European Journal of Neuroscience, pp. 111, 2017 doi:10.1111/ejn.

13527

The effect of age, sex and clinical features on the volume

of Corpus Callosum in pre-schoolers with Autism
Spectrum Disorder: a casecontrol study

Alessia Giuliano,1,2,* Irene Saviozzi,3,* Paolo Brambilla,4,5 Filippo Muratori,6,7 Alessandra Retico2 and
Sara Calderoni7
1
Physics Department, University of Pisa, Pisa, Italy
2
Pisa Division, National Institute for Nuclear Physics, Largo Pontecorvo 3, 56127 Pisa, Italy
3
IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy
4
Department of Neurosciences and Mental Health, Psychiatric Clinic, Fondazione IRCCS Ca Granda Ospedale Maggiore
Policlinico, University of Milan, Milan, Italy
5
Department of Psychiatry and Behavioural Sciences, University of Texas Health Science Center at Houston, Houston, TX, USA
6
Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
7
Department of Developmental Neuroscience, IRCCS Stella Maris Foundation, Pisa, Italy

Keywords: Freesurfer, neurodevelopmental disorders, sex difference, structural MRI, young children

Edited by John Foxe

Received 11 November 2016, revised 31 December 2016, accepted 14 January 2017

Abstract
A growing body of literature has identified volume alterations of the corpus callosum (CC) in subjects with autism spectrum disor-
ders (ASD). However, to date very few investigations have been conducted on pre-school-age ASD children. This study aims to
compare the volume of CC and its sub-regions between pre-schoolers with ASD and controls (CON) and to examine their rela-
tionship to demographic and clinical variables (sex, age, non-verbal IQ -NVIQ-, expressive non-echolalic language, emotional and
behavioural problems, and autism severity). The volume of CC of 40 pre-schoolers with ASD (20 males and 20 females; mean
age: 49  12 months; mean NVIQ: 73  22) and 40 sex-, age-, and NVIQ-matched CON subjects (20 M and 20 F; mean age:
49  14 months; mean NVIQ: 73  23) were quantified applying the FreeSurfer automated parcellation software on Magnetic
Resonance images. No significant volumetric differences in CC total volume and in its sub-regions between ASD and CON were
found using total brain volume as a covariate. Analogously, absence of CC volumetric differences was evident when boys and
girls with ASD were compared with their matched controls. The CC total volume of younger ASD male subjects was found signifi-
cantly larger with respect to matched CON, which is consistent with the atypical growth trajectory widely reported in these young
children. The CC total volume was negatively correlated with autism severity, whereas no association between CC volume and
other clinical variables was detected. If replicated, the indirect relationship between CC volume and autism severity suggests the
involvement of CC in core ASD symptoms.

Introduction
Autism Spectrum Disorders (ASD) are a heterogeneous group of
neurodevelopmental pathologies characterised by early-onset abnor-
malities in social communication and interaction, as well as atypi-
cally restricted and repetitive behaviours and interests (APA, 2013).
Despite the exact etiopathogenesis of idiopathic ASD is not fully
elucidated, recent evidences suggest an interaction between genetic
liability and environmental inuences in producing early alteration
Correspondence: A. Retico, as above.
E-mail: alessandra.retico@pi.infn.it
*These authors contributed equally to this work.
of brain development (Stamou et al., 2013). According to this view,
structural Magnetic Resonance Imaging (sMRI) studies of young
ASD patients have consistently reported abnormalities in total brain
volume as well as in grey and white matter (WM) volume of dis-
crete brain regions (see reviews by Amaral et al., 2008; Bellani
et al., 2013a; Calderoni et al., 2014). Specically, the abnormally
large brains frequently described in ASD toddlers (Courchesne
et al., 2003; Dementieva et al., 2005; Muratori et al., 2012) can
negatively impact on large-scale structural and functional cerebral
connectivity (Lewis & Elman, 2008). Among long-distance WM
structures, the corpus callosum (CC) is the largest commissural tract
in the human brain connecting the left and right hemispheres and it
is thought to be involved in the integration of neural information

2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
2 A. Giuliano et al.
across distant brain regions. For this crucial role, the CC has been
frequently implicated in the pathogenesis of ASD (for recent
reviews, see Frazier and Hardan, 2009; Bellani et al., 2013b),
because problems with processing of multiple source of sensory
information are common in ASD patients (Iarocci & McDonald,
2006), and are possibly sustained by the specic combination of a
local overconnectivity and a long-distance under-connectivity (Kana
et al., 2011). In keeping with this theory, the increased length of
CC bres was directly related to reductions in interhemispheric con-
nectivity and CC size in adults with ASD (Lewis et al., 2013).
Moreover, patients with complete or partial CC agenesis have
shown decits in pragmatic aspects of language processing (Paul
et al., 2003), impaired humour understanding (Brown et al., 2005),
difculty with social world comprehension and interaction (Syming-
ton et al., 2010),and ASD (Paul et al., 2014). Consequently, the CC
has been one of the most investigated WM tracts in ASD patients.
In particular, several neuroanatomic reports have analysed callosal
size in ASD subjects compared with matched controls, but dis-
crepant results were frequently detected. Despite reductions of the
CC volume is a replicated feature of subjects with ASD (Alexander
et al., 2007; Anderson et al., 2011; Duan et al., 2010; Frazier et al.,
2012; Hardan et al., 2009; Keary et al., 2009; McAlonan et al.,
2009; Thomas et al., 2011; Vidal et al., 2006; Waiter et al., 2005),
a number of studies reveal no CC volumetric differences between
ASD patients and controls (Bonilha et al., 2008; Cheng et al., 2011;
Hong et al., 2011; Ke et al., 2008; Mengotti et al., 2011; Toal et al.,
2010; Waiter et al., 2004). In addition, the site of CC reduction may
vary among reports, because in some cases, the whole CC is
involved (Anderson et al., 2011; Duan et al., 2010; Frazier et al.,
2012; Hanaie et al., 2014; Hardan et al., 2009; McAlonan et al.,
2009), whereas in others is limited to the anterior (Alexander et al.,
2007; Keary et al., 2009; Schaer et al., 2013; Thomas et al., 2011),
the posterior (Waiter et al., 2005), or sub-regions of both the ante-
rior and posterior CC (Vidal et al., 2006). These inconsistent nd-
ings may reect the relatively small sample sizes of each study, as
well as methodological differences in scan acquisition parameters,
and measurement techniques (e.g. manual delineation of region of
interest -ROI-, semi-automated tools such as FreeSurfer -Dale et al.,
1999-, or automated whole-brain method like voxel-based morphom-
etry -VBM- Ashburner and Friston, 2000). In an attempt to over-
come these limitations, large open-access ASD data repositories
(e.g. the Autism Brain Imaging Data Exchange -ABIDE-) have
already shared MRI dataset of children, adolescents and adults
previously collected across multiple sites, allowing to increase the
statistical power of results, and possibly to disentangle different sub-
groups of brain endophenotypes (Di Martino et al., 2014). Studies
from the ABIDE cohort on CC volume did not detect statistically
signicant differences between ASD patients and controls when
brain volume was added as a covariate (Lefebvre et al., 2015), or
demonstrated smaller CC volume limited to the central segment in
the ASD group compared with the control group (Haar et al., 2014).
Investigations on the large sample size of the ABIDE dataset have
certainly several advantages, but a possible drawback is the lack of
data on children under 6 years of age that hampers efforts to shed
light on neuroanatomical underpinnings during early stage of ASD.
In wider terms, data on CC volume in samples of pre-schoolers with
ASD (maximum mean age: 6 years) are scarce and difcult to com-
pare to each other because of the differences on sample selection
(age, gender, and clinical features of participants), and CC size
parameters analysed (volume vs. area). Among these studies, both
reduction (Boger-Megiddo et al., 2006; Nordahl et al., 2015) and
absence of differences in CC regions (Riva et al., 2011; Xiao et al.,
2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
2014) between ASD children and controls were detected. Only one
study reported an enlarged CC in infants who develop ASD within
the rst year of life, but differences were no longer apparent by
2 years of age (Wolff et al., 2015).
To explore the brain-behaviour relationship, the possible correla-
tion between CC volume and ASD clinical features has been investi-
gated in some studies. For instance, volume reduction in the CC of
ASD participants has been found to correlate with social decits,
repetitive behaviours and sensory abnormalities (Frazier et al.,
2012), executive function and complex motor tasks decits (Keary
et al., 2009), whereas an increase of CC area was associated with a
better functioning of ASD patients in terms of autism behaviours,
intelligence, and speed of processing (Prigge et al., 2013). However,
a paucity of data on samples all composed by pre-school children
with ASD is again noteworthy.
In this study, we compared the CC volume in pre-schoolers with
ASD and controls carefully matched for age, gender, and non-verbal
IQ (NVIQ) using FREESURFER v5.1 (http://surfer.nmr.mgh.harvard.ed
u/), a widely-used semi-automated brain-segmentation methodology.
By further subdividing the CC into ve anatomically discrete por-
tions (anterior, mid-anterior, central, mid-posterior and posterior
sub-regions, as shown in Fig. 1), we investigated the differences in
the volume for different CC regions and possible correlations with
demographic and clinical variables (gender, age, NVIQ, and expres-
sive non-echolalic language). Lastly, in the ASD group, we assessed
relationship between callosal volume and both core ASD features
and emotional/behavioural problems evaluated respectively through
the Autism Diagnostic Observation Schedule-Generic (ADOS-G;
Lord et al., 2000) and the Child Behaviour CheckList 15 (CBCL
15; Achenbach & Rescorla, 2000).
Methods
Participants
A group of twenty male and twenty female pre-schoolers with ASD
[mean age  SD = 49  12 months; age range = 2870 months]
and a group of 40 control subjects matched by gender, age, and
NVIQ were selected for this case-control study (see Table 1, where
demographic and main clinical features of subjects are summarized).
Fig. 1. Visualisation of the parcelled sub-regions of the corpus callosum
according to the Freesurfer suite. The overlay to a mid-sagittal slice of a
T1w MRI is obtained using 3DSlicer visualization software package (http://
www.slicer.org). L (left); Ant (anterior), Mid-Ant (middle anterior), Centr
(central), Mid-Post (middle posterior), Post (posterior) sub-regions of the cor-
pus callosum.
European Journal of Neuroscience, 111
 Corpus Callosum in ASD children 3

Table 1. Dataset composition and sample characteristics

Subject group, mean  SD (range)

Variable ASD (n = 40) Controls (n = 40)

Age (months) 49  12 (2870) 49  14 (2472)

NVIQ 73  23 (34113) 73  23 (31123)

Age (months) Males (n = 20) Females (n = 20) Males (n = 20) Females (n = 20)

Age (months) 50  10 (3470) 48  13 (2869] 48  13 (2470] 50  16 (2272]

NVIQ 75  23 (40113] 70  23 (34113] 73  23 (32123] 72  24 (31106]

ID (n = 10) no-ID (n = 10) ID (n = 10) no-ID (n = 10) ID (n = 10) no-ID (n = 10) ID (n = 10) no-ID (n = 10)

Age (months) 52  10 (3770) 48  10 (3466) 43  14 (2869) 54  10 (3669) 52  13 (2470) 45  13 (3065) 51  14 (3066) 50  18 (2272)
NVIQ 56  9 (40 70) 94  13 (81113) 51  10 (3465) 89  14 (73113) 54  11 (3268) 92  15 (74123) 52  13 (3168) 93  10 (73106)

ASD, autism spectrum disorders; ID, intellectual disability; NVIQ, non-verbal intelligence quotient; no-ID, without intellectual disability; SD, standard deviation.

Participants in the ASD group were recruited at the Autism Spec- chronological age, productive language skills and functioning level
trum Disorders Unit of IRCCS Stella Maris Foundation (Pisa), a ter- of subjects. These included the Leiter International Performance
tiary hospital and research university in Italy. They were rigorously Scale-Revised (Roid and Miller, 1997), the Grifths Mental Devel-
diagnosed according to the DSM-5 criteria (APA, 2013) by a multi- opment Scale (Grifths, 1984), the Italian version of Wechsler Pre-
disciplinary team including a senior child psychiatrist, an experi- school and Primary Scale of Intelligence (WPPSI, Wechsler, 1973).
enced clinical child psychologist and a speechlanguage pathologist When the tool provides a mental age (MA), IQ was estimated divid-
during 35 days of extensive evaluation, and conrmed by the ing MA by the childs chronological age (CA): [(MA/CA) 9 100].
ADOS-G (Lord et al., 2000) administrated by expert clinicians. To promote greater data homogeneity, we selected the non-verbal
ASD patients were included if their age was between 2 and 6 years IQ scores.
and their NVIQ 30. Exclusion criteria were: (i) anomalies detected The expressive language development of ASD patients and con-
by MRI (ii) neurological syndromes or focal neurological signs; (iii) trols was evaluated according to the ve levels identied by an
signicant sensory impairment (e.g., blindness, deafness); (iv) anam- authoritative consensus group about productive language abilities in
nesis of birth asphyxia, pre-mature birth, or epilepsy; (v) use of any pre-schoolers with ASD (Tager-Flusberg et al., 2009): level 1?Pre-
psychotropic medication; and (vi) potential secondary causes of verbal Communication; level 2?First Words; level 3?Word Com-
ASD determined by high-resolution karyotyping, DNA analysis of binations; level 4?Sentences; level 5?Complex Language. Thus,
Fragile-X, or screening tests for inborn errors of metabolism (plasma all subjects were assigned to one of the conditions above mentioned,
and urine aminoacid analysis, urine organic acid measurement, urine as reported in Table 2. It can be noticed that the language levels are
mucopolysaccarides quantitation, plasma and urine creatine and not equally distributed between individuals with ASD and controls,
guanidinoacetate analysis). with ASD subjects displaying lower language levels.
The control group was composed of 20 pre-schoolers with idio- Within the ASD group, an evaluation of autistic symptoms and of
pathic intellectual disability (ID), and 20 pre-schoolers without intel- emotional/behavioural problems was performed through the ADOS-
lectual disability (no-ID) recruited at the same hospital. Subjects G (Lord et al., 2000) and the CBCL (Achenbach & Rescorla, 2000)
with ID were included within the control group to obtain a match respectively. The ADOS-G is a semistructured, standardized assess-
for NVIQ between patients and controls (Crone et al., 2010), as well ment of social interaction, communication, play, and imaginative
as to increase the size of the data sample under investigation. The use of materials (Lord et al., 2000). Module 1 of the ADOS (de-
diagnosis of idiopathic ID was made after a negative thorough signed for pre-verbal children or children with only single words) or
assessment for underlying causes, including audiometry, thyroid hor- Module 2 (children with consistent phrase speech) was administered
mone disorders, high-resolution karyotyping, DNA analysis of Frag-
ile-X and screening tests for inborn errors of metabolism. The
control group was selected so as to meet the same exclusionary cri- Table 2. Number of subjects by language levels in the ASD and control
teria as the ASD patients -except the criterion (f) specied above groups
with the further requirements of exclusion of ASD diagnosis (per-
formed by a senior child psychiatrist and based on the DSM-5 crite- Number of subjects in each group
ria), and no family history of ASD.
ASD (n = 40) Controls (n = 40)
ASD and ID patients underwent the brain MRI examination as a
completion of the assessment pathway with the aim of excluding Language Males Females Males Females
brain alteration, whereas no-ID subjects performed brain MRI level (n = 20) (n = 20) (n = 20) (n = 20)
because of various reasons (including headache, strabismus, cataract,
seizures with fever, paroxysmal vertigo). Level 1 6 7 3 3
Level 2 4 8 3 6
Level 3 3 0 3 0
Clinical measures Level 4 7 4 6 5
Level 5 0 1 5 6
ASD patients and controls performed a standardized evaluation of
cognitive abilities through a variety of tests, depending on ASD, autism spectrum disorders.

2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, 111
4 A. Giuliano et al.
by trained researchers in this study. ADOS-G provides scores that
are distinct in three domains: Language and Communication-scores,
Reciprocal Social Interaction-scores and Total-scores. The CBCL
15 is a parent-report questionnaire that contains 100 problem
items rated on a 3-point scale: 0 (not true), 1 (somewhat or some-
times true) and 2 (very true or often true). The measure provides
scores for three summary scales (i.e., Internalizing, Externalizing
and Total Problems), seven syndrome scales (i.e., Emotionally Reac-
tive, Anxious/Depressed, Somatic Complaints, Withdrawn, Sleep
Problems, Attention Problems, and Aggressive Behaviour), and ve
DSM-Oriented scales (i.e., Affective Problems, Anxiety Problems,
Pervasive Developmental Problems, Attention Decit/Hyperactive
Problems and Oppositional Deant Problems). In particular, we
restricted our analysis to the CBCL 15 scales that have been
found to be altered in pre-schoolers with ASD compared with both
typically developing controls and peers with other psychiatric disor-
ders (Muratori et al., 2011; Narzisi et al., 2013), i.e. the three sum-
mary scales (Internalizing, Externalizing and Total Problems), two
syndrome scales (Withdrawn and Attention Problems), and the
DSM-Oriented Pervasive Developmental Problems scale.
MRI data acquisition and processing
Structural images of the brain were acquired on a 1.5 T MR system
(Signa Horizon LX, General Electric Medical System) tted with 40
mT/m high-speed gradients. Within the MR protocol for children a
whole-brain fast spoiled gradient recalled acquisition in the steady-
state T1-weighted (T1w) series (FSPGR) were collected in the axial
plane with repetition time 12.4 ms, echo time 2.4 ms, inversion time
700 ms, ip angle of 10, yielding 124 contiguous 1.1 mm axial
slices with an in-plane resolution of 1.1 9 1.1 mm2. Both ASD and
control subjects were sedated with a general anaesthesia with a halo-
genated agent while spontaneously breathing, according to a strict
clinical protocol approved by the institutional review board of the
IRCCS Stella Maris Foundation and performed by a paediatric
anaesthesiologist. The written informed consent from a parent or
guardian of children was obtained.
Brain T1w MRI images were processed and volumetric segmenta-
tion was performed with the semi-automated parcellation approach
implemented in the Freesurfer image analysis suite.
FREESURFER software is well-documented and freely available for
download. The technical details of these procedures are described in
the publication by Fischl (2012), and references therein. Briey, this
processing includes motion correction, removal of non-brain tissue
using a hybrid watershed/surface deformation procedure, automated
Talairach transformation, segmentation of the subcortical white matter
and deep grey matter volumetric structures, intensity normalization,
tessellation of the grey matter white matter boundary, automated
topology correction and surface deformation following intensity gradi-
ents to optimally place the grey/white and grey/cerebrospinal uid
borders at the location where the greatest shift in intensity denes the
transition to the other tissue class. The maps are created using spatial
intensity gradients across tissue classes and are therefore not simply
reliant on absolute signal intensity. The maps produced are not
restricted to the voxel resolution of the original data thus are capable
of detecting submillimetre differences between groups.
We implemented the Freesurfer pre-processing steps on each sub-
ject of the sample, and included manual correction when needed
(e.g. registration, skull stripping and/or white matter editing). The
CC was thus automatically identied and segmented for each sub-
ject. Moreover, through the script mri_cc, Freesurfer automatically
divides the CC into ve segments along its main axis (eigenaxis),
2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
thus ve callosal sub-regions were identied (see Fig. 1): CC-Ante-
rior (CC-Ant), CC-Mid Anterior (CC-Mid-Ant), CC-Central (CC-
Cen), CC-Mid Posterior (CC-Mid-Post) and CC-Posterior (CC-Post).
Total CC volume was calculated as the sum of these ve segment
volumes for each study participant.
Supratentorial total brain volume (TBV) was estimated using
Freesurfer, and includes everything except the cerebellum and the
brain stem. In particular, it includes the volume of the ventricles,
choroid plexus, and vessels also.
Inter-rater reliability
As already stated, once MRI images were parcelled according to the
Freesurfer procedure they were visually assessed by one rater and,
when required, a manual correction was made in the pre-processing
phase. We specically tested the inter-rater reliability in carrying out
this manual step of the image processing. Two raters evaluated the
image segmentation quality. The inter-rater reliability was evaluated
on a representative sub-group of 10 subjects (equally distributed
among ASD and controls, six males and four females) assessed by
all raters. The choice of limiting this analysis to 10 subjects, which
are representative of the whole sample, was exclusively dictated by
practical reasons, as the manual adjustments of Freesurfer segmenta-
tions requires about 34 h per case. The raters were blind to diag-
noses and clinical-demographic features of subjects. Inter-rater
reliability statistical analysis was made on the volumes of the brain
parcels reported in the les aseg.stats generated by Freesurfer. A
paired two-sample t-test was carried out for each volume, consider-
ing P < 0.05 as signicance level.
Statistical analysis of CC volumes
The statistical examination of the CC volumes was performed using
the analysis of variance (ANOVA) test. In this case, the test statistic
follows the Fisher-Snedecor distribution (F-distribution) under the
null hypothesis of equal means between the groups, which variables
are assumed to be normally distributed and to have the same stan-
dard deviation. The probability (P-value) of observing a statistical
value of F greater than that observed was evaluated, considering
P < 0.05 as signicance level to reject the null hypothesis.
The statistical analyses were univariate because each test was per-
formed on a single variable compared between the groups. When
the ANOVA tests were applied on correlated variables, such as the
volumes of CC sub-regions and ADOS-G sub-scores, the signi-
cance level was Bonferroni corrected to take into account the prob-
lem of multiple comparisons.
To test for linear relationships between two variables we used the
Pearsons correlation index.
Results
Inter-rater reliability
The inter-rater reliability test, assessed with a paired-two-sample t-
test on the volumes of the 66 ROIs of 10 subjects, showed a good
degree of concordance among raters. Particularly, no signicant
inter-rater disagreement is detected on CC volumes (CC-Posterior,
P = 0.83; CC-Mid-Posterior, P = 0.27; CC-Central, P = 0.47; CC-
Mid-Anterior, P = 0.19; CC-Anterior, P = 0.16). Once the inter-
rater reliability was demonstrated on the representative sub-group of
10 subjects, the rest of the sample was randomly partitioned
between the two raters and manually corrected by only one of them.
European Journal of Neuroscience, 111
 Corpus Callosum in ASD children 5

Table 3. Total brain volume comparison between ASD and controls (*: sig- Table 4. : Corpus callosum total volume comparison between ASD and
nicance level P < 0.05), accounting for age and gender, where appropriate controls (*: signicance level P < 0.05), accounting for total brain volume
and gender, where appropriate
Males and Females
Males and Females
ANOVA
Subject group, mean  SD (covariates: gender, age) ANOVA
Subject group, mean  SD (covariates: gender, TBV)
Variable ASD (n = 40) Controls (n = 40) F P
Variable ASD (n = 40) Controls (n = 40) F P
TBV (l) 1.08  0.12 1.01  0.13 6.9 0.010*
CC (mL) 2.9  0.6 2.5  0.6 1.28 0.26
Males
Males
ANOVA
Subject group, mean  SD (covariate: age) ANOVA
Subject group, mean  SD (covariate: TBV)
Variable ASD (n = 20) Controls (n = 20) F P
Variable ASD (n = 20) Controls (n = 20) F P
TBV (l) 1.15  0.11 1.05  0.14 6.6 0.014*
CC (mL) 2.9  0.5 2.5  0.6 1.03 0.32
Females
Females
ANOVA
Subject group, mean  SD (covariate: age) ANOVA
Subject group, mean  SD (covariate: TBV)
Variable ASD (n = 20) Controls (n = 20) F P
Variable ASD (n = 20) Controls (n = 20) F P
TBV (l) 1.02  0.10 0.98  0.12 1.1 0.3
CC (mL) 2.8  0.6 2.6  0.6 0.4 0.53
ASD, autism spectrum disorders; SD, standard deviation; TBV, total brain
volume. ASD, autism spectrum disorders; CC, corpus callosum total volume; SD,
standard deviation; TBV, total brain volume.

Total Brain volume

The results of the comparison between the TBV values in the ASD
and control subjects, and in the gender-specic sub-groups, are sum-
marized in Table 3. TBV is higher in ASD when compared with the
controls, when age and gender were considered as covariates
(P = 0.01). This difference is driven mainly by male subjects, where
TBV is found signicantly higher in ASD compared with control
subjects (P = 0.014), when age is considered as covariate; by con-
trast, TBV does not show signicant differences between female
subjects with ASD and matched control subjects (P = 0.3).
Corpus callosum total volume
The results of the comparison between the CC total volumes of the
ASD and control subjects, and in the gender-specic sub-groups, are
summarized in Table 4. The total volume of the CC does not signif-
icantly differ between ASD and controls, when TBV or TBV and
gender are appropriately considered as covariates. This result is
obtained in the whole dataset analysis and preserved both in the
male and in the female sub-groups.
Sub-regions corpus callosum volumes
The results of the comparison between the CC sub-regional volumes
of the ASD and control subjects, and in the gender-specic sub-
groups, are summarized in Table 5. The sub-regional volumes of the
CC tend to be larger in ASD when compared with controls. How-
ever, when appropriate covariates are considered (e.g. TBV and gen-
der), the volumes of CC sub-regions do not differ signicantly
between groups. Figure 1 shows a visualization of the CC sub-
regions segmented by Freesurfer, overlaid to a mid-sagittal slice of a
T1w MRI using 3DSlicer visualization software package (http://
www.slicer.org).
Gender effect on corpus callosum volume
In the analysis of the volumes of CC and its sub-regions, we consid-
ered both the entire sample of subjects and the gender-specic sub-
populations. No signicant gender-specic differences were found in
the ASD vs. control comparison of volumes of the CC and its sub-
regions, as reported in Tables 4 and 5.
Age effect on corpus callosum volume
The CC total volume positively correlates with age in both ASD
(P = 0.046, r = 0.32) and control groups (P = 0.009, r = 0.41). As
the signicance of the correlations is sensibly higher in the CON with
respect to the ASD sample, to better explore the age effect we anal-
ysed the CC total volume differences in two sub-groups of: children
younger than 49 months (named Group 1: age 49 months,
N = 41); children older than 49 months (named Group 2:
age > 49 months, N = 39). The matching for age between ASD and
CON was assessed by performing a two-sample t-test both in younger
and older children sub-groups, and considering P < 0.05 as signi-
cance level to reject the null hypothesis (Group1: P = 0.34 for males
and females, P = 0.33 for males, P = 0.65 for females; Group 2:
P = 0.41 for males and females, P = 0.61 for males, P = 0.67 for
females). Table 6 summarizes the results obtained for the CC total
volume on Group 1 and Group 2. Both in Group 1 and in Group 2 no
signicant differences were found in the analysis of the CC total vol-
ume when ASD are compared with controls and TBV and gender
(where appropriate) were used as covariates (Group 1: P = 0.3; Group
2: P = 0.47). A signicant difference emerged between younger male
children: CC total volume is signicantly larger in the ASD group
compared with controls (P = 0.011), when TBV is used as covariate.
In older male children, no signicant difference is detected

2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, 111
6 A. Giuliano et al.

Table 5. Volumes of CC sub-regions compared between ASD and controls (P = 0.026, r = 0.36, signicance threshold P < 0.05). As shown
(signicance level P < 0.01, with Bonferroni correction), accounting for in Table 7, all ADOS-G scores showed a negative trend correlation
TBV and gender, where appropriate
with the CC total volume, both in the male and in the female sub-
Males and Females
groups. Negative correlation trends were found for Language and
Communication-scores and for Reciprocal social interaction-scores
ANOVA both in male and female sub-groups, but they did not remain signi-
(covariates: cant after the Bonferroni correction (when the signicance threshold
Subject group, mean  SD gender, TBV) becomes P < 0.025). The signicant correlation of the CC total vol-
ume and the ADOS-G Total scores is shown in Fig. 2.
Variable ASD (n = 40) Controls (n = 40) F P

CC-Ant (mL) 0.81  0.20 0.72  0.15 5.4 0.022 Child Behaviour Checklist for ages 1.55 correlation
CC-Mid-Ant (mL) 0.54  0.17 0.60  0.20 1.2 0.2
CC-Cen (mL) 0.43  0.11 0.37  0.12 5.4 0.023 The CC total volume does not show signicant correlation with
CC-Mid-Post (mL) 0.37  0.11 0.32  0.10 4.9 0.03 CBCL 15 scores, in none of the analysed scales: Internalizing
CC-Post (mL) 0.71  0.12 0.64  0.14 5.5 0.022
Problems (P = 0.74), Externalizing Problems (P = 0.87), Total
Males Problems (P = 0.99), Withdrawn (P = 0.80), Attention Problems
(P = 0.40), and DSM-oriented Pervasive Development Problems
ANOVA (P = 0.57), as shown in Table 8.
Subject group, mean  SD (covariate: TBV)

Variable ASD (n = 20) Controls (n = 20) F P Discussion

CC-Ant (mL) 0.79  0.19 0.76  0.13 0.19 0.7 In this study we used a cross-sectional design to examine the vol-
CC-Mid-Ant (mL) 0.58  0.16 0.45  0.18 1.8 0.18 ume of CC in an equal number of male and female pre-schoolers
CC-Cen (mL) 0.46  0.10 0.36  0.12 2.9 0.10 with ASD compared with sex, age, and non-verbal IQ matched con-
CC-Mid-Post (mL) 0.41  0.09 0.31  0.10 4.5 0.04
trols. In addition, we explored whether CC volume is related to cer-
CC-Post (mL) 0.71  0.09 0.65  0.15 0 1
tain demographic and clinical features of the ASD sample. We
Females observed: (i) no group differences in total and sub-region CC vol-
umes; (ii) an increased CC volume in the sub-sample of the younger
ANOVA
Subject group, mean  SD (covariate: TBV)
ASD males compared with age and gender-matched controls; (iii) a
negative correlation between total CC volume and autistic symptoms
Variable ASD (n = 20) Controls (n = 20) F P in the patients group; and (ic) the absence of statistically signicant
association between CC volume and other variables (NVIQ, lan-
CC-Ant (mL) 0.83  0.20 0.67  0.16 5.3 0.027 guage, emotional and behavioural problems).
CC-Mid-Ant (mL) 0.50  0.17 0.54  0.21 1.2 0.2
CC-Cen (mL) 0.40  0.11 0.39  0.11 0 1
CC-Mid-Post (mL) 0.34  0.12 0.33  0.11 0.24 0.6 Preserved CC volume
CC-Post (mL) 0.71  0.14 0.63  0.14 2.0 0.16
Results suggested that, when total brain volume isaccounted for,
ASD, autism spectrum disorders; TBV, total brain volume; SD, standard ASD patients and controls did not signicantly differ in overall CC
deviation; Ant, anterior; Mid-Ant, middle anterior; Cen, central; Mid-Post,
volume. This nding is in agreement with some (Riva et al., 2011;
middle posterior; Post, posterior.
Xiao et al., 2014), but not all reports (Boger-Megiddo et al., 2006;
Nordahl et al., 2015) on young ASD subjects of similar age. In fact,
(P = 0.33). In the female set no differences were found in Group 1 in neuroanatomical investigations on ASD patients, it is crucial to
(P = 0.45) and Group 2 (P = 0.061). compare samples by age to obtain more reliable and consistent
results (Lin et al., 2015). Interestingly, when area was used as mea-
sure parameter (Boger-Megiddo et al., 2006; Nordahl et al., 2015) a
Non-verbal IQ correlation
reduction in CC region was detected, whereas studies using volume
The CC total volume does not correlate with NVIQ neither in the as measurement value (Riva et al., 2011; Xiao et al., 2014) found
ASD (P = 0.7, r = 0.06) nor in the control group (P = 0.6, no CC differences between ASD patients and controls. It is possible
r = 0.08). Moreover, there were no signicant correlations in the that the peculiar shape of CC in ASD individuals accounts for these
male and female sub-groups. apparently discrepant results: according to this view, Duan et al.
(2010) reported a signicant reduction in the callosal length of ASD
subjects suggesting that the decrease found in CC size, especially in
Language correlation
studies using mid-sagittal area, could be attributable to the decrease
The CC total volume does not correlate signicantly with the expres- in the anterior-posterior length, rather than to a homogeneous three-
sive language level neither in the ASD group (P = 0.97, r = 0.006) dimensional reduction. We detected an absence of signicant differ-
nor in the control group (P = 0.33, r = 0.16). Furthermore, no signi- ences in CC sub-region volumes between patients with ASD and
cant correlations were found in the gender-specic sub-groups. controls. Previous studies comprising pre-schoolers did not observe
any differences in the size of CC subdivisions (Nordahl et al.,
2015), or described a reduction in each sub-region of the CC (Duan
Autism Diagnostic Observation Schedule correlation
et al., 2010) in the isthmus (Prigge et al., 2013), in the rostrum and
The CC total volume has a signicant negative correlation with rostral body (Boger-Megiddo et al., 2006). However, a comparison
ADOS-G Total scores in the whole sample of ASD individuals to previous investigations is not possible, because in our study, CC

2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, 111
 Corpus Callosum in ASD children 7

Table 6. Group 1 (age 49 months) and group 2 (age > 49 months) corpus callosum total volume analysis results (*: signicance level P < 0.05), accounting
for TBV and gender, where appropriate

Group 1 (age 49 months; Group 2 (age > 49 months;

n = 41) n = 39)

Males and Females

Subject group, ANOVA (covariates: Subject group, ANOVA (covariates:

mean  SD gender, TBV) mean  std gender, TBV)

ASD (n = 21) Controls (n = 20) F P ASD (n = 19) Controls (n = 20) F P

CC (mL) 2.8  0.6 2.4  0.5 1.1 0.3 2.9  0.5 2.7  0.6 0.53 0.47

Males

Subject group, ANOVA (covariate: Subject group, ANOVA (covariate:

mean  SD TBV) mean  std TBV)

Variable ASD (n = 9) Controls (n = 10) F P ASD (n = 11) Controls (n = 10) F P

CC (mL) 3.2  0.5 2.4  0.3 8 0.011* 2.8  0.5 2.7  0.8 1.01 0.33
Females

Subject group, ANOVA (covariate: Subject group, ANOVA (covariate:

mean  SD TBV) mean  std TBV)

Variable ASD (n = 12) Controls (n = 10) F P ASD (n = 8) Controls (n = 10) F P

CC (mL) 2.5  0.6 2.4  0.7 0.6 0.45 3.1  0.6 2.7  0.4 4.1 0.061

ASD, autism spectrum disorders; CC, corpus callosum total volume; SD, standard deviation; TBV, total brain volume.

Table 7. Correlation of ADOS-G scores with CC total volumes (*: signi-

cance level P < 0.05 for Total scores; P < 0.025 for Language and Commu-
nication-scores and Reciprocal Social Interaction-scores, accounting for
Bonferroni correction)

Correlation analysis: CC and ADOS-G

Pearsons index P

Males and Females

ADOS-G Lang-Com 0.35 0.027
ADOS-G Soc 0.33 0.042
ADOS-G Tot 0.36 0.026*
Males
ADOS-G Lang-Com 0.19 0.42
ADOS-G Soc 0.22 0.35
ADOS-G Tot 0.23 0.32
Females
ADOS-G Lang-Com 0.46 0.048
ADOS-G Soc 0.37 0.11 Fig. 2. Correlation of the CC total volume and ADOS-G Total score in the
ADOS-G Tot 0.42 0.08 entire group of ASD subjects; subjects are represented by different colours
according to gender; a line is plotted for signicant correlation (P < 0.05).
Lang-Com, Language and Communication; Soc, Reciprocal Social Interac- ASD-M, ASD male subjects; ASD-F, ASD female subjects.
tion; Soc, Reciprocal Social Interaction Tot, Total; Tot, Total/Lang-Com,
Language and Communication.
Age effect
Crucially, when our data were separated according to median age
(49 months and >49 months), it became evident that the absence
segmentation was automatically performed with the Freesurfer soft- of CC volume differences between patients and controls was driven
ware which subdivides CC in ve regions on the basis of equally by the older ASD subjects, as younger male individuals with ASD
distant segments along its main axis, whereas the other studies used displayed a statistically signicant increase of CC volume compared
Witelson (1989) subdivisions (Boger-Megiddo et al., 2006, Nordahl with sex and age-matched controls. This result suggests an atypical
et al., 2015, Prigge et al., 2013), or a home-made method (Duan CC growth trajectory in subjects with ASD, characterised by a
et al., 2010). greater development in early ages, followed by a slower rate of

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European Journal of Neuroscience, 111
8 A. Giuliano et al.

Table 8. CBCL 15 scores correlations with Corpus callosum total vol- et al., 2015; Postorino et al., 2015), whereas others showed better per-
umes (*: signicance level P < 0.05) formance of young males on communicative and ne-motor skills,
and better visual receptive abilities in young females (Carter et al.,
Correlation analysis: CC and CBCL 15
2007; Hartley and Sikora, 2009). In addition, restricted, repetitive,
Males and Females Males Females stereotyped behaviour are more common in male toddlers than in
female toddlers (Hartley and Sikora, 2009; Sipes et al., 2011), and
Pearsons Pearsons Pearsons young females exhibited more comorbid psychopathology relative to
index P index P index P matched males (Hartley and Sikora, 2009). Whether and how these
supposed behavioural differences between males and females with
Int 0.05 0.74 0.21 0.39 0.16 0.36 ASD are mirrored at a neuroanatomical level remains a matter of
Ext 0.02 0.87 0.10 0.70 0.01 0.93
Tot 0.0001 0.99 0.11 0.67 0.03 0.86 hypothesis, which requires further exploration. In fact, only few stud-
Withd 0.04 0.80 0.52 0.02* 0.15 0.37 ies have examined this issue in ASD pre-schoolers (Bloss and
Atten 0.11 0.40 0.07 0.76 0.22 0.19 Courchesne, 2007; Nordahl et al., 2011; Retico et al., 2016; Schu-
DSM-PDP 0.08 0.57 0.26 0.29 0.25 0.14 mann et al., 2009 and Schumann et al., 2010; Sparks et al., 2002),
with various and often unreplicated cerebral differences between the
Atten, Attention problems scale; CC, corpus callosum total volume; DSM-
PDP, DSM-oriented Pervasive developmental problems scale; Ext, External- sexes. Only one study of CC sex differences in pre-schoolers with
izing problems scale; Int, Internalizing problems scale; Tot, Total problems ASD was conducted to date (Nordahl et al., 2015): results revealed a
scale; Withd, Withdrawn scale. reduction in total area of the CC in both males and females with ASD
relative to their TD counterparts. Moreover, the analysis of bres pro-
jecting to cortical regions exhibited sex differences, with smaller CC
growth in subsequent ages, resulting in smaller CC in adolescent bres projecting to the anterior frontal cortex in ASD girls relative to
and adult patients (Bellani et al., 2013b). An increase in CC volume boys and smaller bres projecting to the orbitofrontal cortex in ASD
at 6 and 12 months of age in infants who later develop ASD, rela- boys relative to girls.
tive to TD infants, corroborates this hypothesis (Wolff et al., 2015).
Moreover, reduction in CC size was found in a meta-analysis
involving subjects with a mean age of approximately 14 years, and Clinical correlations
was more prominent with increasing age (Frazier and Hardan, Exploratory analyses within the ASD group revealed an inverse cor-
2009). The atypical growth pattern of CC in ASD children is further relation between CC volume and ADOS-G (Lord et al., 2000) total
supported by the different signicance levels found in the correla- scores, suggesting a relationship between the CC volume reduction
tions between CC volume and age. Specically, while a strong posi- and severity of ASD symptoms. This nding is consistent with pre-
tive correlation between CC volume and age was detected in the vious studies reporting a smaller CC volume in ASD pre-schoolers
control group, the correlation is barely signicant in pre-schoolers (Boger-Megiddo et al., 2006; Prigge et al., 2013) and children (Har-
with ASD, suggesting a slow of CC growth in these subjects. The dan et al., 2009) with more severe clinical features. We can specu-
CC neuroanatomical differences found in male pre-school children late that the CC growth trajectory could be different on the basis of
with ASD, but not in female pre-school children with ASD, com- ASD severity: specically, more severely affected children may
pared with controls, are in line with the sexual dimorphism in the have an earlier reduction of CC size, whereas in children with a
brain, identied both in typical (Gilmore et al., 2007), and ASD mild phenotype it begins afterwards. However, this nding requires
young individuals (Retico et al., 2016). However, it is important to conrmation on a broader sample, and furthermore, histological
underline that the cross-sectional nature of this investigation requires studies are warranted to clarify the cellular and molecular underpin-
caution on the interpretation of age-related phenomena, as they do nings of the possible differences in CC volumes between sub-groups
not derive from intra-individual measures of anatomical change. Evi- of patients with ASD. It is worth noting that the volume of CC is
dence of an early atypical CC maturation are conrmed by longitu- not inversely correlated with the DSM-oriented Pervasive Develop-
dinal studies which detected alterations in the trajectory of CC ment Problems scale of the CBCL 1.55, another possible measure
development as early as the rst year of life (Wolff et al., 2015), of ASD severity (Fulceri et al., 2016). The lack of coherence
and no longer present between 3 and 5 years of age (Nordahl et al., between the CBCL and the ADOS can be partly explained by the
2015). Thus, during the rst years of life, the altered CC growth tra- different types of raters required for these two instruments, as well
jectory in ASD individuals seems to reect the total brain volume as by the different items investigated. Specically, the CBCL 1.55
growth trajectory of ASD patients, characterised by an early over- is a quick (it takes 510 min) caregiver questionnaire for a broad
growth with larger brain volumes and a subsequent slowed brain range of behavioural and emotional problems with a scale measuring
growth compared with age-matched TD controls (Redcay & Courch- behaviours that are suggestive of an ASD risk. Conversely, the
esne, 2005). ADOS is the gold-standard observational diagnostic instrument for
ASD, consisting of a semistructured direct observation of the child
Sex differences by a trained clinician designed to assess autism-related behaviours
and it takes about 3045 min to administer.
Among ASD subjects, no signicant sex difference emerged in the In our sample, no signicant correlation was found between CC
volumes both of the entire CC and of its sub-regions, when TBV and volume and non-verbal IQ, in either the ASD or control groups. Pre-
age were considered. Analogously, among control group, no signi- vious reports in ASD patients have suggested that a reduced CC size
cant sex-related difference is found in the volumes of the entire CC or may be linked to a lower IQ (Freitag et al., 2009; Verhoeven et al.,
its sub-regions. Studies on sex differences in phenotypic presentation 2010; Prigge et al., 2013). Moreover, Alexander et al. (2007)
of young children with ASD provided contradictory results. Speci- described a specic sub-group of autistic patients, characterised by
cally, some investigations failed to detect sex difference in the adap- smaller CC volume, reduced WM integrity, poorer intellectual abili-
tive and behavioural functioning of toddlers with ASD (Reinhardt ties and slower processing speed. Callosal sizes were related to IQ

2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, 111

in typical children also: specically, a negative correlation between
callosal thickness and IQ was detected in the paediatric population
(Luders et al., 2014) whereas a positive correlation was reported in
adult samples (Chiang et al., 2009), suggesting again that the rela-
tionship between callosal morphology and clinical features changes
during brain maturation.
Moreover, no statistically signicant relationship was found
between expressive verbal skills and CC volume both in subjects
with ASD and in controls. Despite no other study to date aimed to
assess the correlation between verbal abilities and callosal size, this
topic requires further investigation. In fact, a recent work on TD
young children demonstrated that the anatomical substrates that sup-
port the acquisition of spoken language comprise the splenium of
CC (Swanson et al., 2014).
Conclusions
We conducted a structural assessment of regional volumes of the
CC in a relatively large sample of pre-schoolers with ASD and
carefully matched controls using structural MRI. We identied an
absence of CC volumetric differences in patients with ASD com-
pared with controls. The ASD sub-group of younger males
showed an increased CC volume, whereas smaller CC volume is
associated with a more severe phenotype in ASD patients. These
ndings support the hypothesis of impaired interhemispheric con-
nectivity in at least part of patients with ASD. Future longitudinal
studies that combine multimodal imaging techniques (e.g. struc-
tural MRI, diffusion weighted imaging, resting-state functional
MRI), coupled with genetic and molecular testing may help to
elucidate CC neuroanatomical underpinnings, dene ASD biomark-
ers, and ultimately advance treatment for both boys and girls with
an ASD diagnosis.
Data accessibility
The authors state that the data analysed in this study are archived
in an appropriate repository accessible to the Institutes participat-
ing to this research. They take the responsibility for the integrity
of the data and their preservation. These data will be made avail-
able on request to other researcher interested in additional analy-
sis under specic research agreement to be dened by the
partners.
Conflict of interest
The authors declare that the research was conducted in the absence
of any commercial or nancial relationships that could be construed
as a potential conict of interest.
Funding
This work was partially supported by grant from the IRCCS Stella
Maris Foundation (Ricerca Corrente, and the 591000 voluntary
contributions, Italian Ministry of Health), by the Italian Ministry of
Health and by Tuscany Region with the grant GR-2010-2317873,
and by Bando FAS Salute Sviluppo Toscana (ARIANNA Project
C52I16000020002).
Acknowledgements
The authors are grateful to Dr. Elisa Veronese for assistance in the setup of
the pre-processing pipeline.
2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
European Journal of Neuroscience, 111
Corpus Callosum in ASD children 9
Author contributions
SC and FM designed the study; IS and SC collected the clinical data;
AG, IS and AR carried out data processing and analysis; SC, AR, PB,
and FM interpreted the results; AG, AR, IS, and SC drafted the paper;
PB and FM critically revised the paper.
Abbreviations
ADOS-G, autism diagnostic observation schedule-generic; ANOVA, analysis of
variance; ASD, autism spectrum disorder; CBCL, the Child Behaviour
CheckList; CC-Ant, CC-Anterior; CC-Cen, CC-Central; CC, Corpus Callosum;
CC-Mid-Ant, CC-Mid Anterior; CC-Mid-Post, CC-Mid-Posterior; CC-Post,
CC-Posterior; CON, control subjects; ID, intellectual disability; IQ, intelligence
quotient; MA, mental age; MRI, magnetic resonance imaging; noID, without
intellectual disability; NVIQ, non-verbal IQ; TBV, total brain volume; WM,
white matter.
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