Study design and population

This study was carried out at the Department of Pediatrics, Innsbruck University Hospital, Austria, from May 2012 to March 2015. The survey area, Tyrol, is a state in Western Austria with 680 000 inhabitants and approximately 7000 live births per year. We investigated a group of former preterm infants born between 01/01/2007 and 07/31/2009 at <32 weeks’ gestational age, who were invited to a routine preschool visit at our preterm follow-up clinic, and a control group of same-aged children born at term who were recruited through regional kindergartens or while undergoing routine preoperative screening for common surgical procedures (adenotomy, tonsillotomy) at Innsbruck University Hospital. None of the subjects had congenital malformations or chromosomal abnormalities. Approval of this study by the local ethics committee, IRB Medical University of Innsbruck (UN 4491), was obtained in advance. Written informed consent was obtained from all legal guardians. All children included in the study orally consented to participation.

Perinatal characteristics

Basic perinatal data for each child belonging to the preterm study group were drawn from the routine preterm follow-up database at our institution. To account for gender-and gestational age-specific differences, birth weight z-scores were calculated for every subject by means of the Fenton 2013 Growth Calculator for Preterm Infants (available from http://www.peditools.org/fenton2013) [9]. Classification of smoking during pregnancy was based on self-reporting by mothers. Maternal educational status was classified as less than 12 years or 12 years and more.

Study visit

All examinations were carried out either at Innsbruck University Hospital or at provisional medical posts installed in participating kindergartens between 8 a.m. and 10 a.m. by specifically trained personnel. After a routine clinical examination, weight was measured by means of calibrated medical precision scales and height was determined by a Harpenden stadiometer. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. In order to account for gender- and age-specific differences, BMI z-scores were calculated for each study participant by means of a reference data set[10].

Blood pressure was measured thrice on the right upper arm with the appropriate cuff size after a five-minute resting period in a seated position using an automated oscillometric device.

Intima-media thickness (IMT) was determined ultrasonographically in the dorsal arterial wall of the most caudal 15 mm of the abdominal aorta by means of a high frequency linear array transducer (12L-RS, GE Healthcare) as described previously [7].

Blood samples were collected after a minimum overnight fasting period of eight hours. Fasting glucose, fasting insulin, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, adiponectin and leptin levels were determined at the Central Institute for Medical and Chemical Laboratory Diagnosis (Innsbruck University Hospital). Homeostasis model assessment (HOMA) index was calculated for each subject as fasting insulin [mU/l] times fasting glucose [mg/dl], divided by 405.

All children and their parents were asked to fill out a questionnaire regarding family history of cardiovascular disease (CVD) and childhood nutrition habits. A positive family history of CVD was defined as a diagnosis of coronary heart disease, angina, heart attack, congenital heart disease or stroke in first-degree male relative of child and/or parent under the age of 55 or first-degree female relative of child and/or parent under the age of 65 [11]. Information on childhood nutrition habits was gathered by means of an established standardized food frequency questionnaire (“What do you eat?”, kindly provided by the Robert Koch Institute, Berlin, Germany) [12]. Childhood nutrition was subsequently categorized as unfavorable, neutral or favorable for further analyses.

In children born at term not previously registered at our hospital, basic perinatal data were collected at study visit. In the preterm group, data not available from our routine preterm follow-up data base were also filled in during the study visit. Remaining missing data were classified as “unknown”.

Statistical methods

All statistical analyses were performed by means of SPSS for Windows, version 22 (SPSS Inc.).

In order to ensure representativeness of study samples, both term and preterm study cohorts were compared with reference populations (term: SIDS database Tyrol, birth years 2007–2009; preterm: Innsbruck routine preterm follow-up database, birth years 2007–2009) in regard to gender distribution, gestational age, birth weight and maternal educational status by Pearson’s Chi-Square and Mann-Whitney U test.

Differences in perinatal characteristics as well as characteristics at study visit between term and preterm study groups were determined by means of Pearson’s Chi-Square, Mann-Whitney U or Student’s T-test, depending on type and distribution of the variable analyzed.

Logistic regression analysis was used to determine differences between preterm and term subjects in blood pressure and aortic IMT measurements as well as laboratory markers. To account for skewed distributions or heteroscedasticity of the dependent variable, fasting glucose and insulin levels, HOMA index as well as triglyceride, adiponectin and leptin levels were logarithmically transformed prior to analyses. With regard to covariates and potential confounders, a step-wise approach was used. Model A was adjusted for age at examination and gender, birth weight z-score, smoking during pregnancy, maternal educational status and positive family history of CVD. Model B added current BMI z-scores and childhood nutrition profiles. Odds ratios (OR) and respective confidence intervals (CI) were calculated as OR per standard deviation (SD) increase for all continuous variables.

Prevalence of prehypertension (systolic and/or diastolic blood pressure ≥90th percentile), raised fasting glucose (≥75th percentile) and HOMA index (≥75th percentile), elevated total cholesterol (≥150 mg/dl or 3.9 mmol/l) and LDL cholesterol levels (≥100 mg/dl or 2.6 mmol/l) as well as low HDL cholesterol levels (<40 mg/dl or 1.0 mmol/l) was calculated for preterm and term study groups [13,14,15]. OR and respective CI were determined by logistic regression analysis with respective adjustments in models A and B (see above).

Study population and demographics

A total number of 90 former term and 182 former preterm infants were assessed for eligibility. 89 former term and 93 former preterm infants were included in the study (Fig 1).

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Fig 1. Flow diagram.

Overview of children assessed for eligibility and enrolled in the study as well as data availability with regard to laboratory markers and intima-media thickness in term and preterm study groups. Basic perinatal characteristics, anthropometric data and blood pressure measurements were available for all subjects included in the study.

https://doi.org/10.1371/journal.pone.0168162.g001

Children born at term enrolled in the study were similar to the general Tyrolean population in birth weight and gender distribution. Maternal educational status was higher in the study cohort. Former preterm infants enrolled in the study did not significantly differ from a preterm reference population in gestational age, birth weight, gender distribution or maternal educational status (data available on request).

Population characteristics

Perinatal characteristics as well as characteristics at study visit of all children enrolled are given in Table 1. At study visit, children born preterm had a significantly lower body mass index and BMI z-scores than term controls. A smaller percentage of favorable childhood nutrition profiles was reported in this group.

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Table 1. Perinatal characteristics and characteristics at study visit in former term and preterm infants.

https://doi.org/10.1371/journal.pone.0168162.t001

None of the participants were taking medication with a possible influence on the cardiovascular system, glucose homeostasis or lipid metabolism.

With regard to antenatal administration of corticosteroids, 76.3% of preterm infants received a full course prior to birth and 11.8% received an incomplete course. No corticosteroids were administered in 8.6% of cases; in 3.2% administration was not documented. Postnatal corticosteroids were administered in 14.0% of preterm infants for treatment of bronchopulmonary dysplasia. All findings did not significantly differ from a preterm reference population (p = 1.000 for both analyses).

Blood pressure and aortic intima-media thickness measurements

In comparison to same-aged control subjects born at term, former preterm infants had higher systolic blood pressure readings (Table 2). Results remained similar after adjusting for covariates in all models applied (Table 3). No differences in mean blood pressures were observed (Tables 2 and 3). With regard to diastolic blood pressure, former preterm infants had higher measured values than children born at term (Table 2) and this difference was significant in the fully adjusted model (Table 3). Aortic IMT was similar in both study groups (Tables 2 and 3).

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Table 2. Blood pressure readings, aortic intima-media thickness and laboratory parameters.

https://doi.org/10.1371/journal.pone.0168162.t002

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Table 3. Logistic regression analysis of cardiovascular risk factors in children born preterm in comparison to same-aged controls born at term.

https://doi.org/10.1371/journal.pone.0168162.t003

Glucose homeostasis

Fasting glucose levels were significantly higher in former preterm infants in comparison to children born at term (Table 2), with consistent results after adjusting for covariates in all statistical models used (Table 3). Fasting insulin levels were higher in the preterm group, but this finding did not reach statistical significance in any of the models applied (Tables 2 and 3). HOMA index was significantly higher in the preterm study group (Table 2). Statistical significance was also reached in the fully adjusted model (Table 3).

Lipid profiles

In comparison to children born at term, former preterm infants had significantly higher total cholesterol and LDL cholesterol, but also HDL cholesterol levels (Table 2). Results were similar throughout all statistical models (Table 3). Triglyceride levels were similar in both groups (Tables 2 and 3).

Adipocytokine levels

No differences between groups were found in adiponectin levels (Tables 2 and 3). With regard to leptin, higher levels were observed in the preterm study group in comparison to term controls. This finding reached statistical significance after adjusting for current BMI z-scores, and childhood nutrition profiles (Table 3).

Prevalence of cardiovascular risk factors at preschool age

In order to obtain a better understanding of the clinical relevance of our findings, we also determined the prevalence of manifest abnormalities in blood pressure, glucose and lipid metabolism. In accordance with above-mentioned results, the prevalence of systolic prehypertension was significantly higher in former preterm infants in comparison to term-born controls (23.7% vs. 2.2%; OR [95% CI] 13.8 [3.1–60.9], p = 0.001). Prevalence of diastolic prehypertension was also higher in the preterm group, but this finding did not reach statistical significance. Results were similar after adjustment for covariates in all statistical models.

Raised fasting glucose levels were more frequently observed in the preterm group (28.6% vs. 5.9%; OR [95% CI] 6.4 [1.4–28.8], p = 0.016; similar results in models A and -B). Also, raised HOMA index was more common in former preterm infants (49.4% vs. 27.6%; OR [95% CI] 2.6 [1.0–6.4], p = 0.046; statistical significance also reached in model B).

With regard to hypercholesterolemia, total cholesterol levels above the normal range were significantly more prevalent in the preterm study group (77.4% vs. 52.9%; OR [95% CI] 3.0 [1.3–7.1], p = 0.010); similar results in models A and B). Comparable results were found for elevated LDL cholesterol levels (54.8% vs. 26.5%; OR [95% CI] 3.4 [1.4–8.1]; p = 0.007; similar results in models A and -B). The prevalence of low HDL cholesterol levels was similar between groups (3.7% vs. 9.7%; OR [95% CI] 0.4 [0.07–2.0], p = 0.255; similar results in models A and B).

Due to the small number of small for gestational age newborns in both study groups, a subgroup analysis for these subjects was not undertaken. A separate analysis excluding small for gestational age subjects was conducted and yielded similar results (comparable effect sizes) in all regards.

Blood pressure readings, aortic IMT measurements, fasting glucose and insulin levels, HOMA index, total, LDL and HDL cholesterol levels, triglyceride, adiponectin and leptin levels were not influenced by use of antenatal corticosteroids and did not significantly differ between former preterm infants who did or did not receive corticosteroids postnatally (S1 and S2 Tables).

In preterm-born children heightened risk factor levels were independent of gestational age. For example, mean systolic blood pressure readings were 101, 101, 106, 100, and 101 mmHg in gestational age groups <26 weeks, 26–28 weeks, 29 weeks, 30 weeks, and 31–32 weeks. Mean fasting glucose levels were 81 mg/dl (4.5 mmol/l), 82 mg/dl (4.6 mmol/l), 83 mg/dl (4.6 mmol/l), 87 mg/dl (4.8 mmol/l), and 83 mg/dl (4.6 mmol/l); total cholesterol levels were 158 mg/dl (4.1 mmol/l), 164 mg/dl (4.2 mmol/l), 175 mg/dl (4.5 mmol/l), 176 mg/dl (4.6 mmol/l), and 171 mg/dl (4.4 mmol/l).