Regional Brain Volume Abnormalities and Long-term Cognitive Outcome in Preterm Infants FREE

Infants weighing less than 1500 g at birth now represent 1.5% of all live births in the United States.¹ Although survival rates currently approach 85%, the 12% to 32% prevalence of major neurodevelopmental handicaps in surviving children represents a growing public health concern.^{2 - 3} Preterm infants, even those who have uncomplicated neonatal courses, frequently experience serious cognitive and educational difficulties.⁴ IQ scores are nearly 1 SD (15 points) below the population mean,⁵ more than half of these children require special assistance in school, and nearly 20% repeat a grade in school by age 8 years.⁶ Although the biological determinants of these developmental difficulties are unknown, the neurobehavioral outcome of preterm infants has been reported to worsen with a younger gestational age (GA) at birth.^{2 ,6}

Fetuses of 24 weeks' GA are now viable. The brain at this point in development is a thin shell of tissue surrounding the cerebral ventricles, and virtually all of normal cortical and subcortical architecture has yet to be established. Subsequent physiological stress can seriously disrupt the maturational processes that lay down this architecture. The effects of stress vary by brain region and with the nature and timing of the insult.⁷ The stresses associated with birth early in or prior to the third trimester of gestation are therefore likely to disrupt brain maturation with regional specificity. The ensuing abnormalities in brain structure may then contribute to the long-term cognitive deficits of preterm children.

We measured regional brain volumes on the magnetic resonance imaging (MRI) scans of 25 prematurely born 8-year-old children and 39 group-matched term control children. We hypothesized that brain volumes of the preterm children would differ significantly from volumes of the term controls and that these abnormalities would be regionally specific. We then examined the associations of regional brain volume with cognitive measures to test our hypothesis that abnormalities in brain volume would be associated with deficits in long-term cognitive outcome. Finally, we examined the associations between regional brain volume and perinatal risk factors in the preterm cohort.

This study was performed at the Yale University School of Medicine, New Haven, Conn, and at Brown University School of Medicine, Providence, RI, in 1998-1999. The procedures were approved by the institutional review boards at each site. All children and their parents provided informed written consent and were paid to participate in the study. The MRI scans were performed at Yale.

The preterm cohort consisted of children enrolled in the follow-up component of a multisite trial of indomethacin to prevent intraventricular hemorrhage (IVH).⁸ From the original 440 surviving preterm children in this trial, 370 (84%) have been successfully followed up since birth.⁹ The first 26 children sequentially enrolled in the prevention study were recruited for the imaging study when they reached 8 years corrected age (age from the obstetric due date). The preterm sample, therefore, was not selected on the basis of outcome status or perinatal history.

Term control children, aged 7 to 9 years, were recruited from randomly selected names on a telemarketing list of 10,000 families in the local community. The families were identified by the telemarketing company as having children aged 7 to 9 years and as living in the same general neighborhoods (based on ZIP code) as the preterm children. Families on this list were selected for contact using a random number generator. Introductory letters were followed by recruitment and screening phone calls. Controls were frequency matched with the subjects by continually updating information on accumulating subjects and controls to provide similar distributions of age, sex, maternal education, and minority status (caregiver report of white or not) in the 2 groups.¹⁰ Of the eligible control families contacted, approximately 10% participated.

Newborns weighing 600 to 1250 g were recruited within 6 hours of birth to the IVH prevention trial. All infants were examined with serial cranial echoencephalograms (ECHOs) to evaluate their brains for IVH or ischemia. ECHOs were performed at 5 to 11 hours of age, on postnatal days 2, 4, 5, 7, 14, and 21 and at 40 weeks' GA.⁸ The grading systems for IVH, ventriculomegaly, and periventricular leukomalacia (PVL) are described elsewhere.¹¹

The GA of the infants was determined using a modification of the Ballard scale.¹² Prenatal, perinatal, and neonatal data were obtained prospectively by maternal interviews and from the maternal and neonatal hospital charts. Bronchopulmonary dysplasia was diagnosed if an infant required oxygen supplementation and had an abnormal chest x-ray finding at 28 days of life. Chronic lung disease was defined as oxygen dependence at 36 weeks corrected age. Resuscitation scores quantified the difficulty in resuscitating the infant at birth using an anchored ordinal variable, as previously described.⁸

At 96 months corrected age each child underwent a blinded assessment of neurodevelopmental outcome. IQ was measured with the Wechsler Intelligence Scale for Children-III.¹³ Visuomotor functioning was assessed with the Developmental Test of Visual-Motor Integration (VMI).¹⁴ Psychiatric diagnoses were based on the Kiddie-Schedule for Affective Disorders and Schizophrenia Epidemiologic Version.¹⁵ Behavioral problems were assessed by parent report using the Child Behavior Checklist.¹⁶ Cerebral palsy was diagnosed if hypertonicity, hyperreflexia, and dystonia or spasticity were noted on neurologic examination.

The MRI scans were obtained on all children without sedation using a single 1.5-T scanner (GE Signa; GE Medical Systems, Milwaukee, Wis). Head positioning was standardized using canthomeatal landmarks. A sagittal spoiled gradient recall sequence was obtained for volumetric studies (time to repeat = 24 milliseconds, echo time = 5 milliseconds, 45° flip, frequency encoding superior/inferior, no wrap, 256 × 192 matrix, field of view = 30 cm, 2 excitations, slice thickness = 1.2 mm, 124 contiguous slices).

Morphometric analyses were performed on Sun Ultra 10 workstations using ANALYZE 7.5 software (Biomedical Imaging Resource, Mayo Foundation, Rochester, Minn) with operators blinded to subject characteristics and hemisphere (images were randomly flipped left-right prior to analysis). Large-scale variations in image intensity were removed and then images were reformatted to standardize head flexion, rotation, and tilt prior to region definition.¹⁷ An isointensity contour function was used in conjunction with manual editing to isolate the brain and cerebellum. The brain was divided into hemispheres using a curvilinear plane positioned through standard midline landmarks. Each hemisphere was then divided into 8 anatomical sectors using 1 axial plane containing the anterior commissure–posterior commissure (AC-PC) line and 3 limiting coronal planes—1 tangent to the genu of the corpus callosum, 1 tangent to the anterior border of the AC, and 1 through the PC. The 8 sectors were dorsal prefrontal, orbitofrontal, premotor, subgenual, sensorimotor, parieto-occipital, midtemporal, and inferior occipital cortices (Figure 1). The reliability and validity of related schemes of brain subdivision have been documented.^{18 - 19} The volumes of these cortical subdivisions included both gray and associated white matter but not cerebrospinal fluid. More detailed regional subdivisions were performed to provide samples of relatively pure tissue types. These included samples of cortical gray matter from the amygdala and hippocampus, subcortical gray matter from the basal ganglia nuclei (caudate, putamen, and globus pallidus), white matter from the corpus callosum, and cerebrospinal fluid in the cerebral ventricles. The procedures for defining and subdividing these structures are described elsewhere.^{17 ,20 - 22} Their interrater reliability was assessed on 10 scans. Intraclass correlation coefficients were greater than 0.98 for cortical and ventricle subdivisions, caudate, and putamen; greater than 0.90 for the globus pallidus, hippocampus, and corpus callosum measures; and greater than 0.85 for the amygdala.

A power analysis was first conducted to ensure an adequate sample size for hypothesis testing. Because this study was planned as a pilot study to justify MRI scanning of the entire multisite preterm cohort, we wished to have adequate power (>80%) to detect a large effect size at the 2-sided, .05 level of significance.²³ The size of our sample met these criteria. It risked, however, not detecting more moderate group differences. A greater sensitivity to detect between-group differences would require a larger sample.

We tested 2 a priori hypotheses. Our primary hypothesis was that regional brain volumes would differ between diagnostic groups. This was tested using a multivariate analysis of covariance (MANCOVA) for each set of brain regions. Test statistics were calculated using the type III sums of squares, which calculates an effect in the model as the sum of squares adjusted for any other effects not containing the effect of interest. Height and sex were entered as covariates to control for generalized scaling effects within the brain. The analyses were repeated using total brain volume as a covariate to control for generalized scaling effects. The effects of 2- and 3-way interactions between diagnosis and the covariates were not significant and were therefore not included in the final model. Post hoc P values were Bonferroni-corrected to account for multiple comparisons. Appropriateness of the models was ensured by examining residuals.

Our secondary hypothesis was that the sum of volumes in regions that differ significantly between groups would be associated with full-scale IQ in the preterm children. This was tested using multiple linear regression with sex and height as covariates. Because the number of preterm children diagnosed with any single neuropsychiatric disorder was small, the associations of diagnosis with brain volumes in the preterm children were not assessed.

As exploratory analyses, we examined the associations of regional volumes with perinatal variables in the preterm children using the Pearson product moment correlation coefficient for normally distributed continuous variables (birth weight and maternal age), the Spearman rank order correlation coefficient for ordinal or skewed continuous variables (GA, 5-minute Apgar scores, resuscitation score, number of septic episodes, and number of days receiving ventilatory assistance), or the nonparametric Mann-Whitney U test for dichotomous variables (IVH, bronchopulmonary dysplasia, and chronic lung disease). Two time cutoffs for IVH (within the first 6 hours or the first 5 days of postnatal life) were used in these analyses. The first 5 postnatal days are the period of greatest risk for IVH (all episodes of IVH in this sample occurred within the first 5 days). Those infants with IVH within the first 6 hours of life additionally are known to have prolonged depression of cerebral blood flow, placing their developing brains at increased risk for ischemic injury.²⁵ Data pertaining to certain perinatal variables that have been associated with preterm birth, such as maternal infection, chorioamnionitis, and cytokine levels, were not reliably available and therefore not assessed. We also examined several associations between regional volumes. The effects of sex and height were removed from the regional volumes prior to calculation of association indices. To help correct for multiple comparisons, P<.01 was considered significant for these exploratory analyses. All analyses were performed in SPSS version 9.0 (SPSS Inc, Chicago, Ill), and all P values were 2-sided.

We obtained MRI scans for 26 preterm children and 39 term controls. The scan of 1 preterm child was degraded by motion artifact and excluded from further analyses. Subject characterization is shown in Table 1. As expected, IQ and height were significantly reduced in the preterm children.²⁶ According to parent reports on the Child Behavior Checklist, the preterm children were more withdrawn (P = .03), inattentive (P<.001), aggressive (P = .02), and troubled in their thought processes (P = .04) than the term controls. The cognitive profiles, incidences of neurologic handicaps, rates of neuropsychiatric disorder, and behavioral problems in the preterm children were nearly identical to those found in other follow-up studies of preterm children,^{3 - 4} thus supporting the representativeness of the sample. In addition, demographic, neonatal, and cognitive outcome measures indicated that the cohort was representative of the larger sample of surviving children in the multisite trial from which they were identified. They were similar to other survivors in full-scale IQ (P = .78), performance IQ (P = .41), verbal IQ (P = .96), sex (P = .64), maternal education (P = .24), minority status (P = .21), birth weight (P = .45), 5-minute Apgar score (P = .87), IVH within 6 hours of birth (P = .11), IVH within 5 days of birth (P = .10), bronchopulmonary dysplasia (P = .36), chronic lung disease (P = .20), newborn ventriculomegaly (P = 1.0), PVL (P = .61), indomethacin exposure (P = .94), and spastic quadriplegia (P = .62). The preterm children in this study were born at a slightly older GA than the remaining multisite preterm cohort (mean [SD], 28.7 [1.7] vs 27.9 [2.0] weeks; P<.05) because of the survival of younger fetuses subsequently enrolled in the multisite trial.

Preterm children differed significantly from term controls in regional brain volumes. The MANCOVAs demonstrated significant overall multivariate effects of prematurity in cortex, ventricles, basal ganglia, amygdala and hippocampus, and corpus callosum (multivariate P<.001 for all). The effects of height were not significant in any of the analyses, and significant sex effects were seen only for corpus callosum subregions (P<.03). Post hoc analyses are presented in Table 2 and Figure 1.

The basal ganglia, corpus callosum, amygdala, and hippocampus findings persisted when total brain volume was entered as a covariate in the MANCOVA group comparisons, thus indicating that the reduced size of these structures was disproportionately greater than predicted by the smaller brains of preterm children. The findings persisted when children with a history of IVH were excluded from the analyses, thus indicating that prior hemorrhage was not responsible for the noted size reductions. Inclusion of maternal education, minority status, and indomethacin treatment in the MANCOVAs did not affect the results and therefore were not included in the final model.

The cortical regions that differed significantly between groups in testing of the primary hypothesis were summed. After covarying for the effects of sex and height, this single measure correlated significantly with full-scale IQ (β = .49; P<.001), confirming our secondary hypothesis. Further analyses explored which IQ subscales and individual test items contributed most to this significant association (Table 3, Figure 2). Full-scale, verbal, and performance IQ scores were associated positively with regional volumes, most strongly and consistently with volumes of sensorimotor and midtemporal brain regions. Covarying for maternal education, minority status, and indomethacin treatment in the correlation analyses did not affect the results.

Perinatal Variables. Gestational age correlated significantly with volumes of the left and right sensorimotor cortices and right amygdala (ρ = 0.53-0.56; P<.005). The 5-minute Apgar score correlated with volumes of the right sensorimotor cortex (ρ = 0.48; P<.01), the globus pallidus nucleus bilaterally (ρ = 0.57-0.59; P<.005), and the midbody of the corpus callosum (ρ = 0.50; P<.01). Intraventricular hemorrhage within 6 hours of birth correlated significantly with volumes of the cerebellum (Mann-Whitney U = − 2.74; P<.01) and left caudate nucleus (Mann-Whitney U = − 2.16; P<.01) in the preterm children. The number of septic episodes, birth weight, ventriculomegaly at term, maternal age, number of days receiving ventilatory assistance, and the presence of bronchopulmonary dysplasia or chronic lung disease were not significantly associated with regional volumes. The small number of preterm children with a history of PVL precluded statistical analyses (Figure 3).

Interregional Correlations. Area measurements of the posterior corpus callosum (including the midbody, isthmus, and splenium) were significantly associated with the respective projection areas (sensorimotor, midtemporal, and occipital parcellation units) of the interhemispheric axons contained in those corpus callosum subregions²⁷ (0.47 < r < 0.63; .02 < P < .001). Additionally, the correlation analyses depicted in Figure 4 suggest that in control subjects the volumes of posterior brain regions were positively associated with the volumes of the occiptal horns of the lateral ventricles. These volumes were inversly associated in the preterm cohort, however, suggesting that reduced volumes of the cerebral tissue adjacent to the occipital horns may account for the massive dilation of the neighboring cerebral ventricles in preterm children.

To our knowledge, this is the first quantitative MRI study of the long-term outcome of regional brain volumes in preterm infants and the first to describe significant associations of cognitive outcome with morphological disturbances in brain development in this clinical population. At age 8 years, regional cortical volumes in preterm children were significantly smaller than in term controls. The abnormalities were centered in the sensorimotor cortex but also involved the adjacent premotor, parieto-occipital, subgenual, and midtemporal regions, and the cerebellum. Subcortical gray matter in the basal ganglia, white matter in the posterior corpus callosum, and cortical gray matter in the amygdala and hippocampus were also reduced more than expected from the overall reduction in brain volume. Cerebrospinal fluid in the occipital and temporal horns of the cerebral ventricles was markedly increased in the preterm children. These group differences demonstrate the presence of differential regional vulnerabilities in the developing brains of preterm children. Although this pattern of injury has not been described in previous quantitative studies, certain of its features have been noted in qualitative studies of school-aged preterm children, including enlargement of the occipital horns,²⁸ white matter damage,^{28 - 29} and thinning of the posterior corpus callosum.^{29 - 30}

The volumes of these brain regions in the preterm cohort correlated significantly with IQ measures. Regional volumes also correlated significantly with GA at birth, 5-minute Apgar scores, and IVH within 6 hours of birth. These associations of brain volumes with IQ measures and perinatal variables require replication in light of the multiple comparisons performed. Nevertheless, taken together they suggest that perinatal events produce long-term disturbances in cerebral development and that these disturbances in cerebral development in turn account for cognitive deficits in preterm infants. The prominent involvement of motor portions of the cortex, corpus callosum, and basal ganglia could possibly account for the predisposition to cerebral palsy and other motor disturbances in preterm children.

We cannot say whether the reduced cortical volumes in preterm children preferentially involved either cortical gray or associated white matter because our cortical measures did not distinguish gray from white matter tissue. Nevertheless, the reduced size of the corpus callosum suggests that long white matter–association axons are structurally compromised, particularly the known projections to posterior portions of the callosum from sensorimotor, midtemporal, and parietal cortices,²⁷ the regions where cortical volume reductions in preterm children were largest. These white matter findings are consistent with postmortem and in vivo MRI findings of reduced white matter volume or compromised white matter structural integrity in preterm infants and children.^{28 - 29 ,31 - 36} White matter compromise is not the only abnormality in preterm brain development, however, as reduced basal ganglia, amygdala, and hippocampus volumes indicate that development of subcortical and cortical gray matter structures is also disturbed.

Our findings are generally consistent with a prior quantitative MRI report of reduced cortex and white matter volumes and increased cerebrospinal fluid in preterm infants studied at term.³⁶ Volumes in the subgroup of preterm infants without PVL, however, did not differ from term controls in that study, which stands in contrast to our findings of prominent volume reductions in 8-year-old children who did not have IVH, PVL, or ventriculomegaly as newborns. This disparity suggests that volume abnormalities in children without PVL may become manifest between infancy and childhood.

The pattern of regional cortical vulnerability identified here bears some resemblance to the patterns of lesions associated with asphyxia^{37 - 38} or IVH³⁹ in term infants. These patterns of abnormality have been attributed to disturbances in blood flow and oxygen delivery to regions where metabolism is highest,^{37 ,40} including sensorimotor cortex in term⁴¹ and possibly preterm⁴² infants. In the present study, the prominent reductions in volume of the sensorimotor cortex prompt speculation that the pattern of morphological abnormalities in preterm children may similarly result from regional disruptions in blood flow or nutrient delivery. Several considerations, however, weigh against this possibility in our preterm cohort. First, serial neonatal ECHO examinations indicated that hypoxic injury was rare (1 infant had PVL) and that hemorrhages did not extend into brain parenchyma. Second, the pattern of regional abnormalities was unchanged when children with a history of IVH were excluded from the analyses. Finally, our indices of hypoxia (days receiving ventilatory assistance, bronchopulmonary dysplasia, and chronic lung disease) were not associated significantly with greater long-term volume reductions. These considerations suggest that volume reductions were not likely to have been the consequence of acute, destructive hemorrhagic or hypoxic lesions. It is nevertheless possible that subtler and more chronic disturbances in regional perfusion^{43 - 44} or hypoxia⁴⁵ contributed to the observed abnormalities in brain volume⁴⁶ and cognitive outcome.^{47 - 48}

We are therefore unable to specify precisely what caused the observed morphological abnormalities in our preterm cohort. The prominent volume reductions and the associations of those volumes with poorer cognitive outcome indicate, however, that a premature transition from intrauterine to extrauterine life can profoundly disrupt fetal brain development. The significant association of regional volumes with GA at birth suggests that the disturbance in brain development is proportional to the degree of fetal immaturity when this transition occurs. Longitudinal imaging studies of preterm infants and animal models of preterm birth will help to identify whether, how, and when in development specific physiological stresses associated with prematurity are able to disrupt fetal brain maturation.