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Objective. Low birth weight is associated with increased prevalence of hypertension and cardiovascular disease in adults. The aim of this study was to evaluate genetic and intrauterine environmental contributions to blood pressure (BP) and vascular functions in twins with discordant growth in utero.
Subjects. We studied 31 twin pairs (21 monozygous and nine dizygous), mean age 8 years) with large within-pair differences in birth weight. Among the monozygous pairs, nine had suffered from twin-to-twin-transfusion syndrome (TTTS).
Methods. Apart from BP, we determined diameters and elasticity of the carotid artery and abdominal aorta with ultrasonography, and endothelial function in skin vessels with a laser Doppler technique, before and after transdermal delivery of acetylcholine and nitroglycerin.
Results. Eight of 62 twin subjects had a systolic BP above the 90th percentile in a North-American reference population. Among these, seven/eight were monozygous with a history of poor fetal growth and/or TTTS. In monozygous twin pairs without TTTS, systolic BP and pulse pressure were higher and vascular endothelial function was impaired in the lower birth weight twin. In the TTTS group, the lighter twin had a narrower carotid artery but there was no within-pair difference in arterial elasticity. Pre-eclampsia during the index pregnancy enhanced within-pair differences in BP but abolished within-pair differences in endothelial function.
Conclusions. Severe fetal growth retardation contributes to higher BP, arterial narrowing and endothelial dysfunction in childhood. Pre-eclampsia may act both as an effect modifier and confounder of these associations.
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There is substantial evidence of an inverse relation between birth weight and later blood pressure (BP) [1–4] although recently the strength of the association has been questioned . It is currently a subject of debate whether this association reflects common genetic pathways or results from lasting programming effects because of adverse environmental influences in utero. To assess the various hypotheses, twin studies have been carried out, which focus on birth weight discordances within pairs and subsequent differences in BP [6–14]. However, the findings in these studies have been contradictory. Some ascribe the association with genetic factors [8, 12], whilst others maintain that fetal programming accounts for some of the prevalence of later hypertension [6, 7, 9]. Both or other explanations have also been proposed [10, 11, 13, 14]. As previously pointed out, the differences in results may partly reflect methodological problems, including bias in self-reported birth weights, crossing over of twin identities in various registers, unknown chorionicity and imprecise determinations of BP [13, 15].
Vascular programming has recently been shown in 1-year-old monozygous (MZ) twin infants who had suffered from haemodynamic stress in utero because of twin-to-twin transfusion syndrome (TTTS) . Increased brachial artery stiffness was found in the smaller, but not in the heavier twin. Although no differences in BP were seen shortly after birth, loss of natural arterial elasticity has been proposed to contribute to an increase in BP at an older age .
If fetal programming of BP occurs, we hypothesized that differences could be detected in MZ twins discordant in fetal growth and development. In this study, we measured BP in prepubertal twins with large differences in weight at birth. Amongst MZ pairs, we included those with and without a history of TTTS. Because loss of arterial elasticity and endothelial dysfunction have been associated with fetal growth retardation [16, 18–21] and may predispose to adult hypertension and atheroma formation, we also included measurements of these vascular functions.
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From log-books of all births (n > 60 000) at Karolinska and Danderyd in Stockholm during 1991–1997, we found 35 liveborn same-sexed twin pairs in whom one was appropriate for gestational age (AGA; birth weight = mean ± 2 SD) and the other small for gestational age (SGA; birth weight < mean − 2 SD according to Swedish reference data ). Exclusion criteria were very preterm delivery (gestational age: <32 weeks), congenital syndromes and chronic illness in need of medication. Four families were lost to follow up. The remaining 31 families were invited and 22 accepted to participate in the study. The birth weights and gestational ages of those who declined did not differ significantly from those who participated.
Another twin-cohort, consisting of all consecutive and prospectively diagnosed TTTS pairs in which both survived and were born in Stockholm during 1994–1997, was also included (n = 9 pairs). TTTS is a condition that complicates monochorionic twin pregnancies and is due to an unbalanced blood flow through unidirectional vascular anastomoses in the placenta. It is characterized by fetal growth restriction in the smaller, donor twin and hypervolaemia and heart failure in the heavier, recipient twin. In our study, diagnosis was based on serial antenatal ultrasound examinations according to internationally accepted criteria . Most subjects in our study had mild to moderate disease, as evidenced by dual survival. During pregnancy, six mothers of the TTTS children had been treated with amnioreduction alone or in combination with indomethacin, to reduce the risk of rupture of the membranes and preterm labour. None of the women underwent treatment with laser coagulation of the placental anastomoses. All families gave their informed consent before the investigations and the study was approved by the local Ethics Committee.
Altogether 62 twins (34 girls) were studied at an age of 7.8 (range: 4.6–11.8) years. They were divided into three groups: (i) monozygous twins without TTTS (MZ without TTTS), (ii) monozygous with TTTS (MZ with TTTS) and (iii) same-sexed dizygous (DZ) twins. Chorionicity was established on the basis of histology. In cases of dichorionic placentas (n = 12) or unknown chorion type (n =2), the determination of monozygosity relied on parental reporting of mistaken identity. All parents were healthy Caucasians. In vitro fertilization had been performed in three mothers, resulting in one MZ and two DZ twin pairs. Maternal and pregnancy data are summarized in Table 1.
Table 1. Subject characteristics
| ||MZ-no TTTS (n = 13 pairs)||MZ-TTTS (n = 9 pairs)||DZ (n = 9 pairs)|
| Age (years)||31 (1.4)||30 (1.2)||32 (1.2)|
| Antenatal steroid therapy||5/13*||9/9||1/9*|
| Gestation (weeks)||35 (0.6)||33 (1.2)||37 (0.6)*|
| BW-SDS for AGA-twin||−0.2 (0.3)||−0.3 (0.3)||−0.4 (0.2)|
| BW-SDS for SGA-twin||−2.9 (0.3)*||−1.8 (0.1)||−2.6 (0.1)*|
| BL-SDS for AGA-twin||−0.3 (0.5)||−0.4 (0.4)||−0.9 (0.3)|
| BL-SDS for SGA-twin||−3.1 (0.6)||−1.7 (0.2)||−2.2 (0.3)|
|Current age (weight and height)|
| Age (years)||7.9 (0.7)||6.4 (0.3)||9.1 (0.5)*|
| Weight, AGA-twin (kg)||29.1 (2.5)||22.3 (1.3)||30.1 (2.0)*|
| Weight, SGA-twin (kg)||27.1 (2.9)||21.1 (1.8)||29.1 (2.2)|
| Height, AGA-twin (cm)||130 (4.1)||119 (2.4)||136 (3.5)*|
| Height, SGA-twin (cm)||128 (4.6)||118 (2.5)||135 (4.0)*|
Blood pressure determinations
After resting for 20 min, heart rate and BP were recorded with the subject lying down with the arm at heart level. An automated oscillometric sphygmomanometer (Boso MedicusTM, Jungingen, Germany) was used with an appropriately sized cuff (9 × 28 cm) around the right upper arm. The mean of six consecutive determinations, taken at least at 3-min intervals, was regarded as the subject's BP. The coefficient of variation (CV) was 9.5% for systolic (SBP) and 9.9% for diastolic blood pressure (DBP), i.e. the same as in other paediatric studies [10, 24].
Arterial dimension and stiffness. The mechanical properties of the abdominal aorta (AA) and the left common carotid artery (CCA) were studied with ultrasonography. A computer-generated pair of electronic echo-trackers (DiamoveTM, Teltec AB, Lund, Sweden) was used to measure the end-diastolic diameter (Dd; mm), pulse amplitude of the diameter (ΔD; mm) and relative strain (ratio: ΔD/Dd, %). The minimum diameter possible to measure with the Diamove equipment is 7.8 μm . These diameter data and those of simultaneously measured BPs were computed to yield the stiffness index (β) using the equation:
The use of noninvasively measured BPs in the brachial artery might lead to an underestimation of the true aortic and carotid BPs. However, the consistent use of arm BP in the calculations should not influence the comparisons of the results between and within the twin pairs in our study. The mean value of three recordings, each consisting of about 6–10 consecutive heart cycles, was taken as the subject's reading.
Endothelial function. A laser Doppler (LD) instrument and a micropharmacology system (Perimed AB, Kista, Sweden) were used to measure changes in perfusion during vascular drug provocations in the skin of the dorsum of the hand. The LD signal is proportional to the number and velocity of moving blood cells in the illuminated superficial skin microvessels and expressed in perfusion units (PU) of output voltage (1 PU = 10 mV). The temperature of the LD probe facing the skin was standardized to 32 °C. After adjustment to the room temperature (22 °C) for 20 min, the vascular studies were performed with the child lying supine with both arms beside the body. To study endothelium-dependent vasodilation, perfusion was recorded after transferal of acetylcholine (ACh) across the skin by iontophoresis (anodal current of 0.1 mA for 20 s repeated six times at 60-s intervals). To study endothelium-independent vasodilation, the effect of an exogenous nitric oxide (NO) donor (1% nitroglycerine) was tested on the contralateral hand. Details concerning vascular methods have been described elsewhere .
Data are presented as mean (SEM) values or proportions. Chi-square and anova were used to test for group differences. Mean within-pair differences [95% confidence interval (CI)] were calculated (=value in the lighter twin at birth − value in the heavier twin at birth) and two-tailed paired t-test was used for cotwin comparisons. A P-value of <0.05 was considered significant and a P-value between 0.05 and 0.10 as borderline significant. Within-pair differences in BP, arterial diameter and pulsatile diameter changes and maximum skin perfusion response to ACh (endothelial function) were considered as primary outcome variables. Regression analyses were used to evaluate contributions to within-pair differences from the following risk factors or confounders: group (MZ twins without TTTS, MZ with TTTS and DZ), pre-eclampsia, use of antenatal steroids, gender, current age, weight and height. Initially, stepwise regression was performed. Independent variables with a P-value of <0.20 in this model were entered into a multiple regression model.
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In this 8-year follow-up study, we found that amongst MZ twins without TTTS with discordant fetal growth, the in utero growth-retarded (SGA) twin had significantly higher SBP and impaired endothelial function in childhood, when compared to the cotwin with appropriate birth weight. In addition, amongst MZ twins suffering from TTTS in fetal life, the smaller donor twin showed narrower carotid artery at school-age. Given the fact that familial effects (genetic and postnatal environmental factors) were controlled for, different fetal exposures are likely to be involved in the explanation of these results. Longitudinal studies will be needed to fully understand the significance of these findings in adult life. They may, independently or in combination, contribute to an increased risk for future coronary heart disease and stroke in the affected twin.
Although our focus was on intra-pair differences, we note that in the subgroup of twins with an adjusted SBP above the 90th percentile, seven of eight had suffered from adverse intrauterine influences in terms of severe growth restriction or TTTS. These findings indicate that fetal programming may contribute to later BP in humans, in accordance with some previous studies [6, 7, 9].
The twins of pre-eclamptic mothers did not show higher BPs than those without such a history. However, in pregnancies complicated by pre-eclampsia, the growth-restricted twin had a higher SBP than its cotwin at follow up. Although several twin studies have suggested that the fetoplacental unit is involved in fetal programming [6, 7, 9], there are no other twin-data on the role of pre-eclampsia. Pre-eclampsia is a hypertensive disorder of the pregnant woman, triggered by vascular lesions and endothelial dysfunction of the placenta. It is two to three times commoner in women with twin than in those with singleton pregnancies  and even more common in twin gestations complicated by severe discordancy in fetal growth . Its interaction with twin discordances in birth weight and later differences in BP is intriguing as it decreased rather than increased covariance. Although a definite explanation cannot be given, the effect modification by pre-eclampsia can be interpreted in one of two ways: either it represents an additional environmental disadvantage to unequal placental sharing or it represents a genetic susceptibility triggered by fetal growth restriction.
Fetal exposure to antenatal steroids has been associated with a rise in BP in adolescence . We found no difference in BP between twins who had received such treatment and those who did not. In MZ twins with TTTS, all had been exposed to antenatal steroids and amongst those with high SBP at follow up, there was an equal distribution between donors and recipients. As TTTS and/or antenatal steroids affected both twins in utero, both could have been programmed. In TTTS, humoral factors compensating for anaemia, hypoxia and hypovolaemia in the donor twin fetus reasonably also reaches the recipient twin fetus. Such an interpretation would help to explain why no within-pair differences in BP were found in the subgroup of MZ twins suffering from TTTS.
An interesting hypothesis of the inverse association between birth weight and BP concerns early reduction in arterial compliance. A combination of abnormal haemodynamics and malnutrition accompanying growth restriction in utero has been proposed as a cause of impaired elastin incorporation in the walls of the aorta and large arteries . Neither the present nor other studies in children have shown a clear link between fetal growth restriction, premature stiffening of the aorta and a subsequent rise in BP . However, aortic narrowing has been described amongst schoolchildren born SGA . We have previously found a stiffer carotid artery in schoolchildren with low-birth weight. Present data from TTTS pairs show that poor fetal growth is also associated with narrowing of the carotid artery . From our data and those of others , it seems that disturbed carotid haemodynamics in the fetus could be an important determinant of long-term carotid development and growth.
Systemic impairment of endothelial function has been found in newborn infants, children and young adults with low-birth weight [18, 20, 21]. Endothelial dysfunction predicts later hypertension, accelerated atherosclerosis and coronary heart disease. The present study provides evidence of an intrauterine contribution to later endothelial dysfunction in humans. The lack of a significant difference in endothelial function amongst MZ twins with TTTS may be due to their shorter gestation (four pairs delivered before 32 weeks of gestation) because preterm delivery attenuates developmental programming of the endothelium .
Formula feeding of infants has been associated with a higher BP in children and adolescents [31, 32]. We cannot exclude confounding from different within-pair infant feeding practices amongst the subjects of this study. The routine in our units, however, is to give the smaller twin sibling priority if the amount of breast milk is insufficient for both.
We did not perform an exercise test on these children, which could have unmasked further within-pair differences in BP and heart rate responses. On average, we found 2–3 mm higher resting SBP in the MZ twin sibling suffering from intrauterine growth failure. This is a larger effect than that reported in epidemiological studies [13, 14]. The validity of our findings may be limited to the subgroup of twins with the largest differences in birth weight. In addition, the generalizability of twin studies to the general population is sometimes a concern, especially as twin growth in utero, even under normal conditions, differs from that of singletons. Because prospectively collected reference data on intrauterine twin growth are lacking, our definitions of being small (SGA) or appropriately sized at birth (AGA) were based on reference data for Swedish singletons.
This study has limitations in statistical power and therefore, the results must not be over-interpreted. The total number of twins possible to recruit, permitted detection of a within-pair mean difference of about 0.5 SD or more. Nevertheless, the choice of a well-characterized twin-cohort with large birth weight discordances provided an opportunity to obtain more detailed insights into perinatal contributions to later BP and vascular structure and function.
The birth weight discordance was lower in MZ twins with TTTS, but was greater in MZ without TTTS and DZ twin pairs. This is most likely reflected that all MZ with TTTS, according to the diagnostic criteria, were diagnosed in utero, and that action (delivery with C-section before term) was taken before the condition deteriorated further.
In conclusion, our findings suggest the occurrence of fetal programming of BP, endothelial function and carotid growth. Although twins overall have similar prevalence of hypertension and cardiovascular mortality as singletons, severe complications during pregnancy such as IUGR (intrauterine growth retardation) and TTTS, are much commoner than in singletons. As shown here, this may also affect health after the perinatal period.