NeuroImage 236 (2021) 118067

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NeuroImage
journal homepage: www.elsevier.com/locate/neuroimage

A 16-year study of longitudinal volumetric brain development in males

with autism
Molly B.D. Prigge a,∗, Nicholas Lange b, Erin D. Bigler c,d,e,f, Jace B. King a, Douglas C. Dean III g,h,i,
Nagesh Adluru g, Andrew L. Alexander g,i,j, Janet E. Lainhart g,j, Brandon A. Zielinski a,d,k
a
Department of Radiology and Imaging Sciences, University of Utah, Salt Lake City, UT, USA
b
Department of Psychiatry, Harvard School of Medicine, Boston, MA, USA
c
Department of Psychology and Neuroscience Center, Brigham Young University, Provo, UT, USA
d
Department of Neurology, University of Utah, Salt Lake City, UT USA
e
Department of Psychiatry, University of Utah, Salt Lake City, UT USA
f
Department of Neurology, University of California-Davis, Davis, CA USA
g
Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
h
Department of Pediatrics, University of Wisconsin-Madison, Madison, WI, USA
i
Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
j
Department of Psychiatry, University of Wisconsin-Madison, Madison, WI, USA
k
Department of Pediatrics, University of Utah, Salt Lake City, UT, USA

a r t i c l e i n f o a b s t r a c t

Keywords: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unknown brain etiology. Our knowledge
Autism spectrum disorder to date about structural brain development across the lifespan in ASD comes mainly from cross-sectional studies,
Longitudinal development thereby limiting our understanding of true age effects within individuals with the disorder that can only be
MRI
gained through longitudinal research. The present study describes FreeSurfer-derived volumetric findings from a
Brain volumes
longitudinal dataset consisting of 607 T1-weighted magnetic resonance imaging (MRI) scans collected from 105
Ventricles
Corpus callosum male individuals with ASD (349 MRIs) and 125 typically developing male controls (258 MRIs). Participants were
six to forty-five years of age at their first scan, and were scanned up to 5 times over a period of 16 years (average
inter-scan interval of 3.7 years). Atypical age-related volumetric trajectories in ASD included enlarged gray matter
volume in early childhood that approached levels of the control group by late childhood, an age-related increase
in ventricle volume resulting in enlarged ventricles by early adulthood and reduced corpus callosum age-related
volumetric increase resulting in smaller corpus callosum volume in adulthood. Larger corpus callosum volume
was related to a lower (better) ADOS score at the most recent study visit for the participants with ASD. These
longitudinal findings expand our knowledge of volumetric brain-based abnormalities in males with ASD, and
highlight the need to continue to examine brain structure across the lifespan and well into adulthood.

1. Introduction normalities early in life. As early as 6 months of age, increased extra-

Autism spectrum disorder (ASD) is a lifetime disorder with un-
known neurobiological etiology and poorly characterized lifelong clin-
ical course in most cases. Despite earlier diagnosis and interventions,
many individuals remain functionally impaired throughout their lives
(Howlin et al., 2013). In order to understand the etiology of ASD
and the mechanisms involved and design treatments and therapies
that will improve outcome in children, adolescents and adults, a fur-
ther understanding of brain changes in ASD throughout the lifespan is
needed.
There are a number of research groups focusing on longitudinal
brain development in ASD during infancy and childhood that show ab-
axial CSF is present and remains elevated into early childhood in those
who later develop ASD (Shen et al., 2017; Shen et al., 2013). Longi-
tudinal and cross-sectional studies also show enlarged brain volumes
by 2 years of age (Hazlett et al., 2017; Hazlett et al., 2011) although
this may be attributed to subgroups of children within the disorder
(Nordahl et al., 2011; Ohta et al., 2016). A small (n=18 ASD) longi-
tudinal study reported atypical decreases in gray matter volume during
childhood (Hardan et al., 2009), however, the majority of our knowl-
edge about brain development during childhood and adolescence in ASD
is inferred from cross-sectional research. As a result, some early child-
hood studies report increased brain volumes in ASD (Courchesne et al.,
2003; Lucibello et al., 2019; Sparks et al., 2002) that may normalize

∗
Corresponding author at: Department of Radiology and Imaging Sciences, University of Utah, 729 Arapeen Drive, Salt Lake City, UT 84108, USA.
E-mail address: molly.prigge@hsc.utah.edu (M.B.D. Prigge).

https://doi.org/10.1016/j.neuroimage.2021.118067.
Received 1 November 2020; Received in revised form 24 March 2021; Accepted 12 April 2021
Available online 18 April 2021.
1053-8119/© 2021 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
M.B.D. Prigge, N. Lange, E.D. Bigler et al.
by later childhood (Courchesne et al., 2001) or decrease below typical
samples (Herbert et al., 2003; McAlonan et al., 2005); while studies in
adolescence report enlarged gray matter volumes in ASD (Freitag et al.,
2009; Hazlett et al., 2006). A recent publication of a large cross-sectional
sample (n=456) of ASD and typically developing control (TDC) par-
ticipants age 6-25 years found persistently increased global brain vol-
umes (total, gray matter, white matter, ventricles) in ASD, suggesting
that early brain overgrowth does not normalize and remains larger into
adulthood (Yankowitz et al., 2020). This contrasts with other cross-
sectional studies from adolescence into adulthood showing no atypi-
cal global brain structure in ASD (Aylward et al., 2002; Hallahan et al.,
2009; Lin et al., 2015; Maier et al., 2018), regionally decreased volumes
(Ecker et al., 2012; Toal et al., 2010) or absence of regional gray matter
decrease found in a typical comparison group (Raznahan et al., 2010).
The lack of a clear volumetric brain development trajectory in ASD
could be due to a number of factors. Discrepant volumetric findings can
result from different image processing methods (Katuwal et al., 2016),
error introduced by combining datasets collected from different research
sites and scanner hardware (Martinez-Murcia et al., 2017), between-
site heterogeneity in ASD and TDC samples, unknown sex differences in
brain morphology (Bedford et al., 2020), unknown details about change
in clinical course over time, and differences in analytic methods em-
ployed. Cross-sectional studies infer brain changes from age-related dif-
ferences across individuals. Longitudinal studies, although challenging
to conduct as they require collecting multiple time points of data and
retaining participants, often over years, directly measure change within
individuals and can more accurately describe how the brain changes
during development, maturation, and aging (Farrington, 1991). Longi-
tudinal studies also remove additional sources of variance in the data
due to a consistent site and equipment and allow the ability to track
individual differences in clinical course over time that may be related
to brain changes.
In this paper, we describe volumetric findings from the Interdisci-
plinary Science to Learn about Autism (ISLA) project with all data col-
lected at the University of Utah. This longitudinal study of late brain
development in autism began in 2003 and is currently in its 6th wave of
data collection. Participant ages ranged from childhood to early adult-
hood at study enrollment, providing the strength of an accelerated lon-
gitudinal design and ability to examine brain changes over a large devel-
opmental period in a shorter timeframe than a single longitudinal cohort
(Galbraith et al., 2017; Harezlak et al., 2005; Willett et al., 1998). Our
group previously described volumetric brain changes in a subgroup of
male individuals age 6-35 years, with up to 3 longitudinal scans col-
lected over an 8-year period (n=100 ASD, n=56 TDC). We found vol-
umetric developmental trajectories that differed in our ASD group in
whole brain, white matter, lobes and ventricles (Lange et al., 2015).
Since that publication we have collected two additional imaging time-
points and increased our sample sizes of ASD and TDC participants. The
purpose of this study is to examine how additional longitudinal data-
points enhance our understanding of volumetric brain development in
males with autism and extend our previous study into mid-adulthood.
We also describe volumetric differences between two subgroups of our
ASD sample identified at the most recent time point (those who con-
tinued to meet criteria for ASD based on ADOS-2 algorithm and expert
clinical consensus classification vs those who did not) to examine how
clinical heterogeneity within the disorder is related to longitudinal brain
trajectories.
2. Methods
2.1. Participants
Participants included 105 male individuals with ASD and 125 male
participants without ASD (TDC) recruited from a longitudinal neu-
roimaging study at the University of Utah (see Table 1). A subgroup
2
NeuroImage 236 (2021) 118067
of this sample (n=90 ASD, n=55 TDC) overlaps with the participants
from our previous volumetric publication (Lange et al., 2015). Partic-
ipants were scanned up to 5 times over a 16-year period from 2003 –
2019 with the first wave of data collection between 2003 – 2007. Al-
though new participants were recruited at subsequent timepoints, par-
ticipant retention of those recruited during the initial study period has
remained high. Our study includes 82 individuals with ASD and 52 in-
dividuals with TDC who were enrolled during this initial period. Of this
original cohort, 75 ASD and 47 TDC had at least 2 MRIs, representing
91% retention, and 68 ASD and 36 TDC had at least 3 MRIs (84% and
69% retention respectively). At timepoint 5 (2017 – 2019), 73% of the
initial ASD sample (n=68) and 50% of the TDC sample (n=26) returned
for scanning. Fifty-four percent (54%) of the ASD sample reported psy-
chotropic medication use at some point during the longitudinal study:
antidepressants = 45%, stimulants = 24%, antipsychotics = 9%, anti-
anxiety = 8%, mood stabilizers = 6%, anti-insomnia = 5%, anticonvul-
sants = 1%, multiple medications = 30%. At timepoint 5, two partici-
pants, typically developing at study entry, reported new psychotropic
medication use (anti-insomnia and antidepressant).
At study enrollment, ASD was diagnosed using the Autism Diagnos-
tic Interview-Revised (ADI-R (Lord et al., 1994)), the Autism Diagnos-
tic Observation Schedule (ADOS (Lord et al., 2000; Lord et al., 2012)),
DSM-IV (American Psychiatric Association, 1994) and ICD-10 criteria.
Enrollment of both the ASD and TDC groups has been described previ-
ously (Alexander et al., 2007; Lange et al., 2015; Zielinski et al., 2014).
Participant consent, assent for those age 7 – 18 and parental permission
for all participants under age 18 was obtained at each timepoint. All
study procedures were approved by the Institutional Review Board.
Intelligence (IQ) was assessed at study enrollment and subsequent
timepoints as part of a comprehensive cognitive battery. IQ was assessed
with the Differential Abilities Scale (Elliott, 1990), Wechsler Intelligence
Scale for Children-Third Edition (Wechsler, 1991), Wechsler Adult Intel-
ligence Scale-Third Edition (Wechsler, 1997) or Wechsler Abbreviated
Scale of Intelligence (Wechsler, 1999), depending on participant’s age
and verbal ability, providing indices of Full Scale, Verbal and Nonverbal
IQ. Group means and ranges for the most recent IQ scores obtained from
study participants are presented in Table 1.
ASD Subgroup Classification. At timepoint 5 the ADOS-2 Module 4
(Lord et al., 2012) was administered to all ASD participants (n=82).
Our approach to evaluate Persistence and Remission in ASD was to in-
vestigate dimensional outcome based on symptom severity rather than
categorical DSM 5 diagnoses. We defined remitted ASD as falling below
ADOS-2 algorithm criteria for a current classification of ASD and below
expert clinical consensus for a current classification of ASD based on all
information available about the participant’s current behavior. Our re-
mitted ASD group was comprised of individuals who at the time of the
study entry met full ADI-R, ADOS-2, and DSM criteria for ASD, and thus
had a Lifetime diagnosis of ASD. But at Time 5, the level of their ASD
symptoms fell below the threshold for a current classification of ASD.
2.2. Imaging protocol
At each timepoint, whole-brain T1 weighted magnetic resonance im-
ages were acquired on a Siemens Trio 3.0 T scanner. At timepoint 1,
an 8-channel receive-only RF head coil was used to acquire the sagit-
tal 3D MPRAGE images (TI=1100 ms, TR=1800 ms, TE=2.93 ms, flip
angle=12°, FOV=256 mm, slice thickness=1mm, 160 slices). At time-
points 2 through 5, a 12-channel, receive only RF head coil was used
to acquire the sagittal 3D MPRAGE images (TI=900 ms, TR=2300 ms,
TE=2.91 ms, flip angle=9°, FOV=256 mm, slice thickness=1.2 mm, 160
slices). To control for imaging head coil differences between timepoints
1 and 2, a head coil covariate was included in all analyses.
2.3. Volumetric estimation
Volumes were estimated using the longitudinal stream
(Reuter et al., 2012) in the FreeSurfer image analysis suite v6.0
M.B.D. Prigge, N. Lange, E.D. Bigler et al. NeuroImage 236 (2021) 118067

Table 1
Participant characteristics.

Total number of scans ASD TDC

N=105 N=125

349 258

Mean (sd) Range Mean (sd) Range Group Comparison

Age at first scan (years) 16.4 (8.1) 6.0 – 45.4 22.4 (8.7) 6.0 – 44.4 t=5.4, p<.001
Age across all scans (years) 21.2 (8.7) 6.1 – 47.1 22.5 (8.5) 6.0 – 46.9 t=1.9, p=.05
Number MRIs per person 3.3 (1.5) 1–5 2.0 (1.4) 1–5 t=6.4, p<.001
Interscan Interval (years) 3.7 (1.9) 1.6 – 13.4 3.8 (2.3) 1.5 – 14.7 t=0.5, ns
FSIQ 103.7 (18) 60 - 150 118.3 (11) 87 - 144 t=6.7 p<.001
NVIQ 104.8 (17) 67 – 150 116.7 (13) 87 – 150 t=5.2 p<.001
VIQ 102.7 (19) 61 – 145 116.7 (12) 74 – 149 t=5.8 p<.001

Notes: sd=standard deviation; FSIQ: Full Scale IQ; NVIQ: Nonverbal IQ; VIQ: Verbal IQ.

(http://surfer.nmr.mgh.harvard.edu; (Fischl et al., 2002; Fischl et al.,

2004)). All MRI scans were processed on the same workstation. The
longitudinal pipeline creates a within subject template space and image
from all of the images collected for each participant. This processing
results in increased reliability and statistical power because processing
steps, such as skull stripping, Talairach transforms, atlas registration
and parcellations utilize common information from the within subject
template (Reuter et al., 2012). Although this project included a subset
of MRI images described previously, all images were reprocessed using
the updated FreeSurfer v6.0 and were included in the longitudinal
pipeline for each participant. All images were visually inspected and
edited blind to diagnosis by the lead author (M.P.) under the supervi-
sion of the senior author (B.Z.). A total of 635 MRIs were available for
analysis. After initial FreeSurfer processing, quality control involved
inspection of the following volumetric and surface files in Freeview:
T1, brainmask, wm, aseg, rh.white, lh.white, rh.pial, and lh.pial. A
total of 28 MRIs were excluded: 25 images (8 TDC, 17 ASD) had
segmentation errors resulting from poor image quality due to motion
or image artifacts and 3 images (3 TDC) had FreeSurfer processing
errors. After excluding these images, 8 participants had no usable
MRIs (5 TDC, 3 ASD). Of the remaining 607 datasets described in this
paper, 370 MRIs passed quality control with no editing. The remaining
237 MRIs were reprocessed after manual edits to brainmask.mgz files.
Fifteen MRIs had edits to cross-sectional images only (8 TDC, 7 ASD)
and 156 images (62 TDC, 94 ASD) were reprocessed after edits to
that participant’s base image. After base edits, longitudinal edits were Fig. 1. Longitudinal imaging dataset.
made to 45 images (21 TDC, 24 ASD) and 21 longitudinal images were
edited with no prior base or cross-sectional edits (11 TDC, 10 ASD).
The following regions were examined: whole brain (FreeSurfer variable fects and group∗ age interactions. In our initial analysis, age was mean-
SupraTentorialNotVent), total gray matter (cortical and subcortical), centered at 21 years. In the regions where significant group∗ age in-
total white matter, corpus callosum, thalamus, caudate, cerebellum, teractions were found, we performed a post-hoc analysis to identify
ventricles (sum of lateral, inferior lateral, 3rd , 4th , and 5th ventricles), the age at which the groups differed by re-centering the age variable.
and lobes (frontal, temporal, parietal, occipital). We also examined We also employed mixed-effects models to compare mean brain vol-
ventricle to whole brain ratio and total gray matter to whole brain ume and age related changes between the ASD subgroups identified at
ratio. the most recent study visit. Due to the number of regions being ana-
lyzed, FDR correction at a family wise error rate of p<.05 was applied
3. Statistical analysis (Benjamini and Hochberg, 1995) and all reported findings are corrected
p-values. Between-group differences in demographic variables were ex-
Linear mixed-effects models (Laird and Ware, 1982; Lange and amined using t-tests. R version 3.6.2 (R Core Team, 2019) was used and
Laird, 1989) were employed to describe longitudinal volumetric brain the R package nlme (Pinheiro et al., 2020) for mixed-effects models.
changes over time in the ASD participants in comparison to the TDC
group. This method allowed for the inclusion of individual random in- 4. Results
tercepts and slopes and varying number of data points per participant.
All models contained group, linear age and scanner headcoil effects. 4.1. Participant Characteristics
The following covariates were examined: age2 , group∗ age, group∗ age2
and FSIQ. For each region of interest, best fitting models were chosen At the initial study enrollment (2003 – 2007), there were no signif-
by application of the Akaike Information Criterion (Akaike, 1974). In icant differences between ages of the ASD and TDC participants (ASD
the volumetric models, the ASD group was the reference group, thus n=82, mean age=15.8 years, sd=8 years, range 6 – 45 years; TDC n=52,
volumes and age effects are represented in the intercepts and slopes; mean=16.0 years, sd=6 years, range 6 – 29 years; between group differ-
any differences in our TDC sample are interpreted through the group ef- ence t=.14, p=ns; all males). Participants were recruited at subsequent

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M.B.D. Prigge, N. Lange, E.D. Bigler et al. NeuroImage 236 (2021) 118067

Table 2
Linear mixed effects models of volumetric brain development in ASD.

ASD Intercept Group Effect Age Age2 Group x Age Group x Age2 FSIQ
𝛽 (SE) 𝛽 (SE) 𝛽 (SE) 𝛽 (SE) 𝛽 (SE) 𝛽 (SE) 𝛽 (SE)

Whole Brain 1122.18 -3.9 -4.5∗ ∗ ∗ .04∗ 1.24∗ ∗ -.10∗ ∗ 1.0∗

(9.7) (14.1) (.25) (.01) (.42) (.02) (.41)
Gray Matter Total 633.8 -3.5 -6.3∗ ∗ ∗ .14∗ ∗ ∗ 1.0∗ ∗ -.06∗ ∗ .61∗ ∗
(5.3) (7.7) (.18) (.01) (.30) (.02) (.21)
Gray Matter: Cortical 565.7 -2.3 -6.1∗ ∗ ∗ .14∗ ∗ ∗ .96∗ ∗ -.06∗ ∗ .54∗
(4.9) (7.1) (.17) (.01) (.29) (.02) (.19)
Gray Matter: 66.8 -1.0 -.11∗ ∗ ∗ -.006∗ ∗ ∗ .07∗ -.005∗ ∗ .06∗ ∗
Subcortical (.52) (.76) (.01) (.001) (.02) (.001) (.02)
White Matter 381.9 -2.6 1.08∗ ∗ ∗ -.07∗ ∗ ∗ .21 -.02∗ .37∗
(18.8) (6.0) (.10) (.006) (.17) (.01) (.17)
Ventricles 18.6 -2.0∗ .25∗ ∗ ∗ - -.10∗ ∗ - -
(.73) (.98) (.02) (.03)
Cerebellum 148.3 -.25 -.17∗ ∗ -.02∗ ∗ ∗ .04 - .15∗
(1.4) (2.0) (.05) (.002) (.08) (.06)
Thalamus 16.4 -.07 -.04∗ ∗ ∗ -.002∗ ∗ ∗ .02∗ - .01∗
(.14) (.20) (.005) (.0003) (.009) (.005)
Caudate 8.6 -.24 -.03∗ ∗ ∗ -.0005∗ ∗ - - .01∗
.11 (.16) (.002) (.0001) (.004)
Corpus Callosum 3.56 .05 .01∗ ∗ ∗ -.001∗ ∗ ∗ .008∗ - -
(.05) (.07) (.002) (.0001) (.003)
Lobes
Frontal 213.5 -1.1 -2.6∗ ∗ ∗ .06∗ ∗ ∗ .32∗ -.02∗ ∗ .28∗ ∗
(2.1) (3.1) (.08) (.005) (.13) (.008) (.08)
Temporal 129.0 .39 -.95∗ ∗ ∗ .01∗ ∗ ∗ .15∗ -.01∗ -
(1.2) (1.6) (.03) (.002) (.06) (.004)
Parietal 148.5 -.13 -1.99∗ ∗ ∗ .05∗ ∗ ∗ .39∗ ∗ ∗ -.02∗ ∗ .14∗
(1.4) (2.05) (.06) (.004) (.10) (.007) (.05)
Occipital 59.3 -.02 -.48∗ ∗ ∗ .012∗ ∗ ∗ .12∗ ∗ ∗ -.006∗ -
(.67) (.9) (.02) (.001) (.032) (.002)
TGM over WBV .56 -.001 -.003∗ ∗ ∗ .00008∗ ∗ ∗ .0001 - -
(.001) (.002) (.00007) (.00001) (.0001)
VBR .016 -.0018∗ .0003∗ ∗ ∗ - -.0001∗ ∗ - -
(.0006) (.0008) (.00002) (.00003)
corrected p<.001, ∗ ∗ corrected p<.01, ∗ corrected p<.05; 𝛽: Beta value; SE: standard error; TGM: total gray matter; WBV: whole brain volume; VBR: ventricle to
∗∗∗

whole brain ratio; - effect not included in best fitting model.

timepoints to increase our sample and account for attrition, resulting
in similar age ranges but younger mean age in the ASD participants at
each participant’s first scan and across all scans (see Table 1). A total
of 349 MRIs was collected from the ASD group and 258 MRIs from the
TDC group (see Fig. 1). The ASD group had more MRIs per person but
interscan interval did not differ between groups (see Table 1).
4.2. Longitudinal volumetric development in ASD
Results from the best fitting mixed-effects models are summarized
in Table 2. Linear and non-linear volumetric age-related changes are
presented for the ASD group and significant differences from the TDC
comparison sample are captured in the group effect and group by age in-
teractions. Fig. 2 shows the individual volumetric datapoints and linear
mixed model-derived predicted age curves for each group.
4.2.2. Mean volumetric differences
As evident in Table 2, the only ASD vs TDC mean differences present
at age 21 years (mean centered age) were increased ventricles (p=.04)
and VBR (p=.03) in ASD. Some of the longitudinal differences described
above resulted in volumetric differences between the ASD and TDC
groups at different stages of development. Total gray matter volume
was increased in ASD during childhood up to age 12 years (p=.04, 3.1%
larger in ASD); cortical GM was larger up to age 11 (p=.04, 3.3% larger
in ASD) and subcortical GM up to age 15 (p=.03, 2.9% larger). Frontal
volume was increased in ASD until age 8 (4% larger, p=.02) and parietal
and occipital lobes were increased into adolescence (parietal 3% larger
at age 12, p=.03; occipital 3.4% larger at age 9, p=.03). By age 21 years,
ventricular volume was larger in ASD (p=.03, 11% larger) and by age
36, corpus callosum volume was smaller in ASD (p=.03, 5% smaller).

4.2.1. Volumetric age-related changes
The ASD group showed greater volumetric decline with age com-
pared to the TDC sample in whole brain (linear age∗ group p=.006,
age2 ∗ group p<.001), gray matter (linear age∗ group p=.001, age2 ∗ group
p=.001), and lobar volumes (see Table 2 and Fig. 2). Whole brain
and occipital lobe findings replicate those reported previously in
Lange et al. (2015), yet the gray matter findings and the following
are new to this expanded dataset. Total white matter volume showed
a significant age2 ∗ group interaction only (p=.04). Ventricular volume
and ventricle to brain ratio (VBR) increased at a greater rate (ventri-
cle age∗ group p=.006; VBR age∗ group p=.002) and thalamic volume
decreased at a greater rate in ASD (age∗ group p=.04). Corpus callosum
volume increased at a greater rate in the TDC sample (p=.01; see Fig. 2).
We did not find any ASD vs TDC differences in age-related volumetric
changes in the caudate or cerebellum.
4.2.3. Timepoint 5: ASD ADOS-2 outcome classification and brain volumes
We examined longitudinal volumetric changes between the “remit-
ted ASD” subgroup versus the “persistent ASD” group (i.e., those still
meeting ADOS-2 criteria and expert clinical consensus classification for
ASD based on current behaviors). All of the remitted ASD participants
were under 30 years of age, thus to maintain equal age ranges between
ASD subgroups, we restricted the statistical analysis to those partici-
pants age 30 and younger, resulting in 78 participants (persistent ASD
n=61, remitted ASD n=17). We did not find longitudinal age-related
brain differences between the ASD subgroups but did find persistently
smaller corpus callosum volume in the subgroup with persistent ASD
at timepoint 5 (group effect 𝛽=-.37, p=.03, see Fig. 3). We also found
larger TGM/TBV ratios up until age 18 years in the persistent ASD group
(group effect 𝛽=.01, uncorrected p=.04 at age 18, see Fig. 3; this find-
ing was no longer significant at p<.05 after FDR correction). A greater

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Fig. 2. Longitudinal volumetric trajectories in ASD and TDC.

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M.B.D. Prigge, N. Lange, E.D. Bigler et al.
Fig. 3. Volumetric brain differences in ASD subgroups based on Time 5 classification.
percentage of the persistent ASD group reported psychotropic medica-
tion use at timepoint 5 (54% vs remitted ASD 33%). The persistent ASD
group had significantly lower mean Full Scale and Verbal IQ scores (Per-
sistent ASD: FSIQ 101.9, VIQ 99.2, NVIQ 104.5; Remitted ASD: FSIQ
114.0, VIQ 114.5, NVIQ 110.3; group differences: FSIQ t=3.0, p=.005,
VIQ t=3.4 p=.002, NVIQ t=1.7 p=.09, uncorrected p-values) but con-
trolling for FSIQ did not improve model fit for any of the volumetric
comparisons.
To test whether ASD severity differences were present in these sub-
groups prior to timepoint 5, we compared ADOS calibrated severity
scores (CSS; (Gotham et al., 2009; Hus and Lord, 2014)) obtained at
study entry and ADI algorithm scores. The persistent ASD group had
higher ADOS CSS scores at study entry (persistent ASD CSS=8.6, sd=1.3;
remitted ASD CSS=7.5, sd=1.2; t=3.0, p<.01), but a comparison of ADI
algorithm scores from the “Most Abnormal age 4-5” items showed sim-
ilar Social and Communication scores at age 4-5 (ADI A: persistent
ASD = 18.5, sd=5.5; remitted ASD = 18.7, sd=6.6; t=.08, p=ns; ADI
B: persistent ASD = 9, sd=3; remitted ASD 9.25, sd=3; t=.26, p=ns),
suggesting similar autism behaviors in childhood.
5. Discussion
The inclusion of the 4th and 5th waves of longitudinal imaging data
improve our understanding of brain changes from childhood into adult-
hood in males with ASD. This expanded dataset replicated our previ-
ous study describing whole brain volumetric age-related decline in ASD
above that found in TDC but also newly identified greater age-related
decline in cortical and subcortical gray matter volumes in ASD. We also
found developmental differences in ventricles and the corpus callosum
resulting in larger ventricles and reduced corpus callosum volume in
adulthood in ASD.
Atypical longitudinal gray matter trajectories in ASD were accompa-
nied by enlarged gray matter during early childhood that reached levels
in the typical group by later childhood and adolescence: cortical gray
matter by age 11, subcortical gray matter by age 15. We found signif-
icantly greater volumetric decline with age present in all four lobes;
parietal and occipital lobes remained larger up until late childhood.
Our results suggest nonuniform, regional gray matter volumetric dif-
ferences persistent into late childhood in males with ASD. Atypical vol-
umetric development can be driven by a number of underlying factors,
such as regional thickness and surface area changes (Ecker et al., 2013;
Hazlett et al., 2011). Future work is needed to examine additional struc-
tural changes underlying volumetric gray matter.
Ventricular volume and ventricle to brain ratio increased at a greater
rate in our ASD sample throughout the wide age range studied. This
atypical rate of increasing ventricular volume resulted in significantly
increased volume by 21 years of age. Whereas infants who develop ASD
tend to have increased subarachnoid extra-axial CSF (Shen et al., 2017;
6
NeuroImage 236 (2021) 118067
Shen et al., 2013), older individuals with ASD have now well-replicated
volumetric ventricular differences (Haar et al., 2016; Richards et al.,
2020; Turner et al., 2016; van Rooij et al., 2018). To these convergent
findings we add a very important novel detail: for the first time, using
longitudinal data collected over 16 years, we show evidence that ven-
tricular volume is increasing at an atypical rate in males with ASD.
Although we anticipated finding atypical development of cortical
white matter in ASD, we only found nonlinear cortical white matter dif-
ferences. The relationship between atypically increased ventricles and
age-related changes in gray and white matter remains unknown. In trau-
matic brain injury, ventricular enlargement is believed to be related to
periventricular white matter loss (Bigler et al., 2013). How white matter
abnormalities in ASD reported through other imaging modalities, such
as diffusion tensor imaging (DTI; (Ecker et al., 2016; Itahashi et al.,
2015)), are related to ventricle expansion is not yet known but will be
important to examine as markers of brain health and function across the
lifespan.
Corpus callosum volume increased at a greater rate in TDC and was
smaller in ASD by adulthood (significant at age 36 years and older).
Smaller corpus callosum volume or area is a repeated finding in cross-
sectional studies of individuals as young as 2 years of age through adult-
hood (Boger-Megiddo et al., 2006; Frazier et al., 2012; Freitag et al.,
2009; Hardan, Pabalan, et al., 2009; Levman et al., 2018; Piven et al.,
1997; Wolff et al., 2015) with few exceptions (Kucharsky Hiess et al.,
2015). Besides our previous publication, the only other research groups
to report longitudinal corpus callosum development have been in very
young children (Wolff et al., 2015) or only during late childhood and
early adolescence (Frazier et al., 2012). Interestingly, when we exam-
ined our ASD sample further when grouped by ADOS-2 and expert clini-
cal consensus classification based on current behaviors at their most re-
cent study visit, it became apparent that smaller callosal volumes were
driven by the participants still meeting criteria for ASD, or those with
more prevalent ASD behaviors. These findings underscore the impor-
tance of incorporating the clinical heterogeneity of ASD into imaging
studies (Bedford et al., 2020; Boger-Megiddo et al., 2006; Chen et al.,
2019). Although the microstructural properties of the atypical corpus
callosum volume are unknown, atypical corpus callosum structure and
development has widespread implications for behavioral functioning
that relies on interhemispheric communication. Accordingly, our group
has reported previously on processing speed differences in ASD related
to callosal area or diffusion tensor properties (Alexander et al., 2007;
Prigge et al., 2013). Future investigation into the longitudinal develop-
ment of microstructural properties, such as myelination, that may ex-
plain functional differences are warranted.
There are a number of limitations in our study. The first limitation is
the focus on male participants at the outset of the longitudinal study to
decrease heterogeneity and have adequate power in the face of limited
resources. As a result our findings may not be generalizable to brain
M.B.D. Prigge, N. Lange, E.D. Bigler et al.
structure and development in females. A small pilot sample of ASD and
TDC females was recruited at the 4th wave of data collection, and will
continue to be followed. Studies continue to identify sex differences in
brain morphology (Bedford et al., 2020), thus the inclusion of female
participants is essential to understand how sex may be related to and
independent from brain structural abnormalities reported in previous
studies of ASD individuals. Not all participants were able to complete
all 5 imaging timepoints. Our participant retention of the original cohort
has been 73% but we recruited additional ASD and TDC participants at
later timepoints with fewer visits; our mixed models are able to accom-
modate missing data but more complete longitudinal data will allow
individual tracking. Although we excluded images with significant mo-
tion, it is unknown how small amounts of motion may be biasing our
results. Finally, this analysis is limited to volumetric data and specific
regions. As our data analysis is ongoing, we will continue to examine
other regions and imaging features that will, in conjunction with the
current volumetric data, help describe longitudinal brain development
across the lifespan in ASD.
6. Conclusions
Autism is a heterogenous disorder. As a result, a reliance on cross-
sectional samples, which infer developmental brain changes from age-
related differences across different individuals, can provide contradic-
tory and only indirect information about age-related group differences
and when they appear during development. Longitudinal studies that
examine biological changes within affected individuals in relation to
clinical changes across time are well-poised to help elucidate the devel-
opmental neuropathologies of ASD across the lifespan, potential neu-
robiological subtypes, and brain mechanisms involved in variations in
core features clinical course, severity, and outcome.
Data and Code Statement
The raw imaging data for this publication is available through
the National Database for Autism Research (NDAR) under Collections
#1906 and #2400 (PI: Lainhart). FreeSurfer derived values used in the
analysis will be available through the Associated Study feature for the
NDAR Collections.
Acknowledgements
The research reported in this publication was supported by the Na-
tional Institutes of Mental Health of the National Institutes of Health
under Award Numbers R01MH080826 (JEL) and K08MH100609 (BAZ)
and a core grant to the Waisman Center from the National Institute of
Child Health and Human Development (U54 HD090256). The content
is solely the responsibility of the authors and does not necessarily repre-
sent the official views of the National Institutes of Health. The authors
would like to thank Alyson Froehlich, Jared Nielsen, Jubel Morgan and
Carolyn King for their assistance with data collection.
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