Association Analysis in Children Born from Normal and Complicated Pregnancies—Cardiovascular Disease Associated microRNAs and the Incidence of Prehypertension/Hypertension, Overweight/Obesity, Valve Problems and Heart Defects

The goal was to assess how a history of any kind of pregnancy-related complication altered expression profile of microRNAs played a role in the pathogenesis of diabetes, cardiovascular and cerebrovascular diseases in the peripheral blood leukocytes of children at the age of 3–11 years. The prior exposure to gestational hypertension, preeclampsia, fetal growth restriction, gestational diabetes mellitus, preterm prelabor rupture of membranes or spontaneous preterm birth causes that a significant proportion of children (57.42% to 90.0% specifically) had a substantially altered microRNA expression profile, which might be the origin of a lifelong cardiovascular risk. A total of 23 out of 29 tested microRNAs were upregulated in children born from such complicated gestation. The occurrence of overweight, obesity, valve problems and heart defects even intensified upregulation of microRNAs already present in children exposed to such pregnancy complications. The occurrence of overweight/obesity (miR-92a-3p, and miR-210-3p) and valve problems or heart defects (miR-342-3p) induced microRNA upregulation in children affected with pregnancy complications. Overall, 42.86% overweight/obese children and 27.36% children with valve problems or heart defects had even higher microRNA levels than children with normal clinical findings after complicated pregnancies. In addition, the microRNA expression profile was also able to differentiate between children descending from normal gestation in relation to the occurrence of overweight and obesity. Screening on the base of the combination of 19 microRNAs identified 70.0% overweight/obese children at 90.0% specificity. In general, children after complicated pregnancies, just as children after normal pregnancies, with abnormal findings are at a higher risk of the onset of cardiovascular complications, and their dispensarization, with the aim to implement primary prevention strategies, would be beneficial.


Introduction
The increasing worldwide prevalence of pregnancy-related complications generates an increasing number of children with a lifelong cardiovascular risk .
Recently, we reported that a proportion of children affected with gestational hypertension (GH), preeclampsia (PE), fetal growth restriction (FGR) and gestational diabetes mellitus (GDM) had altered microRNA expression profiles that may contribute to the predisposition of these children to later onset of diabetes mellitus, cardiovascular and cerebrovascular diseases [52,53].
The pivotal goal of the current study was to assess how a history of any kind of pregnancy complication (GH, PE, FGR, GDM, preterm prelabor rupture of membranes or spontaneous preterm birth) might contribute to postnatal alterations of microRNA expression profiles in whole peripheral venous blood (leukocytes).
In addition, the subject of our interest was extended to explore the impact of the actual occurrence of individual abnormal findings (overweight, obesity, prehypertension, hypertension, valve problems and heart defects) on already present abnormal microRNA expression profiles in children born from complicated gestation.
An additional goal of the current study was to determine to what extent individual abnormal findings (overweight, obesity, prehypertension, hypertension, valve problems and heart defects) might influence microRNA expression profiles in children born from normal gestation.
The idea of the determination of potential diabetic/cardiovascular risk in children prenatally or perinatally affected by any kind of pregnancy-related complication was based on the fact that appropriate microRNAs are involved in the inducement and progress of diabetes mellitus, cardiovascular and cerebrovascular diseases (Table S1). We selected 29 microRNAs, which had usually been displaying aberrant expression in plasma/serum/affected tissue samples of adult and elderly patients with signs of diabetes mellitus, cardiovascular and cerebrovascular diseases.
Screening on the base of a combination of all above mentioned microRNAs showed that, at 10.0% FPR, 57.42% of children born from complicated gestation had significantly altered postnatal microRNA expression profile, which may indicate an increased risk of later onset of diabetes mellitus, cardiovascular and cerebrovascular diseases (area under the curve (AUC) 0.777, p < 0.001, sensitivity 61.80%, specificity 88.04%, cut off >0.819967957) (Figure 1).
Subsequently, we performed comparison of microRNA expression profile between particular groups of children with respect to actual findings (normal systolic and diastolic blood pressures vs. prehypertension/hypertension; normal body mass index (BMI) vs. overweight/obesity; normal values of echocardiographic measurements vs. the occurrence of valve problems or heart defects), regardless of prenatal or perinatal exposure to pregnancy complications (gestational diabetes mellitus, gestational hypertension, preeclampsia, FGR, preterm prelabor rupture of membranes and/or spontaneous preterm birth).

No Association between the Presence of Prehypertension/Hypertension and Expression of Examined microRNAs
Overall, 63 out of 503 (12.53%) tested children were confirmed over several visits to have systolic prehypertension/hypertension, and 58 out of 503 (11.53%) tested children had diastolic prehypertension/hypertension.
No association between the occurrence of prehypertension or hypertension (combined systolic and diastolic prehypertension/hypertension and/or isolated systolic or diastolic prehypertension/hypertension defined as systolic and/or diastolic blood pressures equal or above the 90th or 95th percentiles) and the expression of examined microRNAs was identified in children, regardless of the course of gestation of their mothers.
Nevertheless, with regard to a high number of outliers in children with normal BMI values and in children with normal values of echocardiographic measurements, and with regard to a large impact of the course of gestation of mothers on postnatal microRNA expression profile, we finally decided to also evaluate microRNA expression data within the equal groups of children (children born from normal gestation only, and children born from complicated gestation only).

Postnatal Expression Profile of 19 Tested microRNAs Differentiates between Overweight/Obese and Normal Weight Children Born from Normal Pregnancies
Overall, 10 out of 92 (10.87%) tested children born from normal gestation were confirmed to be overweight/obese.

Postnatal Expression Profile of 18 Tested microRNAs Differentiates between Overweight/Obese and Normal Weight Children Born from Complicated Gestation
Overall, 22 out of 411 (5.35%) tested children born from complicated gestation were confirmed to be overweight/obese.
Screening on the base of a combination of all these 18 microRNAs was superior to the performance of single microRNAs, and showed that at 10.0% FPR, 42.86% of overweight/obese children born from complicated gestation had a significantly altered postnatal microRNA expression profile (AUC 0.753, p < 0.001, sensitivity 71.4%, specificity 67.7%, cut off >0.038238759) ( Figure 5).

Discussion
At first, we made comparison of microRNA expression profile between children born from complicated and normal gestation, irrespective of the type of pregnancy-related complication (gestational diabetes mellitus, gestational hypertension, preeclampsia, FGR, preterm prelabor rupture of membranes and/or spontaneous preterm birth). Subsequently, we made a comparison of the microRNA expression profile between particular groups of children with respect to actual findings (normal systolic and diastolic blood pressures vs. prehypertension/hypertension; normal BMI vs. overweight/obesity; normal values of echocardiographic measurements vs. the occurrence of valve problems and/or heart defects), regardless of prenatal and perinatal exposure to pregnancy complications (gestational diabetes mellitus, gestational hypertension, preeclampsia, FGR, preterm prelabor rupture of membranes and/or spontaneous preterm birth). In view of the fact that the prenatal and perinatal exposure to any pregnancy-related complication (gestational diabetes mellitus, gestational hypertension, preeclampsia, FGR, preterm prelabor rupture of membranes and/or spontaneous preterm birth) led to substantial alterations of postnatal microRNA expression profile in a proportion of children, we also compared microRNA expression profile between equal groups of children (children born from normal gestation only and children born from complicated gestation only) with respect to actual findings (the occurrence of overweight, obesity, prehypertension, hypertension, valve problems and heart defects).
Our data may be supported by the data of Chen et al. [55], who recently reported that miR-17-5p is, together with miR-27a/b, a key microRNA playing a role in the pathogenesis of childhood obesity via the regulation of the NLK and RRAS2 genes. Nevertheless, this study was performed using different methodological approach, tissue samples were analyzed using microarray methodology. Similarly, our data are compliant with the data of other investigators, who reported that miR-103a-3p is one of microRNAs implicated in appetite and energy balance control affecting overeating during obesity development including children as well [56,57]. Likewise, Marzano et al. [58] observed upregulation of circulating (serum) miR-92a-3p in obese children, regardless of whether they had been born small for gestational age or appropriate for gestational age.
Circulating (plasma) miR-342-3p was reported, until now, to be associated just with endothelial dysfunction in children, not with other cardiovascular risk factors or other abnormal clinical findings, which is in agreement with our data, even though plasma and whole peripheral blood microRNA gene expression profiles may have different patterns [59].

Participants
The study of a prospective design running within the period of 8/2016-10/2020 involved children of Caucasian descent at the age of 3 to 11 years born from normal pregnancies (n = 92) and pregnancies complicated with gestational diabetes mellitus (n = 118), gestational hypertension (n = 53), preeclampsia (n = 135), fetal growth restriction (n = 35), preterm prelabor rupture of membranes or spontaneous preterm birth (n = 70). The data of children born from normal and complicated gestation are presented in Table 1.
Normal gestation was reported as that one without medical, obstetrical, or surgical complications, where healthy infants with the weight > 2500 g were born after 37 weeks of gestation [52,53].
Gestational diabetes mellitus, glucose intolerance during gestation, was diagnosed following the recommendations of the International Association of Diabetes and Pregnancy Study Groups (IADPSG) [53,60].
Gestational hypertension, hypertension (>140/90 mmHg) with no sign of proteinuria with the first onset after 20 weeks of gestation, was diagnosed following the recommendations of American College of Obstetricians and Gynecologists (ACOG) [52,61].
Preeclampsia was characterized as the occurrence of hypertension and proteinuria (>300 mg/24 h) that appeared firstly after 20 weeks of gestation [52,61]. The severity of preeclampsia was assessed following the recommendations of ACOG [52,61].
FGR, the estimated fetal weight (EFW) below the 3rd percentile or below the 10th percentile for the evaluated gestational age, early FGR (before 32 week of gestation), and late FGR (after 32 week of gestation) were diagnosed based on the current recommendations [52,[62][63][64].
Preterm birth (PTB) was defined as the delivery before 37 weeks of gestation at the occurrence of regular uterine contractions along with cervical changes. Preterm prelabor rupture of membranes (PPROM) was diagnosed when amniotic fluid leakage preceded the onset of labor by at least 2 h [65][66][67]. The exclusion criteria for preterm birth (PTB or PPROM) included gestational hypertension, preeclampsia, gestational diabetes mellitus, significant vaginal bleeding and signs of fetal growth restriction.
More details on the definition of individual pregnancy complications are also available in Supplementary Materials.
Children with inborn defects, chromosomal abnormalities and children born from gestation with other complications were excluded from the study.   Informed consent was gained from all study participants. Two ethics committees (with headquarters in the Institute for the Care of the Mother and Child, and the Third Faculty of Medicine, Charles University) granted the approval with the study (grant no. AZV 16-27761A, long-term monitoring of complex cardiovascular profile in the mother, fetus and offspring descending from pregnancy-related complications, dates of approval: 27.3.2014 and 28.5.2015). All procedures were in agreement with the Helsinki Declaration of 1975, as revised in 2000.

Blood Pressure and Echocardiography Measurements, and Body Mass Index Assessment
Standardized blood pressure (BP) and echocardiography measurements and BMI assessment were performed as previously described [52,53,68]. More details are also available in Supplementary Materials.

Processing of Samples, Reverse Transcription, and Relative Quantification of microRNAs
Processing of samples, reverse transcription and relative quantification of microRNAs were performed as previously described [52,53,69,70]. More details are also available in Supplementary Materials.

Statistical Analysis
Statistical analyses (the Shapiro-Wilk test, Mann-Whitney test (M-W), receivers operating characteristic (ROC) curves, and logistic regression combined with ROC curve analysis) were performed as previously described [52,53,71]. The box plots of log-normalized gene expression values (RT-qPCR expression, log 10 2 −∆∆Ct ) for particular microRNAs are presented. More details concerning statistical analyses and graphical processing are also available in Supplementary Materials.

Conclusions
In conclusion, representative microRNA expression profiles for diabetes mellitus, cardiovascular and cerebrovascular diseases may also be detected in peripheral blood leukocytes of children born from complicated gestation (gestational diabetes mellitus, gestational hypertension, preeclampsia, FGR, preterm prelabor rupture of membranes and/or spontaneous preterm birth). This observation implies that pregnancy-related complications of the mother, regardless of its type, may contribute to the predisposition of affected children to later onset of diabetes mellitus, cardiovascular and cerebrovascular diseases. The presence of overweight/obesity and/or valve problems and heart defects have an additional impact on already altered microRNA expression profile in a group of children previously exposed to pregnancy-related complications. MicroRNA expression profiles are also able to differentiate between children with abnormal and normal findings (BMI) born from normal gestation. Consecutive studies are required to confirm the findings of this study.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/21/ 8413/s1. Figure S1: Aberrant microRNA expression profile in children descending from complicated pregnancies irrespective of the type of pregnancy-related complication (gestational diabetes mellitus, gestational hypertension, preeclampsia, fetal growth restriction, preterm prelabor rupture of membranes, and/or spontaneous preterm birth).; Figure S2: Aberrant microRNA expression profile in overweight/obese children irrespective of the course of gestation of their mothers (normal and complicated pregnancies altogether).; Figure S3: Aberrant microRNA expression profile in children with valve problems or heart defects irrespective of the course of gestation of their mothers (normal and complicated pregnancies altogether).; Figure S4: Aberrant microRNA expression profile in overweight/obese children descending from normal pregnancies.; Figure S5: Aberrant microRNA expression profile in overweight/obese children descending from complicated pregnancies.; Figure S6: Aberrant microRNA expression profile in children with valve problems or heart defects descending from complicated pregnancies.; Table S1: The role of studied microRNAs in the pathogenesis of diabetes mellitus and cardiovascular/cerebrovascular diseases.; Supplementary Material-Methods.