Arterial Stiffness and Carotid Intima‐Media Thickness in Children Exposed to Smokeless Tobacco in Fetal Life

Abstract

Background

Arterial stiffening and increased intima‐media thickness can be seen as early as childhood and are associated with increased risk of cardiovascular events in adult life. The authors hypothesized that exposure to prenatal smokeless tobacco (Swedish snus) without additional nicotine exposure after the breastfeeding period would be associated with increased arterial stiffness and intima‐media thickening in preschool children.

Methods and Results

This was a longitudinal follow‐up cohort study of children aged 5 to 6 years exposed to high doses of nicotine in utero. Women exclusively using snus and unexposed controls were enrolled in early pregnancy (gestational age range, 6–12 weeks). Exposure data were collected during and after pregnancy with questionnaires from both groups. For this study, only children of women using >48 mg nicotine per day during their entire pregnancy were included in the exposure group. Outcomes were determined in 40 healthy children (21 exposed to snus in utero). Ultrasonography of the common carotid artery was used to determine carotid intima‐media thickness and calculate arterial stiffness index from the relationship between pulsatile changes in arterial diameter and arterial pressure. Children exposed to snus in fetal life had higher carotid stiffness (median 4.1 [interquartile range (IQR), 2.4–5] versus 2.9 [IQR, 2.1–3.5]; P=0.014) than tobacco‐free controls. Carotid strain (relative diameter change) was lower in children exposed to snus (mean 16% [SD, 5.7%] versus 21% [SD, 6.6%]) than in controls (P=0.015). Carotid intima‐media thickness did not differ significantly between children exposed to snus and controls.

Conclusions

Exposure to snus during fetal life was associated with a stiffer carotid artery in preschool children.

Nonstandard Abbreviations and Acronyms

cIMT

carotid intima‐media thickness

EDD

end‐diastolic diameter

ESD

end‐systolic diameter

Clinical Perspective.

What Is New?

• Unlike previous studies on the association between maternal smoking in pregnancy and arterial damage in offspring, the current study pinpointed perinatal exposure to smokeless tobacco, ie, nicotine.

• Preschool children who were exposed to high doses of nicotine in utero had reduced carotid arterial strain and stiffer carotid arteries.

What Are the Clinical Implications?

• Exposure to prenatal nicotine may have implications for the risk of cardiovascular events in later life.

• This finding adds to current recommendations that women should abstain from all forms of tobacco and nicotine products during pregnancy.

Early stiffening of the arterial wall is a marker of increased risk for cardiovascular disease. Increased arterial wall stiffness and thickening of its intima and media layers are considered precursors of vascular disease, with well‐described associations with hypertension, stroke, coronary heart disease, and myocardial infarction. ¹ , ² , ³ The origins of arterial wall stiffening are multifactorial and strongly related to age. ⁴ , ⁵ Signs of vascular aging may appear as early as childhood. ⁶ , ⁷ , ⁸

In individuals exposed to cigarette smoke, vascular aging is accelerated. ³ , ⁸ Smoking is an important modifiable and independent risk factor for cardiovascular disease. ⁸ , ⁹ Studies of parental smoking and secondhand smoke have reported arterial wall changes and thicker intima media in exposed children. ⁶ , ¹⁰ , ¹¹ Subclinical arterial wall changes arising early in childhood are suggested to follow a trajectory into adulthood. ¹² , ¹³ , ¹⁴

Studies of children whose mothers smoked during pregnancy have proposed adverse and long‐lasting prenatal programming effects, such as thicker arterial walls and increased arterial stiffness. ⁶ , ¹⁰ However, these studies have not been able to rule out the confounding of secondhand smoke exposure. In addition, the discrimination between acute and chronic exposure has also been complicated as most children have been exposed to secondhand smoke continuously during their childhood.

In Sweden, the prevalence of smoking is lower than in many other European countries, but the total tobacco consumption is comparable to other countries. The explanation for this incongruity is a widespread use of smokeless tobacco (Swedish snus). Snus lacks the numerous combustion toxins abundant in cigarette smoke but delivers high doses of nicotine.

Pregnant women using snus expose their fetuses to nicotine without any of the combustion toxins. After birth, the exposure may continue through breastfeeding but once it has ceased there would be no further exposure during childhood. We used this unique exposure situation to evaluate whether there is a lasting association between prenatal and perinatal nicotine exposure and long‐lasting impairment of arterial wall properties. Our hypothesis was that exclusive prenatal and perinatal snus exposure was associated with increased arterial stiffness and a thicker intima media in preschool children.

Methods

Data Availability and Ethical Approval

The anonymized data that support the findings of this study are available from the corresponding author upon reasonable request. This study was approved by the regional ethical review board in Stockholm, and written consent was obtained from all patients.

Study Design

Participants in this cohort study originated from a larger national study of perinatal smokeless tobacco exposure. ¹⁵ The women in the original snus study cohort were enrolled early in pregnancy (gestational week 6–12) when signing in at antenatal clinics in different parts of Sweden. The women were followed carefully during the entire pregnancy and after delivery with questionnaires on snus brand, number of standardized pouches per day, exposure to secondhand smoke, and use of drugs, alcohol, and medication.

From the original cohort, families with healthy children at the age of 5 to 6 years and who resided in Umeå or Stockholm were invited. Only children of mothers with a high daily snus dose (>48 mg nicotine per day) during the entire pregnancy were included as exposed participants. The amount of nicotine was estimated from the reported use of standardized pouches. Children from the original cohort who were reported as completely unexposed to any form of tobacco or nicotine before or after birth were included as controls. Exclusion criteria were preterm birth, low birth weight, multiple birth, malformations, chronic health issues, exposure to cigarette smoke or secondhand smoke during childhood, or maternal use of alcohol, drugs, or any other source of tobacco or nicotine during pregnancy. If the child had an acute infection with or without fever, the assessment was postponed for at least 4 weeks.

A total of 56 children were eligible for inclusion and 12 were lost because of moving from the area or loss of address. Two families declined participation, and 2 more were lost due to last‐minute cancellation. The final cohort consisted of 21 snus‐exposed children and 19 tobacco‐free controls.

Clinical and Vascular Assessment

Families were scheduled to attend the pediatric cardiology department either in Stockholm or Umeå during the daytime. Information on participant's medical history, medication, and breastfeeding data and parent's medical history was obtained by a questionnaire. The parents were asked to report their medical diagnoses and were specifically asked whether they had a history of hypertension with medical treatment. Perinatal data were collected with records from the Swedish Medical Birth Register.

Standardized operational procedures were used for all of the assessments. All investigators including the trained nurses were blinded to the groups. Height and weight were measured with the child either undressed or in light clothing. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared and the BMI z score was calculated using the World Health Organization reference data from 2007 for ages 5 to 19 years. ¹⁶

Estimation of arterial stiffness included blood pressure (BP) values. After 15 minutes of calm adaptation in the examination room, an oscillometric device (Dinamap Compact TS, Critikon) was used to measure systolic BP and diastolic BP in the right upper arm of the seated child. Three consecutive measurements of BP with at least 1 minute in between were obtained, and the mean of the 3 measurements was calculated for all of the children.

Measurement of Intima‐Media Thickness in the Carotid Artery

The child was examined in the supine position with the neck slightly extended using a high‐frequency linear array ultrasound probe specialized for vessel structure (Philips vasculature probe, 15 MHz) in B‐mode with a simultaneous ECG recording. The right carotid artery was examined first in the short‐axis view and thereafter in the long‐axis view where the carotid bulb was identified. The intima media was visualized proximal to the bulb on the distant wall of the vessel and recorded in a loop of 3 consecutive R‐R intervals.

The ultrasound examination was stored on a server for later analysis with a semiautomatic measurement with the software Arterial Health Pack (Siemens). A segment of 10 mm was analyzed, and the maximum, mean, and SD of the intima media were measured. The mean of 3 measurements was used for analysis. The measurements were conducted in accordance with the recommendations and guidelines for measuring carotid intima‐media thickness (cIMT) in children by the Association of European Pediatric Cardiology. ¹⁷ Relative cIMT was calculated according to the formula: [2×IMT]/carotid end‐diastolic diameter (EDD) multiplied by 100 and presented as percentage.

Measurement of Carotid Artery Size and Calculation of Arterial Wall Strain

The size of the vessel was measured in systole and diastole, identified from the ECG, and measured from leading edge to leading edge of the adventitia in B‐mode, long‐axis view. The mean of 3 measurements of end‐systolic diameter (ESD) and EDD was used for analysis.

The carotid artery strain was calculated as the relative pulsatile diameter change (delta D) according to the formula:

[ESD–EDD]/EDD multiplied by 100 and presented as percentage.

Calculation of Carotid Stiffness

The stiffness index (beta) ¹⁸ for the carotid artery was calculated using the formula:

Ln [systolic BP/diastolic BP]/[(ESD–EDD)/EDD].

Statistical Analyses

The results are reported as mean (SD), number (percentage), or median (interquartile range [IQR]). All variables were tested for normality with Shapiro‐Wilks test. For group comparison we used Students t test and χ² test. The non‐Gaussian–distributed variables were compared using the Mann‐Whitney U test. Due to the small sample size, only univariate descriptive statistics were used.

A P value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS for Windows version 25 (IBM).

Results

Study Cohort

Compared with tobacco‐free controls, snus‐using mothers did not differ in age, weight, height, or BMI. The mean duration of breastfeeding did not differ between groups, but breastfeeding <6 months was more common in the snus group (67%) than in controls (32%, P=0.027). Cohort characteristics are displayed in Table 1.

Table 1.

Maternal and Neonatal Characteristics of the Study Cohort

Total (N=40)	Controls (n=19)	Snus in pregnancy (n=21)	P value
Age, y	29.7 (5.8)	32.5 (4.1)	0.09
Parental hypertension, n (%)	0	4 (19%)	0.07
Prepregnancy weight, kg	66.1 (10.1)	73.0 (16.7)	0.14
Height, cm	166.6 (4.0)	165.9 (4.6)	0.67
Prepregnancy BMI, kg/m2	25.0 (3.62)	26.6 (5.78)	0.33
Breastfeeding duration, mo	8.3 (4.7)	5.7 (4.1)	0.08
Breastfeeding duration <6 mo, n (%)	6 (32%)	14 (67%)	0.03*
Infant birth weight, g	3695 (314)	3497 (590)	0.20

Data are mean values (SD) if not stated otherwise.

BMI indicates body mass index.

^*Statistically significant, P value<0.05.

The children of snus‐using mothers did not differ significantly from tobacco‐free control children regarding sex, birth weight, age, or weight and height and BMI at follow‐up. There were 3 individuals in each group with a BMI >90th percentile (Table 2).

Table 2.

Anthropometry of 5‐ to 6‐Year‐Old Children Exposed to Snus in Fetal Life and Unexposed Controls

Total (N=40)	Controls (n=19)	Snus in utero (n=21)	P value
Girls, n (%)	10 (52)	9 (43)	0.54
Age at examination, mo	70 (8.2)	73.6 (7.6)	0.16
Weight, kg median (IQR)	21.0 (19–23)	23.3 (19–28)	0.11
Height, cm	115.4 (7)	119.2 (7.5)	0.11
BMI, kg/m2	16 (1.1)	16.8 (1.8)	0.09
BMI, percentile	62.4 (24.6)	69.6 (22)	0.33

Data are mean (SD) if not stated otherwise.

BMI indicates body mass index.

There was no difference in city of residency between the 21 snus‐exposed children (18 residing in Umeå, and 3 in Stockholm) and the 19 tobacco‐free controls (12 residing in Umeå, and 7 in Stockholm, P=0.15).

Blood Pressure

Children prenatally exposed to snus had a higher mean systolic BP than control children (98.7 mm Hg [SD, 7.1 mm Hg] versus 93.3 mm Hg [SD, 5.8 mm Hg], P=0.013). There was no statistically significant difference in diastolic BP. The BP findings have been reported in a previous study. ¹⁹

Carotid IMT

The overall cIMT was 0.416 mm (SD, 0.062 mm) and did not differ significantly between the snus‐exposed children and the tobacco‐free controls. In addition, there was no difference between the groups when relating the absolute cIMT measurement with the dimension of the carotid artery (Table 3).

Table 3.

BP, Heart Rate, Dimensions of Carotid Artery, Strain, Arterial Stiffness Index, and cIMT in Controls and in Children Exposed to Prenatal Snus

Total (N=40)	Controls (n=19)	Snus in utero (n=21)	Mean difference (95% CI)	P value
Blood pressure,
Systolic BP, mm Hg	93.3 (5.8)	98.7 (7.1)	−5.4 (−9.6 to 1.2)	0.013†
Diastolic BP, mm Hg	54.6 (4.5)	56.1 (5.2)	−1.4 (−4.5 to 1.7)	0.36
Heart rate, mean (SD)
ECG, beats per min	91 (11)	90 (12)	0.9 (−6.8 to 8.5)	0.81
Carotid dimensions
Carotid systole, mm, mean (SD)	6.2 (0.5)	6.4 (0.5)	−0.2 (−0.5 to 0.1)	0.16
Carotid diastole, mm, mean (SD)	5.2 (0.4)	5.6 (0.6)	−0.4 (−0.8 to 0.1)	0.019†
Carotid delta D, mm, mean (SD)	1.07 (0.31)	0.87 (0.24)	0.2 (0.03 to 0.38)	0.023†
Strain
Strain, %	21 (6.6)	16 (5.7)	5 (1 to 8.9)	0.015†
Intima media
cIMT, mm	0.408 (0.048)	0.422 (0.073)	−0.014 (−0.054 to 0.026)	0.49
cIMT SD*, mm	0.048 (0.015)	0.048 (0.014)	0 (−0.01 to 0.01)	0.97
cIMT relative, %	15.8 (2)	15.2 (2.4)	0.52 (−0.9 to 1.9)	0.46

BP indicates blood pressure; cIMT, carotid intima‐media thickness; delta D, diameter pulse amplitude; and HR, heart rate.

^*SD for each measurement.

^† Statistically significant, P value<0.05.

Children with a family history of hypertension (n=4) had thicker intima media (mean difference, 0.064 mm) than children without a family history of hypertension (P=0.049).

Carotid Diameters and Strain

Carotid EDD was significantly larger in snus‐exposed children (5.6 [0.6] versus 5.2 [0.4] mm) than in tobacco‐free controls (P=0.019). Carotid ESD did not differ significantly between snus‐exposed and control children. The mean difference between ESD and EDD (ie, delta D) was smaller in snus‐exposed children (0.87 mm [SD, 0.24 mm] versus 1.07 mm [SD, 0.31 mm]) than in controls (P=0.023). The mean carotid strain (ie, the relative pulsatile diameter change) was lower in snus‐exposed children (16% [SD, 5.7%] versus 21% [SD, 6.6%]) than in controls (P=0.015) (Figure 1).

Figure 1. Carotid artery strain in children with prenatal snus exposure (n=21) and tobacco‐free controls (n=19).

Mean and 95% CI.

Carotid Stiffness Index

The stiffness index was higher in snus‐exposed children, with a median of 4.1 (IQR, 2.4–5.1) versus 2.9 (IQR, 2.1–3.5) in controls (P=0.014) (Figure 2). We found no significant associations between carotid stiffness index and birth weight, breastfeeding duration, current weight or height, sex, or parental history of hypertension.

Figure 2. Stiffness index (beta) in prenatal snus‐exposed children (n=21) and tobacco‐free controls (n=19).

Median and interquartile range.

Discussion

In the current study of perinatal smokeless tobacco exposure, we report that preschool children with snus‐using mothers during pregnancy had a 41% higher arterial stiffness index compared with tobacco‐free controls. The significantly increased carotid stiffness index in children exposed to snus in utero reflected smaller pulsatile changes of the carotid arterial wall despite higher pulse pressures and larger end‐diastolic carotid artery diameters than in unexposed control children. In contrast, cIMT did not differ between the 2 groups.

Our measurements are congruent with earlier reports from 5‐ to 10‐year‐old controls, ²⁰ , ²¹ who demonstrated a stiffness index (beta) of 2.9 to 3.0. Arterial stiffness reflects a smaller arterial capacity to respond to pressure changes, either as a defect fetal deposition of elastin in the arterial wall, ²² or due to accelerated loss of elasticity. Arterial stiffness has been reported to be associated with several parameters such as hypertension, obesity, duration of breastfeeding, and smoking. ²³ , ²⁴ , ²⁵ , ²⁶ Stiffer arterial walls have previously been demonstrated in adult smokers and are considered both an acute and chronic effect of smoking. ²⁴ Decreased carotid artery distensibility has been shown in 5‐year‐old children with smoking parents. ⁶ A Swedish study on long‐term snus use in adult men reported increased arterial stiffness and endothelial dysfunction compared with nontobacco‐using controls. ²⁷ The use of electronic cigarettes and nicotine tablets have also been associated with an acute and increased arterial stiffness, and adverse effects of nicotine per se have been considered as part of the underlying mechanism. ²⁸ , ²⁹ , ³⁰

We found no difference in cIMT between the snus‐exposed children and controls. The mean cIMT found (0.42 mm) corresponds well with other studies using the same procedure protocol and semiautomatic measuring. In 1 study of Swedish preterm children of the same age, the cIMT mean was 0.38 mm, ²⁰ in another study of healthy children aged 1 to 10 years, the cIMT mean was 0.42 mm, ³¹ and in a large (N=697) study of children (aged 7–17 years), the cIMT range was 0.44–0.48. ³²

Reports about maternal smoking and intima‐media wall changes in offspring are scarce and partly incongruent. Intimal wall changes of the umbilical arteries ³³ and in the aorta have been described in newborn babies following maternal smoking during pregnancy. ³⁴ Adolescents exposed to secondhand smoke had a thicker intima media in the aorta and carotid artery, ³⁵ and a study of 5‐year‐old children exposed to smoke demonstrated thicker intima media of the carotid artery. ⁶ On the other hand, a study of 8‐year‐old children with prenatal and postnatal cigarette smoke exposure did not show any changes of cIMT. ³⁶ As these studies all focus on smoking, they could not separate potential nicotinic effects from other potentially harmful substances following tobacco combustion.

The pathophysiological process of atherosclerosis is thought to be promoted by cigarette smoke through contribution of inflammation, insulin resistance, dyslipidemia, thrombosis, and endothelial dysregulation. ⁹ , ³⁷ Nicotine can at least partly be responsible for these changes, and nicotine affects autonomic regulation with raised BP as a consequence. In support of a causal relationship, there are numerous animal studies demonstrating arterial wall changes after exposure to nicotine. ³⁸ , ³⁹ , ⁴⁰ , ⁴¹

The common carotid artery is a large elastic vessel with a wall made up of multiple elastic layers consisting of 2 scaffolding proteins: elastin and collagen. The regulated balance between these 2 can be altered by inflammatory changes and increased luminal pressure where collagen is favored and elastin is degraded, finally leading to a stiffer wall. One study in monkeys reported that prenatal nicotine exposure disturbs the normal production of elastin during fetal life and leads to thicker and stiffer pulmonary arterial walls. ⁴²

The involvement of nicotine in the process of arterial wall changes is evident, but the process is complex, and the interaction between arterial stiffness, arterial wall thickening, BP, and autonomic control is not fully understood. ⁴³ , ⁴⁴

The autonomic nervous system and the endothelial function regulate vascular tone together, and the autonomic nervous system is involved in BP control via the renin‐angiotensin‐aldosterone system and baroreceptor loop. ⁴⁵ The autonomic nervous system thus seems to be of major importance, and it is highly susceptible to nicotine actions. We have previously demonstrated altered autonomic balance in children with prenatal nicotine exposure both in infancy and in preschool age. ¹⁹ , ⁴⁶ Importantly, although there is a trajectory from these early subclinical arterial wall changes to adult cardiovascular disease, the alterations may be modifiable and accessible for preventive actions.

To our knowledge, there are no previous studies of maternal smokeless tobacco and vascular wall changes in offspring. This study of women using Swedish snus, a smokeless tobacco with a long tradition in Sweden, is therefore the first to allow separation of nicotinic effects. Snus is oral moist tobacco packaged in small pouches that are put under the upper lip. It contains high doses of nicotine but low concentrations of nitrosamines, pesticides, and metals compared with other tobacco forms. Furthermore, and importantly, it contains none of the combustion toxins found in cigarette smoke.

This study has some limitations, most importantly the small number of participants with limited power to discover small‐moderate group differences or dose‐response relationships. Although we adopted strict exclusion criteria for secondhand smoke and collected information on the use of other drugs and medications during pregnancy, confounding by other sources of pollutants, as well as socioeconomic confounding cannot be excluded. The nicotine exposure was self‐reported, and the study did not include any testing of nicotine metabolites. However, the validity of self‐reported intakes of snus have been verified in studies of metabolites in urine. ¹⁵

The study also has strengths, such as the prospective design with repeated surveys, including weekly dose of nicotine during pregnancy and a long follow‐up time of 6 years. The cohorts were carefully chosen with a small age span and strict exclusion criteria including preterm birth, small for gestational age, cardiovascular malformations, health problems, and most importantly secondhand smoke. All examinations were performed by one experienced sonographer with the same set of ultrasound machines and all analyses were blinded for the group and performed according to recommended procedure protocols for vascular assessments.

In conclusion, preschool children exposed to snus in utero exhibit stiffer carotid arteries than unexposed children. Although these changes are asymptomatic during childhood, they may have implications for future follow‐up and risk of cardiovascular events in adult life. The findings also add to the recommendation that pregnant women should abstain from all types of tobacco and nicotine products.

Sources of Funding

This work was supported by research grants from Swedish Medical Research Council (521–2009‐4884), Swedish Council for Working Life and Social Research (209–1619), Swedish Heart Lung Foundation, the Samaritan Foundation, Odd Fellows Grand Lodge Sweden, and the Childhood Foundation of the Swedish Order of Freemasons.

Disclosures

None.