Cerebral Cortex July 2015;25:1897–1905
doi:10.1093/cercor/bht431
Advance Access publication January 31, 2014
Brain Growth Gains and Losses in Extremely Preterm Infants at Term
Nelly Padilla1,4, Georgios Alexandrou1, Mats Blennow2,3, Hugo Lagercrantz1,3 and Ulrika Ådén1,3
1
Department of Women’s and Children’s Health, 2Department of CLINTEC, Karolinska Institutet, Stockholm, Sweden,
3
Department of Neonatology, Karolinska University Hospital, Stockholm, Sweden and 4Department of Maternal-Fetal Medicine
and Neonatology (ICGON) and Hospital Clínic, Institut D’investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona,
Spain
Address correspondence to Nelly Padilla, Neonatal Research Unit, Q2:07, Astrid Lindgren Children’s Hospital, S-171 76 Stockholm, Sweden.
Email: nelly.padilla@ki.se
Premature exposure to the extrauterine environment negatively
affects the brains’ developmental trajectory. Our aim was to deter-
mine whether extremely preterm (EPT) infants, with no evidence of
focal brain lesions, show morphological brain differences when com-
pared with term-born infants. Additionally, we investigated associ-
ations between perinatal factors and neuroanatomical alterations.
Conventional magnetic resonance imaging was acquired at term-
equivalent age (TEA) from 47 EPT infants born before 27 weeks of
gestation, and 15 healthy, term-born controls. Automatic segmenta-
tion and voxel-based morphometry-Diffeomorphic Anatomical Regis-
tration through Exponentiated Lie algebra (DARTEL) were used.
Compared with controls, EPT infants displayed global reductions in
cortical and subcortical gray matter, brainstem, and an increased
cerebrospinal fluid volume. Regionally, they showed decreased
volumes of all brain tissues, in particular cortical gray matter. In-
creased volumes of cortical gray and white matter were observed in
regions involved in visual processing. Increasing prematurity, intra-
ventricular hemorrhage grade I–II, and patent ductus arteriosus lig-
ation were associated with decreased volumes and had a particular
effect on the cerebellum. Concluding, EPT infants without focal brain
lesions had an altered brain growth at TEA that particularly affected
the gray matter, and varied when it came to the presence of perinatal
risk factors. Brain growth gains in EPT infants may be related to a
longer extrauterine experience.
Keywords: brain growth, extremely preterm birth, perinatal risk factors,
voxel-based morphometry
Introduction
The brain of the extremely preterm (EPT) infant grows and
develops in a completely different way than it would in the
womb. In addition, these babies are exposed to extrauterine
stimuli, which may affect the processes of brain maturation,
and as a result, how the brain gets wired (Lubsen et al. 2011).
Altered connectivity of the developing brain may result in cog-
nitive and neuropsychiatric impairment (Skiold et al. 2012;
Marret et al. 2013; Serenius et al. 2013). It is, therefore, very
important to develop a greater understanding of the structural
mechanisms responsible for neurodevelopmental problems,
so that effective early intervention programs can be devised.
Most of the earlier studies have focused on infants born later
than 27 weeks of gestation, and information on the structural
consequences of EPT birth is scarce. Moreover, most of the
studies in the field report altered the brain development of
preterm children in childhood, whereas our study focuses on
the early brain development of EPT infants up to their
term-equivalent age (TEA; term age). Similar studies have
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previously reported significant reductions in deep gray matter
volumes (Inder et al. 2005), gray matter structures, and promi-
nent compromise of the white matter (Parikh et al. 2013) in the
presence of several clinical complications. Using diffusion
magnetic resonance imaging (MRI), we have previously
described altered white matter organization in the centrum
semiovale and the corpus callosum of EPT infants with diffuse
excessive high signal intensities (DEHSIs; Skiold et al. 2010)
and a specific pattern of functional connectivity at term age
(Fransson et al. 2007, 2011). Taken together, the body of litera-
ture suggests different patterns of early brain development in
the anatomy and function of the EPT infant. However, brain in-
juries such as high-grade intraventricular hemorrhage (IVH)
and periventricular white matter lesions are very common in
these infants, and the relative contribution of prematurity itself
is unclear. We therefore studied EPT infants without evidence
of focal brain lesions at TEA, based on neonatal ultrasound or
MRI, by employing complementary techniques in order to
extend previous findings in this population.
Voxel-based morphometry (VBM; Ashburner and Friston
2000) is an automated procedure for quantifying changes from
a voxel-by-voxel analysis of MRI data. It has been used to
examine the effects of preterm birth during childhood (Kesler
et al. 2008; Padilla et al. 2011), adolescence (Nosarti et al.
2008; Nagy et al. 2009), and early adulthood (Allin et al. 2004).
Only one study including preterm infants at term age has used
a VBM approach to detect gray matter volume differences of
the hippocampus (Lodygensky et al. 2008). We aimed to inves-
tigate volumetric differences in brain tissues, on a whole-brain
level, by using VBM in EPT infants examined at term age. We
hypothesized that EPT infants at term age would show differ-
ences in brain morphology on a global and regional level
when compared with term infants, even without evidence of
focal brain lesions being detected by neonatal ultrasound or
conventional MRI. We used automated segmentation and VBM
improved by a Diffeomorphic Anatomical Registration through
Exponentiated Lie algebra (DARTEL) algorithm (Ashburner
2007), respectively. Our secondary objective was to examine
the effect of potentially adverse perinatal factors on brain
volumes.
Materials and Methods
Subjects
Eligible subjects for this study were all EPT infants (<27 weeks of ges-
tation) born in the Stockholm region between 1 January 2004 and 31
December 2008, who successfully underwent T1- and T2-weighted (w)
MRI at term age. We excluded infants with any grade of periventricular
leukomalacia (PVL) or IVH grade III and IV on neonatal ultrasound
 and focal brain lesions (e.g. cysts and infarctions), moderate and
severe white matter abnormalities (defined qualitatively) (Inder et al.
2003), or persistent ventricular dilatation on MRI examination at term
age. A group of healthy, term-born infants was recruited from the ma-
ternity ward and was also scanned at term age. At term age, 190 EPT
infants successfully underwent imaging. Of these infants, 19 (9.7%)
did not have structural MRI acquisition, meaning that 177 MRI cases
were assessed for good quality assurance. The most common reasons
for poor quality studies were: the presence of motion artifacts in 62
(35.02%) patients, incomplete coverage of the brain in 38 (21.46%)
patients, and blurring of the gray and white matter interfaces in 15
(8.47%) patients. Fifteen (8.47%) MRI cases were excluded for clinical
reasons. Two of these patients had noncystic PVL, one had cystic PVL,
seven had IVH grades III and IV (six had qualitatively defined white
matter abnormalities, with one categorized as severe and five as mod-
erate) (Inder et al. 2003), and five had posthemorrhagic ventriculome-
galy. Two of the 15 infants who were excluded were classified as small
for gestational age. A control group of 21 healthy, term-born infants
underwent MRI, and six (28.57%) of these were excluded due to
motion artifacts. Our final sample comprised 47 EPT infants of 177
(26.55%), who all had MRI images suitable for segmentation and volu-
metric analyses, together with 15 term infants of 21 (71.4%). The
regional ethical review board in Stockholm granted ethical approval
for the study and written consent was obtained from the parents of the
infants.
MRI Data Acquisition
All imaging was performed on a Philips Intera 1.5-T MRI system
(Philips International, Amsterdam, The Netherlands). The convention-
al MRI protocol consisted of a sagittal T1-w turbo spin-echo sequence,
an axial inversion recovery sequence, and an axial T2-w sequence.
Details of the sequence parameters have previously been published
(Skiold et al. 2012). The 3-dimensional T1-w gradient-echo images
were acquired with an echo time of 4.6 min, a repetition time of 40
min, a flip angle of 30°, a voxel size of 0.7 × 0.7 × 0.1 mm, and an field
of view of 180 mm.
Qualitative Assessment of the MRI Studies
Structural scans (T1- and T2-w images) were assessed by a neuroradiol-
ogist to ensure that they were free of gross abnormalities. Qualitative
white matter abnormalities were defined based on a previously pub-
lished scoring system (Inder et al. 2003). This system assessed 5 separ-
ate items: Abnormal white matter signal, reduced white matter volume,
cystic changes, myelination/thinning of the corpus callosum, and ven-
tricular dilatation. White matter abnormalities were further classified
by the composite scores of these 5 categories (ranging from 5 to 15)
into: No white matter abnormalities (score 5–6), mild white matter ab-
normalities (score 7–9), moderate white matter abnormalities (score
10–12), or severe white matter abnormalities (score 13–15).
Automatic Brain Segmentation and Voxel-Based Morphometry
The prior manual steps included reorientation of the original T1-w
images in the plane of anterior–posterior commissures and removal of
nonbrain tissue components using the Brain Extraction Tool (Smith
2002). Structural images were then segmented into tissue classes using
unified segmentation (Ashburner and Friston 2005), as implemented
in the “new segment” option of the SPM v8 software, Wellcome Depart-
ment, University College (London, UK), running on MATLAB v7.5
(MathWorks, Natrick, MA, USA). For guiding segmentation, we used
tissue probability maps from preterm infants scanned at term age
(Kuklisova-Murgasova et al. 2011). A quantitative evaluation of the seg-
mentations was performed, and the Dice coefficient (Dice 1945) was
calculated showing an agreement of 0.87 ± 0.02 for cortical gray
matter, 0.86 ± 0.02 for white matter, 0.79 ± 0.03 for deep gray matter,
0.84 ± 0.02 for cerebellum, and 0.83 ± 0.01 for brainstem (see Sup-
plementary Material for further details on automatic brain segmenta-
tion and validation).
The segmented brain tissues (see Supplementary Fig. 1) were
spatially normalized by using DARTEL. Finally, all images were
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modulated and smoothed with a full width at half-maximum of 3-mm
Gaussian kernel. We used these smoothed brain tissue images to
conduct the statistical analyses, as outlined below (see Supplementary
Material for further details on VBM). Global brain tissue volumes (in
cm3) were extracted from the segmented/normalized/modulated
images of each subject with the Easy Volume toolbox (Pernet et al.
2009).
Neonatal Complications and Brain Tissue Volumes
To investigate the effect of the neonatal variables on brain tissue
volumes within the preterm group, 2 subgroups were formed based on
the presence or absence of the following neonatal variables: Qualitat-
ively defined mild white matter abnormalities; DEHSI; IVH grades I–II;
patent ductus arteriosus (PDA); PDA with medical treatment (ibupro-
fen); retinopathy of prematurity grade >2; and bronchopulmonary dys-
plasia, defined as the need of oxygen at 36 weeks of gestation and
clinical diagnosis of sepsis, with or without positive blood culture.
Additional analyses were performed based on: the degree of immatur-
ity (≤25+6 and 26+0–26+6 weeks); any use of mechanical ventilation
versus nasal continuous positive airway pressure (nCPAP), and PDA
ligation versus PDA with medical treatment. We also studied corre-
lations between brain tissue volumes and days on ventilatory support
(mechanical ventilation and nCPAP) in the EPT group.
Statistical Analysis
Studied variables were tested for normality and homogeneity before
each analysis. Quantitative data were analyzed with Student’s t-test,
and qualitative data with Pearson’s χ 2 or Fisher’s exact test. The Mann–
Whitney U-test was applied for non-normally distributed data. The stat-
istical significance level was set at P < 0.05. All statistical analyses were
computed using the SPSS, version 20.0 (SPSS, Inc., Chicago, IL, USA).
Global Brain Volumes
To assess differences in global volumetric measures between the
groups, a general lineal model analysis (multivariate) was performed
with all brain volumes as dependent variables and group status as the
independent factor. The data were tested to ensure that no data as-
sumptions were violated. The covariates used were total intracranial
volume (TIV = all brain tissues, including cerebrospinal fluid [CSF]), to
control for generalized scaling effects, and postmenstrual age at the
time of the scan. Owing to the fact that variations in CSF volumes could
affect the TIV in the EPT infants, we also conducted analyses control-
ling for the total volume of the cerebral parenchyma (CPAR), including
all the brain tissues, but excluding CSF, in addition to postmenstrual
age at imaging. For the model, we used a Type III sum of squares,
which is invariant with respect to unequal samples. The same model
was used to investigate the effect of the neonatal variables on brain
tissue volumes between the preterm groups. In this case, analyses
were adjusted for gestational age at birth and days on mechanical venti-
lations as appropriate.
Voxel-Based Morphometry Analysis
For VBM analysis, t-test group comparisons were performed to evalu-
ate the brain volume differences between groups. In the EPT group, a
multiple regression analysis was performed to evaluate the relationship
between brain tissue volumes and days on ventilatory support (mech-
anical ventilation and nCPAP). We restricted the statistical analyses to
areas with a minimum probability value of 0.25 (absolute threshold
masking), avoiding possible edge effects around tissue borders. We
analyzed the following contrasts: Patients greater than controls and
controls greater than patients. Analyses were adjusted for CPAR. TIV,
age at MRI, and gender did not account significantly for variations in
the model and were not included in subsequent analyses. We used the
same previously defined covariates in the VBM analyses between
preterm groups. Only clusters >10 voxels are reported. All statistical
images were subjected to family-wise error correction for multiple
comparisons. For visualization purposes, the significant clusters were
 displayed at uncorrected P < 0.001 in order to identify the affected growth was affected in the presence of all of these perinatal
structures and networks. risk factors. The neonatal variables that significantly differed
between preterm groups were used as covariates in all analyses
(Supplementary Table 4). Of note, areas of increased cortical
Results gray matter were detected in the medically PDA treated group,
Table 1 summarizes the neonatal characteristics of the preterm
and term cohorts. The neonatal morbidity of the preterm
group is reported in Table 2. Table 2
Characteristics of the preterm infants
Global Volume Data
Characteristic Cohort (N = 47)
Global brain volume measurements are summarized in
Antenatal steroids (%) 47 (100)
Table 3. Overall, EPT infants had lower brain volumes than Apgar 5 min, mean (SD), range 7.49 (1.89) (3–10)
term infants, and we specifically found that cortical gray CRIB score, mean (SD), range 5.09 (3.37) (1–11)
matter, deep gray matter, and brainstem volumes were signifi- Days on CPAP, days, mean (SD), range 36.91 (13.91) (1–64)
Days on ventilator, mean (SD), range 11.78 (13.3) (0–47)
cantly lower. The CSF volume was significantly higher in the Postnatal corticosteroid therapy (%) 5/43 (11.6)
preterm group. It is noteworthy that the differences in deep Bronchopulmonary dysplasiaa (%) 17/42 (40.4)
Sepsis (clinical diagnosis) 27/43 (62.7)
gray matter and brainstem volumes remained significant after PDA (%) 36/47 (76.6)
adjustment for CPAR and the exclusion of infants with mild Treated PDA: ibuprofen (%) 21/36 (58.3)
white matter abnormalities. Treated PDA: surgery (%) 15/36 (41.6)
Intraventricular haemorrhage grade I (%) 12/47 (25.5)
Intraventricular haemorrhage grade II (%) 7/47 (14.90)
Whole-Brain Voxel-Based Morphometry Analysis No white matter abnormalities (score 5–6) (%) 23/47 (49.0)
Mild white matter abnormalities (score 7–9) (%) 24/47 (46.80)
The results of this section are presented in Supplementary Diffuse and excessive high signal intensities (%) 23/47 (48.9)
Table 1. Compared with the term group, EPT infants exhibited Retinopathy of prematurity stage ≥3 (%) 14/47 (29.8)
Retinopathy of prematurity stage ≤2 (%) 17/47 (36.1)
significant regional volume changes in all brain tissues. The
cortical gray matter reductions were bilaterally distributed in CPAP, continuous positive airway pressure; CRIB, clinical risk index for babies; PDA, patent ductus
the preterm group (Fig. 1A). These reductions predominantly arteriosus.
a
occurred in the cortical temporal lobe, extending superiorly to Defined as the need of oxygen at 36 weeks of gestation.
the perirolandic cortex, inferiorly to the orbito-frontal regions,
and medially to the entorhinal cortex. Regions in the left insula
also showed a decreased volume. The results were not altered Table 3
when infants treated with postnatal steroids (n = 5) were ex- Cerebral tissue volumes (in cm3) for prematures and term-born infants

cluded from the analysis. Smaller clusters of white matter de- Brain Preterm Term Statistic Statistic (P-value) Statistic
crements were observed in areas adjacent to the gray matter volumes (N = 47) (N = 15) (P-value) adjusted for age (P-value)
volumes at scan ICV
decreases, primarily affecting the temporal cortex and, to a unadjusted
minor extent, the angular gyrus and perirolandic area bilater-
Intracranial 465.13 468.89 t = −0.50
ally (Fig. 1B). Notably, larger gray matter and white matter volume (46.84) (25.28) (0.61)
volumes were detected bilaterally in the occipital, parietal, and Cortical gray matter
frontal cortices (Fig. 1C,D). The preterm group also displayed Mean 171.18 176.54 t = −1.05 F = 7.29 (0.009) F = 3.78
(SD) (18.90) (11.91) (0.29) (0.05)
areas of decreased volume in the deep gray matter, cerebellum, Range 135.98– 155.21–
and brainstem (Fig. 2). 214.13 195.01
White matter
Mean 146.92 150.06 t = −0.73 F = 2.52 (0.11) F = 3.78
Perinatal Risk Factors: Global and Regional Brain (SD) (15.48) (10.09) (0.46) (0.05)
Range 118.97– 131.66–
Volumes 190.69 166.09
The most immature infants, those with low-grade IVH and also Cerebrospinal fluid
with PDA who needed surgical ligation, had significant Mean 86.46 80.15 t = 2.10 (0.03) F = 9.08 (0.004) F = 6.77
(SD) (10.01) (10.37) (0.01)
reduced global and regional brain volumes (Supplementary Range 69.96– 63.56–
Tables 2 and 3) in several regions (Fig. 3). Notably, cerebellar 115.51 99.12
Deep gray matter
Mean 29.12 30.42 t = −1.87 F = 11.98 F = 4.25
(SD) (2.71) (1.82) (0.06) (0.001) (0.04)
Table 1 Range 24.32– 27.47–
37.80 33.41
Characteristics of the study groups
Cerebellum
Mean 26.61 27.35 t = 0.12 (0.37) F = 1.95 (0.16) F = 0.17
Preterm (n = 47) Term (n = 15) Statistic (P-value)
(SD) (2.97) (2.12) (0.68)
Perinatal Range 20.12– 24.70–
Gestational age at delivery 25.67 (1.09) 38.85 (0.35) U (<0.001) 34.70 31.89
Range 23.14–26.86 38.42–39.85 Brainstem
Male/female ratio 21/26 8/7 Fisher’s test 0.767 Mean 4.70 5.25 t = −4.32 F = 45.84 F = 36.62
Birth weight (g) 838.96 (152.83) 3712 (302.88) U (<0.001) (SD) (0.41) (0.31) (<0.001) (<0.001) (<0.001)
Range 528–1161 3075–4100 Range 3.86–5.98 4.73–5.88
Corrected age at scan (weeks) 40.85 (1.02) 40.54 (0.52) t = −1.514 (0.13)
Range 38.14–43.28 39.85–41.85 Note: Unadjusted brain volumes were compared by using a Student’s t-test. Unadjusted volumes
were compared by using a multivariate analysis of covariance.
Note: Results are expressed as mean (SD) or median (range). ICV, intracranial volume (sum of all brain tissues); CPAR, cerebral parenchyma (excluding
U, Mann–Whitney U-test; t, Student’s t-test. cerebrospinal fluid).

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 Figure 1. Regional volumetric differences (warm colors) in the EPT infants compared with term infants (A) gray matter decreases, (B) white matter decreases, (C) gray matter
increases, and (D) white matter increases. The color bar represents the t-score. Display orientation: right = right. Differences are mapped on the render of one preterm infant.
when compared with the group without PDA (P < 0.005).
Although we could not find global brain volume differences
between infants on mechanical ventilation in comparison with
those using nCPAP, a cluster of decreased cortical gray matter
volume in the left primary motor cortex (cluster = 475 voxels,
P = 0.04) was identified on a regional level (not shown). Sig-
nificant negative correlations were observed between days on
mechanical ventilation and brain volumes in deep gray matter
(r = 0.51, P < 0.001), cerebellum (r = 0.57, P < 0.001), and
brainstem (r = 0.53, P < 0.001) (Supplementary Fig. 2). With
respect to other medical conditions, we could not find any
differences in global or regional level brain volumes between
the preterm groups.
Discussion
The main findings of this study are that when the EPT infants
were compared with the term infants, they showed significant
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global reductions in brain tissue, accompanied by a signifi-
cant increase in CSF volume. On a regional level, the preterm
infants showed decreased volumes of all brain tissues, in par-
ticular cortical gray matter. An unexpected finding of the study
is that the EPT infants displayed increased cortical gray matter
and white matter involving vision-related brain areas. Finally,
increasing prematurity, low-grade IVH, and PDA ligation were
associated with different patterns of brain volume reductions
and had a particular effect on the cerebellum. Our study
extends previous findings and supports the notion that EPT
birth induces deviations in the developmental trajectory of the
brain at term age, even in the absence of focal brain pathology.
Global Brain Volumes
We found decreased global volumes of cortical gray matter,
deep gray matter, and brainstem. This is similar to a previous
report (Parikh et al. 2013), which also found smaller global
brain volumes, but with a major involvement of the white
 Figure 2. Sagittal, coronal, and axial slices showing regional volumetric differences in the EPT infants, compared with term infants, in (A) deep gray matter, (B) cerebellum, and
(C) brainstem. The color bar represents the t-score. Display orientation: right = right. Results are superimposed on the smoothed corresponding image of one preterm infant.
matter. However, this might be due to the fact that their study
population had more clinical complications than ours. More-
over, 25% of the infants in that study were small for their gesta-
tional age, a condition that is known to affect white matter
maturation in preterm infants at term age (Lepomaki et al.
2013). In the current study, major volume reductions were
found in the deep gray matter and brainstem of the EPT
infants. The volumetric differences in the deep gray matter per-
sisted even when conventional MRI sequences failed to ident-
ify any white matter abnormality in infants. Our results
confirm previous findings that the deep gray matter is specifi-
cally vulnerable after preterm birth and is particularly affected
in more immature infants (Inder et al. 2005). In this study, EPT
infants had significant reductions in the whole-brainstem
volume. At this early age, the brainstem is part of a vertical-
integrative hierarchical system, along with the limbic and
cortical systems, and is involved in the process of regulatory
functions, such as arousal, attention, and emotion (Geva and
Feldman 2008).
The global volume reductions found in our study were prob-
ably not due to acute or hypoxic injury, as there were no signs
of brain abnormalities caused by these conditions. It is more
than likely that the present results can be explained by the
extreme preterm infants (mean gestational age of 25.6 weeks)
being born during a critical period of brain development,
when synaptic density neuronal wiring is increasing in all the
cortical regions (Zecevic 1998), and the thalamo-cortical pro-
jections are waiting within the subplate zone to reach the corti-
cal plate (Kostovic and Judas 2010). In addition, at the time of
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EPT birth, the many cortical interneurons in the human fetus
are been generated in the proliferative zones and are still
migrating to the cerebral cortex (Letinic and Rakic 2001).
Altogether, the EPT birth per se probably leads to altered con-
nections at multiple anatomical levels, from the cortex to the
deep gray matter and brainstem, with subsequent developmen-
tal disturbances and trophic consequences reflected by volume
alterations (Volpe 2009a, 2009b; Kostovic and Judas 2010; Ball
et al. 2012).
Regional Analyses: Voxel-Based Morphometry
We found that when we compared the EPT infants with the
term infants in the control group, they showed extensive
regions of reduced gray matter in several brain areas that have
previously been described in more mature infants studied in
childhood and adolescence. These areas included: the cortical
temporal lobe bilaterally (Kesler et al. 2008; Nosarti et al. 2008;
Nagy et al. 2009), precentral and postcentral gyri (Peterson
et al. 2003; Allin et al. 2004; Kesler et al. 2008); orbito-frontal
cortex (Thompson et al. 2007; Nagy et al. 2009; Ball et al.
2012); amygdala (Peterson et al. 2000; Kesler et al. 2008); para-
hippocampal gyrus (Kesler et al. 2008); hippocampus (Peter-
son et al. 2000; Kesler et al. 2008; Nagy et al. 2009), and left
insula (Nosarti et al. 2008). The additional involvement of sub-
cortical gray matter sites, including the deep gray matter (an
interface between neocortical and lower-level systems), cer-
ebellum, and brainstem, confirms the increase susceptibility of
the gray matter in the preterm brain (Dean et al. 2013; Malik
et al. 2013).
Cerebral Cortex July 2015, V 25 N 7 1901
 Figure 3. Sagittal, coronal, and axial slices showing regional volumetric differences between preterm infants under specific neonatal conditions. The color bar represents the
t-score. Display orientation: right = right. Results are superimposed on the smoothed corresponding image of one preterm infant.
Structures involved in auditory, language, and somatosen-
sory processing were recognized. We also identified a structure
that has not previously been reported in EPT infants at term
age, namely the entorhinal cortex, a gateway between neocor-
tical association areas and the hippocampal system (Fransen
2005). In adolescents born preterm, entorhinal thinning has
been associated with impaired cognitive function as a result of
aberrant cortical maturation (Skranes et al. 2012). Further
studies should focus on the entorhinal cortex and its correlation
with cognition in EPT samples. The decreased regional volumes
found in our study may be due to compromised neurogenesis
(Malik et al. 2013), disturbances of the dendritic arbor, and
synapse formation of cortical neurons (Dean et al. 2013).
Increased volumes were detected in regions known to be in-
volved in visual processing, contrary to previous studies (Pe-
terson et al. 2003; Shah et al. 2006; Thompson et al. 2007).
However, the infants in these studies were more mature than
the preterm infants included in our study and experienced
several clinical complications. The nature of the increase in
gray and white matter is difficult to interpret as the VBM ap-
proach provides a measure of volume, but provides no infor-
mation on the cytoarchitectural structure. Increases in gray and
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white matter volumes may reflect an abnormal or delayed
pruning program, as has been previously suggested (Nosarti
et al. 2008). However, the visual cortex represents a particular
region of the brain that matures earlier than other areas of the
cortex (Tzarouchi et al. 2009) and has a functional relevance in
EPT infants at term age (Fransson et al. 2007, 2011). In this
respect, increased volumes in our preterm cohort may corre-
spond to advanced maturational aspects in the visual regions,
due to longer extrauterine visual experiences during a critical
period, compared with term infants. In fact, both classic
(Wiesel 1982; Bourgeois et al. 1989) and modern studies (Tsu-
neishi and Casaer 2000; Gimenez et al. 2008) support the
theory that neural activity driven by visual experience is essen-
tial for shaping the early rudimentary cortical connectivity pat-
terns (Penn and Shatz 1999). Further studies that follow up
functional aspects are needed to define the developmental cor-
relates of these findings.
Perinatal Risk Factors
In addition to the degree of immaturity, the presence of IVH
grade I–II and surgically ligated PDA were associated with
 reduced brain growth. The regional gray matter was also af-
fected by the severity of respiratory illness. Overall, brain
volume reductions preferentially affected gray matter struc-
tures and, in particular, the cerebellum, as mentioned above.
The other perinatal factors that were studied had no significant
effect on cerebral structure at term age.
In this study, we included infants with IVH grade I–II as
these hemorrhages are usually considered to be benign. Our
findings of reduced brain volumes are in agreement with pre-
vious MRI studies (Vasileiadis et al. 2004) and may reflect the
influence of the low-grade IVH on developmental processes
related to suppression of cell proliferation (Del Bigio 2011)
and microglia activation (Supramaniam et al. 2013). These
findings may help to explain the impaired developmental out-
comes reported in preterm infants with low-grade IVH at
different ages (Klebermass-Schrehof et al. 2012).
The adverse effect of PDA ligation may be due to disturbed
cerebral circulation. Infants who needed surgical ligation faced
a higher risk of affected brain growth than those who only re-
ceived medical treatment. There seem to be adverse effects of
the PDA ligation per se, where intraoperative or postoperative
physiological alterations may affect cerebral hemodynamics
(El-Khuffash et al. 2013), potentially causing further injury to
an already vulnerable brain. It is, however, surprising to note
that the infants in our cohort who only received medical
treatment (ibuprofen) for PDA had preserved brain volumes
compared with those without. We speculate that the anti-
inflammatory effect of ibuprofen might be protective for brain
growth, which is in line with previous findings where ibupro-
fen showed neuroprotective effects attenuating neuronal
damage (Wixey et al. 2012).
The neuropathological basis of the decreased regional cer-
ebellar volume in our study still needs to be elucidated. Of
note, the gestational age of the EPT neonates included in our
research corresponds to a phase of rapid cerebellar develop-
ment, representing a period of particular vulnerability. It is,
therefore, likely that the suppression of neuronal proliferation
(Volpe 2009a, 2009b), trophic interactions (Tam et al. 2011),
and a compromised cerebellar perfusion (Limperopoulos et al.
2005) might specifically affect the growth of the cerebellum.
Impaired cerebellar growth accounts for subsequent develop-
mental problems, which indicate the need for heightened clini-
cal surveillance and early target interventions. Remarkably, the
presence of DEHSI detected in 48.9% of our preterm cohort at
term age was not associated with brain volume alterations. We
have previously found that the presence of DEHSI in EPT
infants at term age was not related to abnormal neurologic
outcome at 30 months (Skiold et al. 2012). These results
suggested that DEHSI is a prematurity-related transient
phenomenon.
Our study benefits from including a unique cohort of EPT
infants without major brain alterations on neonatal ultrasound
or MRI at term age and a mean gestational age lower than pre-
viously published cohort studies. However, some methodo-
logical concerns must be considered. We are aware that tissue
segmentation in preterm infants at term is a challenging task,
owing to the developmental characteristics of the preterm
brain. Furthermore, the segmentation is limited in smaller cer-
ebral structures, owing to the smaller volumes and a lower
signal-to-noise ratio than in older infants. To minimize these
limitations, we only used good quality T1-w images. For guid-
ing segmentation, we used a larger number of tissue
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probability maps, together with an extraclass tissue map for
background, to provide better modeling of CSF and other non-
brain voxels and further to improve the tissue classification.
Additionally, the tissue probability maps used here were based
on a sample of preterm neonates scanned at term age. We also
used the DARTEL algorithm to provide a better intersubject
registration.
In conclusion, we have shown that at TEA, and in the
absence of focal brain lesions, EPT infants have an altered
global and regional brain growth pattern, involving decreases
and increases of brain tissue volumes. Gray and white matter
gains in regions known to be involved in visual processing
suggest maturational brain changes driven by precocious
visual experiences in EPT infants. The degree of immaturity,
IVH grade I–II, and surgically ligated PDA were associated
with global and regional decreased brain growth that preferen-
tially affected gray matter structures, in particular the cerebel-
lum. Our study suggests that structural alterations may be used
as structural markers to identify children at risk earlier, and
that this may prevent adverse outcomes following EPT birth.
Longitudinal studies, including functional follow-up data,
would help to determine the significance of the volumetric
brain alterations found in the EPT infants in our study.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxford-
journals.org/.
Funding
This work was supported by the following grants: Swedish
Medical Research Council (2011–3981), the regional agree-
ment on medical training and clinical research (ALF 20120450)
between Stockholm County Council and Karolinska Institutet,
Marianne and Marcus Wallenberg Foundation (MMW
2011.0085), EU Seventh Framework (223767), Linnea and
Josef Carlsson´s Foundation, Swedish Order of Freemasons in
Stockholm, Swedish Medical Society, Swedish Brain Foun-
dation, and Sällskapet Barnavård. N.P. was supported by a
Sara Borrell post-doctoral fellowship (CD09/00263), Instituto
de Salud Carlos III, Spain.
Notes
We thank the families and infants who took part in the study, together
with the MR physics group and MR radiology staff at Astrid Lindgren
Children’s Hospital for their valuable technical assistance. Conflict of
Interest: None declared.
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