atherosclerosis in patients with subclinical hypothyroidism and are a good predictor for coronary heart disease development among individuals with elevated TSH levels. Nevertheless, a significant downregulation of miR-146a expression was observed in acute ischemic stroke. A down-regulation of miR-146a was suggested to be self-protective with the aim to decrease the consequences of acute cerebral ischemia injury via the upregulation of FbxllO expression, which protects neurons from ischemic death. Since we observed the upregulation of miR-146a-5p in both groups of children with normal and abnormal postnatal clinical findings, born from pregnancies complicated by gestational hypertension (26.09% vs. 12.9%), we conclude that a protective role of miR-l46a-5p exists at least in a certain risk group of children with the aim to reduce inflammation and its negative consequences. MiR-l8la and miR-l8lb have genes localized on chromosomes lq32.l and 9q33.3. MiR-l8la generates several mature microRNAs involving miR-181a-5p, miR-l8la-3p and miR-l8la2-3p. The miR-l8l family plays a key role in both acute and chronic inflammatory disease states such as atherosclerosis, type 2 diabetes, and obesity. Nevertheless, contradictory data are reported concerning expression levels of miR-181 a in various pathological conditions. Decreased expression of miR-181 a observed in monocytes of obese patients was associated with the metabolic syndrome and coronary artery disease. However, another study reported that upregulation of miR-l8la-5p in adipocytes upregulated insulin-stimulated AKT activation and reduced TNFa-induced insulin resistance. Moreover, increased hepatic miR-l8la impaired glucose and lipid homeostasis by silencing sirtuin 1 in non-alcoholic fatty liver disease. Several contradictory data are also reported concerning serum levels of miR-181 a in diabetic patients. While serum levels of miR-l8la-5p were decreased in obese and diabetic patients, circulating levels of miR-l8la were increased in type 1 diabetic children and adolescents. Nevertheless, circulating miR-l8la-5p levels were increased in patients with ischaemic stroke, transient ischaemic attack and acute myocardial infarction. Our study revealed upregulation of miR-l8la-5p in a proportion of children with both normal and abnormal clinical findings descending from gestational hypertension pregnancies (39.13% and 25.81%). In view of the inconsistency between studies, the role of miR-l8la-5p is not clear. Nevertheless, we conclude that increased levels of miR- l8la-5p in whole peripheral blood of children prenatally exposed to gestational hypertension indicate predisposition to development of cardiovascular diseases.

MiR-l95 gene is located on the chromosome 17r13.1 and generates two microRNAs, miR-195-5p and miR-l95-3p. MiR-l95 is increasing in cardiac hypertrophy, and cardiac miR-l95 upregulation results in heart failure. MiR-l95 is also a powerful regulator of the aortic extracellular matrix. Administration of antagomiR-l95 leads to significant elevation of elastin and collagens in the murine aorta, but has no effect on survival and aortic diameter size. Nevertheless, in plasma samples an inverse correlation between miR-l95 and the presence of abdominal aortic aneurysms and aortic diameter was observed. Surprisingly, miR-l95 plasma levels were decreased in abdominal aortic aneurysms. On the other hand, aortic stenosis caused by leaflet calcification of the bicuspid aortic valve was associated with down-regulation of miR-l95. The mechanism of miR-l95 action is not completely understood. We observed upregulation of miR-l95-5p in a proportion of children with normal clinical findings born from pregnancy with gestational hypertension (34.78%). We conclude that upregulation of miR-l95-5p may have rather protective role than harmful effect.

MiR-210-3p, encoded by a gene located on chromosome 1 lpl 5.5, is the most prominent microRNA consistently stimulated under hypoxic conditions. Upregulation of miR-2lO levels was detected in placentas of women with preeclampsia, a condition that is characterized by hypoxia resulting from inadequate blood supply to the placenta. Several microRNAs involving miR-2lO are involved in atherosclerotic plaque formation through the regulation of endothelial apoptosis. Nevertheless, some authors reported that upregulated levels of miR-2lO positively correlated with the level of endothelial cell apoptosis, while others found that miR-2lO suppression only leads to induction of endothelial cell apoptosis and cell death in hypoxia. Another study showed that upregulation of miR-2lO had cytoprotective effects, mainly in cardiomyocytes and the skeletal muscle. Increased serum levels of miR-210 may indicate early stages of atherosclerosis obliterans and heart failure. MiR-210 also regulates angiogenesis in response to ischemic injury to the brain. Upregulation of miR-210 may activate the Notch signalling pathway, which probably contributes to angiogenesis after cerebral ischemia. We observed a strong positive correlation between gene expression of miR-210-3p in whole peripheral venous blood of children born from pregnancies with preeclampsia and/or FGR and the pulsatility index in the ductus venosus. We conclude that children with postnatal upregulation of miR- 210-3p represent a group in risk of development of cardiovascular and cerebrovascular diseases, since increased pulsatility index in the ductus venosus is an indicator of a poor perinatological outcome. MiR-342-3p is encoded by a gene located on chromosome 14q32.2. MiR-342-3p is considered as an obesity-associated microRNA, since it positively regulates adipogenesis by suppressing QBP2 and releasing the key adipogenic regulator C/EBRa. A set of upregulated microRNAs expressed in peripheral blood mononuclear cells involving miR-342-3p was shared among patients with type 1 diabetes mellitus, type 2 diabetes mellitus and gestational diabetes mellitus. Urinary exosomal miR- 342 was also expressed at significantly elevated levels in type 2 diabetes mellitus patients. However, miR-342-3p downregulation was observed in endothelial cells isolated from lung and heart tissues of type 2 diabetes mellitus mice. Downregulation of miR-342 was also observed in T regulatory cells of patients with type 1 diabetes mellitus. Moreover, decreased plasma miR-342-3p represent a potential biomarker of children aged 5-10 years with endothelial dysfunction. Our study demonstrated downregulation of miR-342-3p in a proportion of children with abnormal postnatal clinical findings that were born from pregnancy with early preeclampsia (termination of gestation before 34 weeks) (26.92%). Therefore, we conclude that this group of children is a group with high risk, which would benefit from implementation of early primary prevention strategies and long-term follow-up.

Brief Description of Drawings

Fig. 1: Analysis results for miR-21-5p according to Example 1

Fig. 2: Analysis results for miR-26a-5p and miR-195-5p combination according to Example 2

Fig. 3: Analysis results for miR-21-5p according to Example 2

Fig. 4: Analysis results for miR-23a-3p according to Example 2

Fig. 5: Analysis results for miR-26a-5p according to Example 2

Fig. 6: Analysis results for miR-103a-3p according to Example 2 Fig. 7: Analysis results for miR-l25b-5p according to Example 2

Fig. 8: Analysis results for miR-l95-5p according to Example 2

Fig. 9: Analysis results for miR-342-3p according to Example 2

Fig. 10: Analysis results for miR-20a-5p according to Example 3

Fig. 11 : Analysis results for miRNA combination according to Example 4

Fig. 12: Analysis results for miR-l-3p according to Example 4

Fig. 13: Analysis results for miR-l7-5p according to Example 4

Fig. 14: Analysis results for miR-29a-3p according to Example 4

Fig. 15: Analysis results for miR-l26-3p according to Example 4

Fig. 16: Analysis results for miR-l33a-3p according to Example 4

Fig. 17: Analysis results for miR-l46a-5p according to Example 4

Fig. 18: Analysis results for miR-l8la-5p according to Example 4

Fig. 19: Analysis results for miRNA combination according to Example 5

Fig. 20: Analysis results for miR-l-3p according to Example 5

Fig. 21: Analysis results for miR-l7-5p according to Example 5

Fig. 22: Analysis results for miR-29a-3p according to Example 5

Fig. 23: Analysis results for miR-l26-3p according to Example 5

Fig. 24: Analysis results for miR-l33a-3p according to Example 5

Fig. 25: Analysis results for miR-l46a-5p according to Example 5

Fig. 26: Analysis results for miR-l8la-5p according to Example 5

Fig. 27: Analysis results for miR-342-3p according to Example 6

Fig. 28: Analysis results for miRNA combination according to Example 7

Fig. 29: Analysis results for miR-l03a-3p according to Example 7

Fig. 30: Analysis results for miR-l33a-3p according to Example 7

Fig. 31 : Analysis results for miRNA combination according to Example 8

Fig. 32: Analysis results for miR-l-3p according to Example 8

Fig. 33: Analysis results for miR-20a-5p according to Example 8

Fig. 34: Analysis results for miR-l03a-3p according to Example 8

Fig. 35: Analysis results for miR-l33a-3p according to Example 8

Fig. 36: Analysis results for miR-20b-5p according to Example 9

Fig. 37: Analysis results for miRNA combination according to Example 10

Fig. 38: Analysis results for miR-l7-5p according to Example 10

Fig. 39: Analysis results for miR-l26-3p according to Example 10

Fig. 40: Analysis results for miR-l33a-3p according to Example 10 Examples of carrying out the invention

Example 1

Postnatal screening of miR-21-5p identifies children at increased cardiovascular risk in a group of children born of physiological pregnancy who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings in cardiac echocardiography)

Although the Kruskal-Wallis test did not show a statistical difference for miR-2l-5p (p = 0.171) between the group of children born of physiological pregnancy with abnormal and normal postnatal findings, subsequent ROC analysis revealed upregulation of miR-2l-5p (AUC 0.649, p = 0.013) in children with abnormal postnatal findings (sensitivity 28.95% at specificity 90.0%).

Table 1 : Analysis results for miR-21 -5p according to Example 1

Example 2

The postnatal screening of the combination of miR-26a-5p and miR-195-5p identifies children with increased cardiovascular risk in a group of children born of pregnancy complicated by gestational hypertension who have normal postnatal findings (normal blood pressure (BP), normal BMI and normal cardiac echocardiography)

The postnatal screening based on the combination of miR-26a-5p and miR-l95-5p showed the highest precision in identifying children with normal postnatal findings who were exposed to gestational hypertension during prenatal life (AUC 0.717, p = 0.001, sensitivity 86.96%, specificity 52.0 %, cut off value >0.246824). A postnatal screening based on a combination of miR-26a-5p and miR-l95-5p identified 34.78% of children at increased cardiovascular risk at 10.0% false positivity in the group of children born from a pregnancy complicated by gestational hypertension who had normal postnatal findings. Single microRNA biomarkers upregulated in children born of pregnancy complicated by gestational hypertension who had normal postnatal findings (miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-l03a-3p, miR-l25b-5p, miR-l95-5p and miR-342-3p) were used for finding the optimum combination.

Table 2: Analysis results for miR-26a-5p and miR-195-5p combination according to Example 2

Table 3: Analysis results for miR-21 -5p according to Example 2

Table 4: Analysis results for miR-23a-3p according to Example 2

Table 5: Analysis results for miR-26a-5p according to Example 2

Table 6: Analysis results for miR-103a-3p according to Example 2

Table 7: Analysis results for miR-125b-5p according to Example 2

Table 8: Analysis results for miR-195-5p according to Example 2

Table 9: Analysis results for miR-342-3p according to Example 2

Example 3

Postnatal screening of miR-20a-5p identifies children at increased cardiovascular risk in a group of children born from pregnancy complicated by gestational hypertension who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings in cardiac echocardiography)

Using the Kruskal-Wallis test compared to the control group, a group of children born of pregnancy complicated by gestational hypertension that had abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings found in cardiac echocardiography) had increased levels of miR-20a-5p (p = 0.041). ROC analysis showed that postnatal screening miR-20a-5p (AUC 0.678, p = 0.005) identified a proportion of children at increased cardiovascular risk in the group of children born from a pregnancy complicated by gestational hypertension who had abnormal postnatal findings (sensitivity 25.81% at 10.0 % false positivity).

Table 10: Analysis results for miR-20a-5p according to Example 3

Example 4

Postnatal screening of the combination of miR-l-3p, miR-29a-3p, miR-126-3p, miR-133a-3p and miR-181a-5p identifies in a group of children born from pregnancy complicated by gestational hypertension, who have normal postnatal findings (normal BP, normal BMI and normal finding in echocardiography of the heart), children at increased cardiovascular risk

A postnatal screening based on the combination of miR-l-3p, miR-29a-3p, miR-l26-3p, miR-l33a-3p and miR-l8la-5p showed the highest accuracy in identifying children with normal postnatal findings who were exposed to gestational hypertension during prenatal life (AUC 0.803, p<0.00l, sensitivity 82.61%, specificity 74.0%, cut off value >0.224754). A postnatal screening based on a combination of miR-l-3p, miR-29a-3p, miR-l26-3p, miR-l33a-3p and miR-l8la-5p identified at 10.0% false positivity in the group of children born from a pregnancy complicated by gestational hypertension, who have normal postnatal findings, 47.83% of children at increased cardiovascular risk. Table 1 1 : Analysis results for miRNA combination according to Example 4

Table 12: Analysis results for miR-1 -3p according to Example 4

Table 13: Analysis results for miR-17-5p according to Example 4

Table 14: Analysis results for miR-29a-3p according to Example 4

Table 15: Analysis results for miR-126-3p according to Example 4

Table 16: Analysis results for miR-133a-3p according to Example 4

Table 17: Analysis results for miR-146a-5p according to Example 4

Table 18: Analysis results for miR-181 a-5p according to Example 4

Example 5

Postnatal screening of the combination of miR-l-3p, miR-17-5p, miR-29a-3p, miR-126-3p, miR- 133a-3p, miR-146a-5p and miR-181a-5p identifies children at increased cardiovascular risk in a group of children born from a pregnancy complicated by gestational hypertension having an abnormal postnatal finding (overweight/obesity, prehypertension/hypertension and/or abnormal finding in cardiac echocardiography)

Postnatal screening based on a combination of all 7 microRNA biomarkers (miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and miR-l8la-5p) showed the highest accuracy in identifying children with abnormal postnatal findings who were exposed to gestational hypertension during prenatal life (AUC 0.801, p<0.00l, sensitivity 70.97%, specificity 76.0%, cut off value >0.353489). Postnatal screening based on a combination of miR-l-3p, miR-l7-5p, miR-29a-3p, miR- l26-3p, miR-l33a-3p, miR-l46a-5p and miR-l8la-5p identified at 10.0% false positivity, in the group of children born from a pregnancy complicated by gestational hypertension, who have an abnormal postnatal finding, 38.71% of children at increased cardiovascular risk.

Table 19: Analysis results for miRNA combination according to Example 5

Table 20: Analysis results for miR-1 -3p according to Example 5

LR = Likelihood ratio

Table 21 : Analysis results for miR-17-5p according to Example 5

Table 22: Analysis results for miR-29a-3p according to Example 5

Table 23: Analysis results for miR-126-3p according to Example 5

Table 24: Analysis results for miR-133a-3p according to Example 5

Table 25: Analysis results for miR-146a-5p according to Example 5

Table 26: Analysis results for miR-181 a-5p according to Example 5

Example 6

Postnatal screening of miR-342-3p identifies children at increased cardiovascular risk in a group of children born from a pregnancy complicated by early preeclampsia who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings in cardiac echocardiography). MiR-342-3p (AUC 0.665, p = 0.016) represents a marker that is able to differentiate between a group of children with abnormal postnatal clinical findings who were exposed to early preeclampsia and a control group of children during prenatal life (sensitivity 26.92% at 10.0% FP), which would not be revealed if solely the Kmskal -Wallis test (p = 0.097) was used for statistical analysis.

Table 27: Analysis results for miR-342-3p according to Example 6

Example 7

Postnatal screening of the combination of miR-103a-3p and miR-133a-3p identifies children with increased cardiovascular risk in a group of children born from a pregnancy complicated by severe preeclampsia who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings in cardiac echocardiography)

Postnatal screening based on the combination of miR-l03a-3p and miR-l33a-3p showed the highest accuracy in identifying children with abnormal postnatal clinical findings who were exposed to severe preeclampsia during prenatal life (AUC 0.637, p = 0.015, sensitivity 70.21%, specificity 56.0%, cut off value >0.429299). A postnatal screening based on the combination of miR-l03a-3p and miR-l33a-3p identified 21.28% of children at increased cardiovascular risk at 10.0% false positivity in the group of children born of a pregnancy complicated by severe preeclampsia with abnormal postnatal findings.

Table 28: Analysis results for miRNA combination according to Example 7

Table 29: Analysis results for miR-103a-3p according to Example 7

Table 30: Analysis results for miR-133a-3p according to Example 7

Example 8

Postnatal screening of the combination of miR-l-3p, miR-20a-5p, miR-103a-3p and miR-133a-3p identifies children at increased cardiovascular risk in a group of children born from a pregnancy complicated by late preeclampsia who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal findings in cardiac echocardiography),

Postnatal screening based on the combination of 4 microRNA biomarkers (miR-l-3p, miR-20a-5p, miR-l03a-3p and miR-l33a-3p) showed the highest accuracy in identifying children with abnormal postnatal findings who were exposed to late preeclampsia during prenatal life (AUC 0.701, p<0.00l, sensitivity 59.46%, specificity 72.0%, cut off value >0.391116). The postnatal screening based on the combination of miR-l-3p, miR-20a-5p, miR-l03a-3p and miR-l33a-3p identified at 10.0% false positivity (FP), in a group of children born from a pregnancy complicated by late preeclampsia with abnormal postnatal findings, 32.43% of children at increased cardiovascular risk. Table 31 : Analysis results for miRNA combination according to Example 8

Table 32: Analysis results for miR-1 -3p according to Example 8

Table 33: Analysis results for miR-20a-5p according to Example 8

Table 34: Analysis results for miR-103a-3p according to Example 8

miR-103a-3p

Table 35: Analysis results for miR-133a-3p according to Example 8

Example 9

Postnatal screening of miR-20b-5p identifies children at increased cardiovascular risk in a group of childrem born from a pregnancy complicated by mild preeclampsia who have normal postnatal findings (normal BP, normal BMI and normal cardiac echocardiography)

MiR-20b-5p (AUC 0.689, p = 0.022) is the only biomarker that differentiates between a control group of children and a group of children with normal postnatal clinical findings born from a pregnancy complicated by mild preeclampsia (sensitivity 36.36% at 10.0% FP). However, a difference in miR- 20b-5p gene expression (p = 0.264) between the two groups would not be detected if only the Kruskal- Wallis test was used for statistical analysis.

Table 36: Analysis results for miR-20b-5p according to Example 9

Example 10

Postnatal screening of the combination of miR-17-5p, miR-126-3p and miR-133a-3p identifies children with increased cardiovascular risk in a group of children born from a pregnancy complicated by fetal growth restriction who have abnormal postnatal findings (overweight/obesity, prehypertension/hypertension and/or abnormal finding in cardiac echocardiography)

Postnatal screening based on the combination of miR-l7-5p, miR-l26-3p and miR-l33a-3p showed the highest accuracy in identifying children with abnormal postnatal clinical findings diagnosed as FGR fetuses during prenatal life (AUC 0.710, p = 0.002, sensitivity 63.64%, specificity 78.0%, cut off value >0.305781). The postnatal screening based on the combination of miR-l7-5p, miR-l26-3p and miR- l33a-3p identified at 10.0% false positivity, in a group of children born of a pregnancy complicated by fetal growth restriction and having an abnormal postnatal finding, 40.91% children with increased cardiovascular risk.

Table 37: Analysis results for miRNA combination according to Example 1 0

Table 38: Analysis results for miR-17-5p according to Example 10

Table 39: Analysis results for miR-126-3p according to Example 10

Table 40: Analysis results for miR-133a-3p according to Example 10

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Claims

1. A method of predicting a risk of onset and development of cardiovascular disease for a child born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction, or in a child born from a physiological pregnancy having an abnormal postnatal clinical finding, said method comprising the steps of:

- wherein the aberrant regulation of the said at least one microRNA marker exceeding the reference cut-off value, or below the reference cut-off value, respectively, indicates the risk of onset and development of cardiovascular disease for the child.

2. The method according to claim 1, wherein the sample of blood is a sample of whole peripheral venous blood.

3. The method according to any one of the precedings claims, wherein the statistical analysis is based on receivers operating characteristic (ROC) curves, or on logistic regression and receivers operating characteristic (ROC) curves.

4. The method according to any one of the precedings claims, wherein the statistical analysis further includes at least one parameter selected from: presence or absence of abnormal echocardiogram finding; presence or absence of prehypertension/hypertension, presence or absence of high BMI.

5. The method according to any one of the precedings claims, wherein the child is born from a physiological pregnancy and has an abnormal postnatal finding, wherein the microRNA marker is miR- 2l-5p.

6. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has a normal postnatal clinical finding, wherein the microRNA markers are miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-l03a-3p, miR-l25b-5p, miR-195- 5p and/or miR-342-3p.

7. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has a normal postnatal clinical finding, wherein a combination of miR-26a-5p and miR-l95-5p is used as the microRNA marker.

8. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has an abnormal postnatal clinical finding, wherein the microRNA marker is miR-20a-5p.

9. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has a normal postnatal clinical finding, wherein a combination of miR-l-3p, miR-29a-3p, miR-l26-3p, miR-l33a-3p and miR-l8la-5p is used as the microRNA marker.

10. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has a normal postnatal clinical finding, wherein the microRNA markers are miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and/or miR-l8la-5p.

11. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has an abnormal postnatal clinical finding, wherein a combination of miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and miR-l8la-5p is used as the microRNA marker.

12. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by gestational hypertension and has an abnormal postnatal clinical finding, wherein the microRNA markers are miR-l-3p, miR-l7-5p, miR-29a-3p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p and/or miR-l8la-5p.

13. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by preeclampsia, wherein the microRNA marker is miR-l33a-3p.

14. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by severe and/or late preeclampsia, wherein the microRNA marker is miR-l33a-3p.

15. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by early preeclampsia and has a normal postnatal clinical finding, wherein the microRNA marker is miR-l33a-3p.

16. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by early preeclampsia and has an abnormal postnatal clinical finding, wherein the microRNA marker is miR-342-3p.

17. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by severe preeclampsia and has an abnormal postnatal clinical finding, wherein the microRNA markers are miR-l03a-3p and/or miR-l33a-3p, or a combination of miR-l03a-3p and miR- l33a-3p is used as the microRNA marker.

18. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by late preeclampsia and has an abnormal postnatal clinical finding, wherein a combination of miR-l-3p, miR-20a-5p, miR-l03a-3p and miR-l33a-3p is used as the microRNA marker.

19. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by late preeclampsia and has an abnormal postnatal clinical finding, wherein the microRNA markers are miR-l-3p, miR-20a-5p, miR-l03a-3p and/or miR-l33a-3p.

20. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by mild preeclampsia and has a normal postnatal clinical finding, wherein the microRNA marker is miR-20b-5p.

21. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by fetal growth restriction and has an abnormal postnatal clinical finding, wherein a combination of miR-l7-5p, miR-l26-3p and miR-l33a-3p is used as the microRNA marker.

22. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by fetal growth restriction and has an abnormal postnatal clinical finding, wherein the microRNA markers are miR-l7-5p, miR-l26-3p and/or miR-l33a-3p.

23. The method according to any one of the claims 1-4, wherein the child is born from a pregnancy complicated by late preeclampsia, wherein the microRNA marker is miR-l-3p.

24. The method according to any one of the preceding claims, wherein determining risk of cardiovascular disease includes determining the risk of cardiovascular disease in sports physical activity.

25. A set of molecular markers for predicting a risk of cardiovascular disease of a child born from a pregnancy complicated by gestational hypertension, preeclampsia and/or fetal growth restriction, or in a child born from a physiological pregnancy having an abnormal postnatal clinical finding, which consists of molecular markers with aberrant expression selected from miR-l-3p, miR-l7-5p, miR-20a- 5p, miR-20b-5p, miR-2l-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-l03a-3p, miR-l25b-5p, miR-l26-3p, miR-l33a-3p, miR-l46a-5p, miR-l8la-5p, miR-l95-5p, miR-2lO-3p, miR-342-3p.