Early origins of adult disease: Low birth weight and vascular remodeling

1

Developmental programming of cardiovascular risk in intrauterine growth

restricted twin fetuses and aorta intima thickness

Silvia Visentin1, Enrico Grisan

2, Vincenzo Zanardo

1, Daniele Trevisanuto

1, Elisa

Veronese2, Francesco Cavallin

1; and Erich Cosmi

1

1Department of Woman and Child Health, Maternal Fetal Medicine Unit, University

of Padua, Padua; Italy

2Department of Information Engineering, University of Padua, Padua; Italy

Reprint requests and corresponding author:

Erich Cosmi, MD, PhD

Director of Maternal and Fetal Medicine Unit

Assistant Professor of Obstetrics and Gynecology

University of Padua School of Medicine, Padua; Italy

Via Giustiniani No 3, 35128, Padua, Italy

Tel. +39-339-8146745; Fax +39-049-8211842

E-mail: [email protected]

2

ABSTRACT

We aimed to test the hypothesis that aorta intima thickness (aIT) in intrauterine

growth restricted (IUGR) twin fetuses shows higher values compared to normal

twins, thus defining an increased cardiovascular risk in individuals that reflect genetic

factors sharing the same womb. Gender as well as chorionicity was considered.

A prospective study performed on twins from January 2009 to July 2011. Twins were

classified in three groups: IUGR fetuses with an estimated fetal weight (EFW) < 10th

percentile and umbilical artery pulsatility index (PI) > 2 SD (Group A), fetuses with

EFW < 10th

percentile with normal Doppler (Group B) and fetuses with an EFW

appropriate for gestational age (Group C). aIT was measured at a median gestational

age of 32 weeks. Values were compared among groups and between each twin with

its co-twin considering gender and chorionicity.

Twenty-five fetuses were classified as Group A, 36 B, and 95 as C. Median aIT was

0.9 mm in Group A, 0.7 mm in B, and 0.6 mm in C (P< 0.0001). There was a

statistically difference between aIT of the twin and its co-twin in Group A and B

(p<0.0001). Gender as well chorionicity did not correlate with aIT.

This study highlights that IUGR fetuses with Doppler abnormalities showed higher

values of aIT, being intermediate in IUGR twins with normal Doppler, supporting a

genetic predisposition to cardiovascular risk independently to gender and chorionicity

Key Worlds: Aorta intima thickness, cardiovascular risk, chorioinicity, Doppler

velocimetry, gender, IUGR, twin,

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INTRODUCTION

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Recently, our group showed that fetal aortic intima-media thickness (aIMT), a

known marker of endothelial dysfunction and atherosclerosis, had higher values in

intrauterine growth restricted (IUGR) fetuses and same infants after a median follow-

up of 18 months compared to appropriate for gestational age (AGA) (Cosmi et al.

2009; Lo Vasco et al. 2011, Zanardo et al. 2011)

The effect of IUGR on vascular function, assessed using markers of endothelial

damage such as aIMT, has never been studied in twin pregnancies.

Monozygotic and dizygotic twins provides a unique opportunity to mimic a

scientific experiment to study IUGR and, reflecting nutritional stresses within a

similar genetic fetal background, to distinguish between genetic and environmental

causes of phenotypic variations in human population (Muhlhusler Beverly et al.

2011). In fact, monozygotic twins share identical genes, whereas dizygotic twins

share on average 50% of their segregating genes.

Moreover, the role of the chorionicity as well as gender in the evaluation of the

cardiovascular risk in twin pregnancies is still not known (Philips et al 2001).

The aim of the present study was to assess aorta intima thickness (aIT) in

IUGR twin fetuses to test the hypothesis that IUGR twins might have a predisposition

to vascular dysfunction independently to their co-twin and environmental factors.

Gender and chorionicity were considered among the groups.

MATERIALS AND METHODS

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Study Definition

A prospective study in twin pregnancies was performed in the Maternal and

Fetal Medicine Unit of the University Hospital of Padua from January 2009 to July

2011. The IRB and Ethical committee of the University hospital approved the study

and written informed consent was obtained from each patient before enrollment.

All women with twin pregnancies attending the clinic were enrolled in the

study either at the time of the first trimester ultrasonography exam performed to

assess chorionicity or during the third trimester if the patient was referred from other

centers. Chorionicity was diagnosed at 10-14 weeks of gestation on the basis of the

presence or absence of the “T” or lambda sign in twins (Carroll et al. 2002)

and was

confirmed histologically after delivery. Gestational age was calculated on the basis

of the first day of the last menstrual period and confirmed by ultrasound measurement

of the fetal crown-rump length (CRL) (Drumm et al. 1977), during I trimester

ultrasonography. Dichorionic diamniotic and monochorionic diamniotic twin

pregnancies were both eligible for the study. Exclusion criteria were: unknown last

menstrual period and chorionicity, triplet pregnancy, twin-to-twin transfusion

syndrome or related conditions, monochorionic monoamniotic twin pregnancies, first

trimester discrepancy of CRL between the pair of twins > 5 days, fetal and placental

abnormalities, maternal history of diabetes, gestational hypertension, preeclampsia,

thyroid/adrenal problems, or clinical chorioamnionitis. Consumption of alcohol

and/or nicotine and use of drugs such as ritodrine or corticosteroids (except for fetal

lung maturation) were also considered exclusion criteria.

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Twin fetuses were divided into three groups: the first was characterized by

IUGR fetuses, defined as fetuses with an EFW < 10th

percentile and with umbilical

artery (UA) Doppler velocimetry Pulsatility index (PI) > 2 standard deviations

(Group A); the second characterized by IUGR fetuses with normal Doppler

velocimetry (Group B); and the third by appropriate for gestational age (AGA)

fetuses, defined as having EFW between the 10th and 90

th percentiles and normal

Doppler velocimetry (Group C) (Blondel 2002).

Standard tables for singleton pregnancies were used to define birth weight

percentiles for gestational age (Hadlock et al. 1985)

All participating women were followed monthly if the pregnancy was

dichorionic diamniotic and fortnightly if the pregnancy was monochorionic

diamniotic according NICE guidelines (2009). All pregnancies characterized by

intertwin growth discordance > 25%18

or by selective IUGR (sIUGR) with or

without Doppler alterations were evaluated weekly when UA PI was higher than 2

standard deviation, and twice weekly in case of Doppler alterations progression. Both

arterial and venous compartment (UA, middle cerebral artery (MCA) and ductus

venosus (DV) were evaluated and estimated fetal weight (EFW) was assessed

fortnightly (NICE 2009)

Timing of delivery was decided on Doppler velocimetry alterations and

gestational age according to the clinical practices of the Department and International

guidelines (Miller et al 2008, Mari 2009, Gratacos et al. 2007).

Ultrasound Measurements

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In all fetuses aIT values was measured at a median gestational age of 32 weeks

(range 30-34), during routine III trimester ultrasound in a coronal or sagittal view of

the fetus at the arterial wall of the most distal 15 mm of the abdominal aorta sampled

below the renal and above the iliac arteries. All measurements were taken using a

high-resolution ultrasound machine equipped with a 3.5-5 MHz linear array

transducer (Antares, Siemens Medical Solutions, Mountain View, CA).

Abdominal aIT was measured placing the calipers at the leading edge of the

blood–intima interface and at the end of the inner portion of the intima vessel (Figure

1). We chose this measurement instead of aIMT, as a recent study of our group on

fetal aorta sampled after intrauterine fetal demise showed an increase of the intima of

the aorta in the IUGR stillbirth (Lo Vasco et al. 2011). All images were stored

digitally for off-line analysis. All measurements were performed in the same

ultrasound scan. Three aIMT measurements were taken and the arithmetic median

value was considered. All images were taken during the last phase of the cardiac

cycle to minimize variability (Cosmi et al. 2009).

Two expert operators (E.C., S.V.) performed all the measurements and intra and

inter-observer agreements were calculated, being 0.88 and 0.86, respectively.

Aorta intima thickness values were evaluated among the groups and compared

between the IUGR twin and its AGA co-twin.

Maternal data, birth weight, neonatal umbilical artery pH and base excess

values were available at birth. Amniotic fluid for kidney functionality was available

in several for some twins, and the results are beyond the aim of this study.

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Statistical analysis

Categorical data were expressed as numbers and percentages, and continuous data as

median and interquartile range (IQR). Categorical data were compared among the

three groups using Fisher's exact test, whereas continuous data using the Kruskal-

Wallis test.

Correlation between continuous data was evaluated using Spearman's rank

correlation.

A median regression model was estimated to identify the effect of the three groups on

aIT, gender and chorionicity, adjusting for potential confounders.

A p-value less than 0.05 was considered significant.

Statistical analysis was performed using R 2.12 (R Development Core Team

2010. R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-

project.org/.).

RESULTS

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Seventy-eight women met the inclusion criteria for the study. There were 156 twins;

among them 110 were dichorionic diamniotic and 46 monochorionic diamniotic. No

differences were observed between the groups as regards maternal age, parity,

pregnancy complications and mode of delivery. The rate of primiparous women was

similar among the groups. All women delivered by caesarean section.

The median gestational age at delivery was the same among the groups

(median 35 weeks, p=0.50).

Anthropometric and clinical characteristics of the groups are reported in Table

1.

Twenty-five twin fetuses were IUGR with Doppler abnormalities (Group A),

36 were IUGR with normal Doppler (Group B), and 95 AGA (Group C).

Distribution of IUGR co-twins did not differ between dichorionic diamniotic

and monochorionic diamniotic twin fetuses (p=0.29).

The median birth weight was 1.665 gr (IQR 1.181-1.930) for Group A, 2.130

gr (IQR 1.680-2.290) for Group B, and 2.290 gr (IQR 1.810-2.520) for Group C

showing a statistically significant difference among groups (p<0.0001).

The median aIT was 0.9 mm (IQR 0.8-1.1) in Group A, 0.7 mm (IQR 0.6-0.8)

in Group B, and 0.6 mm (IQR 0.5-0.7) in Group C (p<0.0001, Figure 2). Adjusting

for potential confounders (sex, birth weight, and birth length), aIT was statistically

lower in Group C compared to Group A (p=0.0004) and B (p=0.01), whereas there

was no statistical difference between Group A and B.

Considering each couple of twins, only AGA and IUGR co-twin with Doppler

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abnormalities showed a significative difference in aIT values of 0.30 mm (IQR 0.20-

0.50) (p<0.0001).

Birth weight was inversely correlated with aIT (r=-0.22, p=0.006; Figure 3).

Moreover, there was no difference in aIT considering gender [0.8 mm and 0.9

mm in Group A and B for females and male, respectively (p= 0.30); 0.5 mm and 0.6

mm in Group C for females and males, respectively (p=0.47)].

Aorta intima thickness did not differ between dichorionic diamniotic

and monochorionic diamniotic twins [0.8 mm in Group A and B for dichorionic and

monochorionic diamniotic twins respectively (p= 0.59); 0.5 mm and 0.6 mm in

Group C for dichorionic and monochorionic diamniotic, respectively (p=0.89)].

Umbilical artery pH and base excess were comparable among the groups

(p=0.63, p=0.52).

DISCUSSION

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The present study highlights that aorta intima values were thicker in twins with

IUGR and UA PI > 2 SD irrespective of gender and chorionicity.

In this study twins were chosen as represent a natural model of IUGR and

allow studying fetal nutritional stress in individuals sharing the same womb

(Muhlhusler Beverly et al. 2011). Moreover aIT was compared between each twin

with its co-twin and correlated to gender and chorionicity to evaluate their influence

on vascular damage.

Intima media thickness is considered one of the earliest morphological markers

of plaque formation and atherosclerosis in IUGR fetuses and neonates (Cosmi et al.

2009, Lo Vasco et al. 2011, Zanardo et al 2011, Jarvisalo et al. 2001).

Using high-resolution ultrasound to study aIMT, Skilton et al (2008) reported

that it was significantly higher in IUGR than in AGA newborns and found this value

an accurate, feasible and sensitive marker of atherosclerosis risk. Since no

confounding factors connected to childhood and adulthood was implicated, aIMT

appeared to delineate fetal contribution to later cardiovascular disease.

Koklu et al. (2007) evaluated the potential use of aIMT to study high-risk

neonates and concluded that it can help to identify precocious asymptomatic vascular

dysfunction in IUGR newborns.

Cosmi et al (2009) assessed aIMT in IUGR fetuses and in the same infants

after a mean follow up of 18 months and found that values were inversely related

EFW and still present at the end of follow-up. Consistent with the finding that

atherosclerosis first begins to develop in the aortic intima; it is possible that IUGR

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and Doppler abnormalities are correlated to altered vascular function causing possible

endothelial damage (Lo Vasco et al. 2011, Zanardo et al. 2011).

These results suggest that IUGR is a potential marker of atherosclerosis

development and may be associated with an increased risk of adult onset diseases (Lo

Vasco et al. 2011, Jarvisalo et al. 2001, McGill et al. 2000). Moreover, microscopic

observation of abdominal aortic walls of stillbirth IUGR fetuses confirmed that there

is intima thickening and the presence of inflammatory elements, such as

macrophages, activated endothelial cells and fibroblastoid cells. Hence, at variance

with previous studies, we measured aIT considering the lumen-intima borders from

the leading edge of the blood–intima interface to the end of the inner portion of the

intima vessel, instead of the distance between the lumen-intima and the media-

adventitia edges.

Although Barker (2000, 2006) and other investigators (Bateson et al. 2004,

Eriksson et al. 2007, Osmond et al. 2007, Johansson et al. 2005, McMillen et al.

2005) hypothesis has demonstrated that small size at birth is linked with long-term

adverse health effects, until recently little was known as to whether these associations

extend to twins and 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.

The present work tries to fill this knowledge gap, since, to our knowledge,

there is no study comparing twin fetuses to investigate the “fetal origins hypothesis”

through measure of aIT.

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Twin model in this study offered the opportunity to evaluate the influence of

low fetal weight and Doppler velocimetry in fetuses that share the same genetic

influence and approximately the 55% of maternal environment on a cardiovascular

marker represented by aIT (Withfield et al. 2001).

As far as we are aware, there are no reports in the literature concerning twin

fetuses classified as IUGR with or without Doppler alterations in which

cardiovascular risk was assessed using aIT. IUGR without Doppler alterations is

considered a group whose risk is probably misunderstood or underestimated. In this

group aIT appeared lower than IUGR co-twins with Doppler abnormalities but higher

than AGA, thus hypothesizing that there might exist a grading of endothelial damage

in which low birth weight and Doppler alterations constitute the major predictors.

The important role for twins regarding the fetal origins hypothesis consists in

testing the intra-pair differences which can assess the role of genetic confounding in

the association between fetal growth and later health outcome even if in literature

seems to be some confusion about how within-pair analyses should be used (Dwyer

et al. 2002).

In this study the IUGR twin had increased aIT with respect to its AGA co-twin.

These results in twins suppose that an increased arterial thickness in one twin respect

to its co-twin might suggest a genetic predisposition of endothelial damage which is

probably present already in fetuses with IUGR from intrauterine life and could play a

role in programming adult disease as suggested by Barker (2000, 2006) and other

investigators (Bateson et al. 2004, Eriksson et al. 2007, Osmond et al. 2007,

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Johansson et al. 2005, McMillen et al. 2005), irrespective of chorionicity, as

demonstrated in this study.

In the present study considering Doppler parameters aIT was lower in

monochorionic diamiotic and dichorionic diamniotic Group B twins, which did not

presented velocimetry alterations respect Group A. Moreover chorionicity and gender

did not seem to influence fetal markers of cardiovascular risk such as aIT, never

considered by other studies, EFW and velocimetry alterations.

The main contribution of this study is the correlation of aIT between pairs of

twins, and the analysis of twins according chorionicity and gender. Moreover, aIT

measurement might be feasible and easily performed by skilled operators as showed

good intra-inter observer variability. The study is limited by the lack of follow-up

during infancy and childhood of these twins, that is being currently carried on, but

whose outcome will require several years.

In conclusion, even in twin pregnancies aIT has been found to be higher in

IUGR fetuses than AGA.

Our findings in twins confirm that aIT should be considered a marker of

cardiovascular risk in IUGR fetuses with Doppler abnormalities, while IUGR twins

with normal Doppler share an intermediate risk. The absence of correlation with

chorionicity and gender strengthens the hypothesis that genetic factors might play a

role in the aforementioned risk.

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