Atypical brain structure and function in young adults exposed to disaster‐related prenatal maternal stress: Project Ice Storm

Studies have shown that prenatal maternal stress (PNMS) affects brain structure and function in childhood. However, less research has examined whether PNMS effects on brain structure and function extend to young adulthood. We recruited women who were pregnant during or within 3 months following the 1998 Quebec ice storm, assessed their PNMS, and prospectively followed‐up their children. T1‐weighted magnetic resonance imaging (MRI) and resting‐state functional MRI were obtained from 19‐year‐old young adults with (n = 39) and without (n = 65) prenatal exposure to the ice storm. We examined between‐group differences in gray matter volume (GMV), surface area (SA), and cortical thickness (CT). We used the brain regions showing between‐group GMV differences as seeds to compare between‐group functional connectivity. Within the Ice Storm group, we examined (1) associations between PNMS and the atypical GMV, SA, CT, and functional connectivity, and (2) moderation by timing of exposure. Primarily, we found that, compared to Controls, the Ice Storm youth had larger GMV and higher functional connectivity of the anterior cingulate cortex, the precuneus, the left occipital pole, and the right hippocampus; they also had larger CT, but not SA, of the left occipital pole. Within the Ice Storm group, maternal subjective distress during preconception and mid‐to‐late pregnancy was associated with atypical left occipital pole CT. These results suggest the long‐lasting impact of disaster‐related PNMS on child brain structure and functional connectivity. Our study also indicates timing‐specific effects of the subjective aspect of PNMS on occipital thickness.


| INTRODUC TI ON
Prenatal maternal stress (PNMS) is a critical risk factor for offspring neurodevelopment (Chan et al., 2018).Structural magnetic resonance imaging (MRI) studies in humans show larger posterior parietal gray matter density in early adolescents exposed to prenatal stressful life events than controls (McQuaid et al., 2019).On the other hand, no gray matter volume (GMV) differences were detected between infants exposed to prenatal depression and controls (Lugo-Candelas et al., 2018).Prenatal anxiety (Qiu et al., 2013) and psychosocial stress (Moog et al., 2021) have been shown to be associated with decreased neonate hippocampal GMV.These studies indicate that the volumetric alterations are region-specific and may differ by the type of PNMS and by offspring age at assessment.The long-lasting effects of PNMS on GMV in young adulthood have been less explored.
Surface area (SA) and cortical thickness (CT) are considered two primary constituent components of cortical GMV (Wierenga et al., 2014).Two previous studies showed that prenatal depression was associated with increased SA of the middle frontal area (El Marroun et al., 2016) and decreased CT of the frontal and temporal areas (Lebel et al., 2016), which raises a question whether SA and CT contributes differently to the GMV variation.To date, only one study showed SA, but not CT, contributing to the variation in the association between prenatal stressful life events and mid-dorsolateral frontal GMV in young adults (Marecková et al., 2019).Given that the trajectories of GMV, SA, and CT develop in a brain region-specific manner (Wierenga et al., 2014), further research is warranted.
Atypical brain structure may lead to atypical brain function (J.Wang et al., 2017).The associations between prenatal anxiety, depression, or stressful life events and offspring brain function have been examined by resting-state functional MRI (rs-fMRI).For example, lower amygdala-prefrontal resting-state functional connectivity (rs-FC) was observed in neonates exposed to prenatal depression compared to non-exposed neonates (Posner et al., 2016), and preterm neonates exposed to prenatal depression and/or anxiety displayed lower rs-FC between the amygdala and the thalamus, the hypothalamus, and the peristriate cortex compared to nonexposed preterm neonates (Scheinost et al., 2016).Prenatal anxiety was associated with decreased amygdala-parietal rs-FC (Donnici et al., 2021) in children, and prenatal stressful life events were associated with decreased amygdala-medial prefrontal rs-FC (Humphreys et al., 2020) in neonates, and increased medial temporal-pregenual rs-FC in adults (Favaro et al., 2015).However, it remains unclear whether atypical brain structure is also accompanied by atypical rs-FC in young adulthood.
Compared with abundant research implicating prenatal stress in atypical brain structure and function, stress occurring during preconception has been less well studied.One possible mechanism underlying long-term effects of prenatal stress on offspring brain development is the impact of maternal glucocorticoids and placental stress regulation on the developing fetal nervous system, which may also apply to the effect of preconception stress.A review of rodent studies on the impact of preconception stress on offspring brain development (Keenan et al., 2018) found that exposure to preconception stressors (e.g., overcrowding, temperature, and pain stress) was linked to altered neurochemistry (Huang et al., 2010), neuroendocrine functioning (Zaidan et al., 2013) and brain morphology (Bock et al., 2016) in the offspring.In humans, an individual's hormonal response to a stressor can last for months; for example, salivary cortisol levels of people were significantly elevated following a severe ice storm in eastern Ontario in 1998 when they were assessed 1 month after the storm compared to unexposed people (Anisman et al., 2001).It has also been shown that stress during preconception typically persists into pregnancy; for example, depressive and anxious symptoms in the preconception phase are closely associated with those during pregnancy, with the symptom profiles remaining largely unchanged from preconception into pregnancy (Kee et al., 2021;Rinne et al., 2022).As such, there is support for expanding research on prenatal stress to preconception stress exposure.
Animal research shows that fetal brain ontogeny and prenatal stress-related biological and psychological processes are modulated in a gestational age-specific manner (Charil et al., 2010).Human studies also show that the consequences of PNMS on offspring brain structure and function vary with the timing of in-utero exposure.For example, anxiety assessed at 19 weeks, but not at 25 or 31 weeks of gestation, was associated with reduced GMV of the cerebellum, prefrontal, medial temporal, postcentral, and occipital regions in children (Buss et al., 2010).In addition, higher cortisol levels during the 2nd, but not the 3rd, trimester of gestation were associated with weaker hippocampal-cingulate rs-FC and stronger hippocampaltemporal rs-FC in infants (Scheinost et al., 2020).Unfortunately, prior studies typically examined only one or two arbitrary time points during gestation.It thus remains unclear whether different timings of exposure moderate the associations between PNMS and offspring brain structure and function.
Natural disasters have been studied as a natural experiment in PNMS research as these events are independent of parental characteristics.The 1998 ice storm is one of the worst disasters in Canadian history, leaving more than 1.5 million households without power for

Significance
Prenatal maternal stress affects brain structure and function in childhood.There is less evidence about whether this effect extends into young adulthood.To address this gap, our study examined a natural disaster as the source of maternal stress.Primarily, we found that, compared to non-exposed controls, disaster-exposed young adult offspring had larger volumes and higher functional connectivity of the anterior cingulate cortex, the precuneus, the left occipital pole and the right hippocampus.Our results suggest long-lasting effects of prenatal maternal stress and close relationships between brain structure and functional connectivity.
as long as 45 days in the coldest months of the year.We launched the world's first prospective longitudinal natural disaster-related PNMS cohort (Project Ice Storm) by recruiting women who were pregnant during this crisis, or became pregnant within the following 3 months.This cohort is unique in its precise timing of the onset and length of exposure to the ice storm in each woman, and because we assessed three aspects of the mothers' stress experiences comprehensively, including objective hardship, subjective distress, and cognitive appraisal.At age 19, their children underwent structural MRI and rs-fMRI scanning.
The primary aim of the current study was to determine whether brain GMV differs between young adults with and without prenatal ice storm exposure, and whether the brain regions with betweengroup GMV differences are also accompanied by between-group differences in rs-FC.To this end, we used a group of age-and sexmatched healthy controls without prenatal ice storm exposure, obtained from the Autism Brain Imaging Data Exchange and the Attention Deficit Hyperactivity Disorder-200 datasets.We also sought to explore the relative contributions of SA and CT to the GMV alterations by examining between-group differences in the GMV, SA, and CT.Our secondary aim was to determine, within the Ice Storm group, whether the GMV, SA, CT, and rs-FC showing between-group differences are associated with levels of PNMS, and whether these associations are moderated by the timing of in-utero exposure.

| Ice Storm group
A series of severe ice storms struck southern Quebec, Canada from January 5 to January 9, 1998.Following the ice storm, we contacted physicians who deliver babies in the four regional hospitals of the Montérégie, an area southeast of Montreal that was most affected by the crisis, and asked for their help in contacting eligible women.
On June 1, 1998, 1144 postal surveys were mailed to women identified by their physicians as being pregnant on January 9, 1998 or who became pregnant within the following 3 months.The survey assessed their levels of PNMS experiences in three aspects: objective hardship, subjective distress, and cognitive appraisal.The initial Project Ice Storm cohort consisted of 224 women who responded to the first questionnaire in June 1998; of these, 176 agreed to further contact.Around 19 years later, 39 young adult offspring (21 females) underwent structural MRI and rs-fMRI scanning.
Socioeconomic status was measured with the Hollingshead scale (Hollingshead, 1975)

| Objective hardship
In June 1998, the severity of objective hardship experienced by the women was assessed based on four dimensions of disaster exposure: Threat (e.g., injuries), Loss (e.g., loss of personal income), Scope (e.g., duration without electricity), and Change (e.g., temporary shelter) (Bromet & Dew, 1995).A total score (Storm32) was calculated by summing scores across all four dimensions; see (Laplante et al., 2007) for the scoring.

| Subjective distress
In June 1998, maternal subjective distress was assessed using a validated 22-item French version (Brunet et al., 2003) of the Impact of Event Scale-Revised (IES-R) (Weiss et al., 1997) which describes post-traumatic stress disorder (PTSD) symptoms of intrusive thoughts, hyperarousal, and avoidance.An IES-R total score of 33 is a cut-off for probable PTSD (Creamer et al., 2003).Log-transformed values of the total score were used in the current analyses due to its skewed distribution.

TA B L E 1 Demographics of Ice Storm participants and controls.
.472 In

| MRI data acquisition
T1w and rs-fMRI scan parameters are presented in Tables S1 and   S2.T1w and rs-fMRI raw images with artifacts or motion corruption were manually inspected: one Ice Storm rs-fMRI scan was excluded (Figure S1).

| MRI data preprocessing
fMRIPrep 1.5.7 (Esteban et al., 2019) was used for preprocessing.The T1w image was corrected for intensity non-uniformity with N4Bias-FieldCorrection (Tustison et al., 2010), distributed with ANTs 2.2.0 (Avants et al., 2008), and used as T1w-reference throughout the workflow.The T1w-reference was then skull-stripped with a Nipype implementation of the antsBrainExtraction.sh workflow (from ANTs), using OASIS30ANTs as target template.Brain tissue segmentation of GM, WM, and CSF was performed on the brain-extracted T1w using fast FSL 5.0.9 (Zhang et al., 2001).Volume-based spatial normalization to the MNI space was performed through nonlinear registration with antsRegistration (ANTs 2.2.0), using brain-extracted versions of both T1w reference and the T1w template.For each of the bloodoxygen-level-dependent (BOLD) runs found per subject, the following preprocessing was performed.First, a reference volume and its skull-stripped version were generated using a custom methodology of fMRIPrep.A deformation field to correct for susceptibility distortions was estimated based on fMRIPrep's fieldmap-less approach.
The deformation field is that resulting from co-registering the BOLD reference to the same-subject T1w-reference with its intensity inverted (Huntenburg, 2014;S. Wang et al., 2017).Registration is performed with antsRegistration (ANTs 2.2.0), and the process regularized by constraining deformation to be nonzero only along the phase-encoding direction, and modulated with an average field map template (Treiber et al., 2016).Based on the estimated susceptibility distortion, a corrected echo-planar imaging reference was calculated for a more accurate co-registration with the anatomical reference.The BOLD reference was then co-registered to the T1w reference using flirt FSL 5.0.9 (Jenkinson & Smith, 2001) with the boundary-based registration (Greve & Fischl, 2009) cost-function.
Co-registration was configured with nine degrees of freedom to account for distortions remaining in the BOLD reference.Head-motion parameters with respect to the BOLD reference (transformation matrices, and six corresponding rotation and translation parameters) are estimated before any spatiotemporal filtering using mcflirt FSL 5.0.9 (Jenkinson et al., 2002).The BOLD time-series were resampled onto their original, native space by applying a single, composite transform to correct for head-motion and susceptibility distortions.
The BOLD time-series were then resampled into the MNI space.Automatic removal of motion artifacts using independent component analysis (ICA-AROMA) (Pruim et al., 2015), was performed on the spatially-normalized, preprocessed BOLD on MNI space time-series after removal of non-steady state volumes and spatial smoothing with an isotropic, Gaussian kernel of 6 mm FWHM.In addition, we conducted WM and CSF signal removal from the BOLD time series and temporally bandpass filtering (>.01 Hz).

| FreeSurfer analysis
fMRIPrep preprocessed T1w images in MNI space were automatically segmented using FreeSurfer 7.1.1(Fischl, 2012) and its library tool recon-all, and cortical and subcortical volumes were calculated.
SA was calculated at the pial level and represents the area of vertex on the gray matter surface, calculated as the average of the area of the tessellated triangles touching that vertex.CT at each vertex was measured by calculating the shortest distance from the white matter to the pial surface (Kelly et al., 2013).GMV, SA and CT were obtained based on the Destrieux Atlas (Destrieux et al., 2010).
The ANCOVA results were Bonferroni-corrected for the number of brain regions.

| Statistical analysis within Ice Storm group
To examine the association among the three aspects of PNMS and GMV, SA, CT, and mean z-values showing between-group differences, partial correlations were run controlling for offspring sex.
To examine whether timing of exposure moderates the association between PNMS and GMV, SA, CT, and mean z-values showing between-group difference, moderated regression analyses were conducted using SPSS PROCESS macro (Hayes, 2017)  thalamus, bilateral hippocampus, precuneus, the left occipital pole, the left postcentral gyrus, and the right lateral occipital gyrus (Figure 1).Cluster details are presented in Table 2.There were no instances of smaller GMV in Ice Storm participants compared to controls.had larger GMV of the left occipital pole with medium-to-large effect size (p = .001, 2 p = .113)and larger CT of the left occipital pole with large effect size (p = .00006, 2 p = .192),both of which met the Bonferroni-corrected threshold.In contrast, the SA of the left occipital pole tended to be smaller in Ice Storm participants with small effect size (p = .076, 2 p = .031),failing to survive the Bonferroni correction.

| Associations between PNMS and GMV
The nine brain regions showing between-group GMV differences at the corrected threshold (shown in Figure 1) were further examined in the subsequent statistical analyses within the Ice Storm group.
We found that more negative maternal cognitive appraisal was associated with larger right lateral occipital gyrus GMV (r = −.325,p = .047,Figure S2a) and larger left occipital pole GMV (r = −.355,p = .029,Figure S2b) in the young adults, but these associations did not survive the Bonferroni correction.

| Timing of exposure moderates associations between PNMS and CT
Only the left occipital pole CT was further examined in the subsequent statistical analyses within the Ice Storm group, given that it is the only variable showing a between-group difference at the corrected level.
We observed an interaction between maternal subjective distress and timing of exposure for the left occipital pole CT (p = .0006).
When probing this interaction, we found that, when mothers were exposed to the ice storm before preconception day −18 (n = 6), the higher the mothers' subjective distress level, the thicker the left occipital pole; when the exposure occurred after pregnancy day 98 (n = 16), the higher the mothers' subjective distress level, the thinner the left occipital pole (shown in Figure 2).

| Between-group rs-FC differences
Relative to controls, Ice Storm participants had Bonferroni-corrected higher rs-FC from the ACC to the right occipital pole, from the left occipital pole to the left paracingulate, from the precuneus to the left middle temporal gyrus, and from the right hippocampus to the right postcentral gyrus (Figure 3).See Table 4 for the details of the clusters.

| Associations between PNMS and rs-FC
The four rs-FC showing between-group differences at the corrected threshold (shown in Figure 3) were further examined in the subsequent statistical analyses within the Ice Storm group.

F I G U R E 2
Timing moderates the association between maternal subjective distress and left occipital pole thickness in young adult offspring when controlling for sex (p = .0006).When the ice storm exposure occurred before preconception day −18, the higher the mothers' subjective distress level, the thicker the left occipital pole; when the exposure occurred after pregnancy day 98, the higher the mothers' subjective distress level, the thinner the left occipital pole.In addition, when mothers had low subjective distress levels (≤log-transformed score of 1.14, original scale score of 2.13), the later in gestation the exposure to the ice storm, the thicker the left occipital pole; when mothers experienced high subjective distress levels (≥log-transformed score of 2.39, original scale score of 9.91), the earlier in gestation the exposure to the ice storm, the thicker the left occipital pole.The dark blue line represents low maternal subjective distress; the bright blue line represents moderate maternal subjective distress, and the red line represents high maternal subjective distress.Low, moderate and high subjective distress lines are represented at the 16th, 50th, and 84th sample percentiles of maternal subjective distress levels, respectively, which were at log-transformed scores of .69,2.08, and 3.20 (original scale scores of .99,7.00, and 23.53), respectively.The regions of significance (p < .05)are represented by the black vertical lines and the gray shadings.*p < .05;CT, cortical thickness; L, left; OP, occipital pole.
We found that lower maternal objective hardship levels tended to be associated with higher rs-FC from the precuneus to the left middle temporal gyrus (r = −.331,p = .046,Figure S3), but this association did not survive the Bonferroni correction.

| DISCUSS ION
To our knowledge, this is the first study examining young adults' whole-brain structure and functional connectivity as a function of PNMS deriving from a natural disaster.There were three primary As noted above, larger left thalamic volume was observed in Ice Storm participants compared to controls.The thalamus acts as a relay center for multiple sensory modalities between other subcortical nuclei and the cerebral cortex (Yoshii, 2021).Morphological microglial activation was observed in the thalamus of rodents after acute stress (Sugama et al., 2007).In humans, early childhood maltreatment has been associated with increased left thalamic volume in adolescents and young adults (Liao et al., 2013).We speculate that the enlarged thalamic volume observed in the current study might be influenced by in-utero exposure to the ice storm.The subsequent lack of association with the degree of severity of PNMS may reflect ceiling effects or insufficient statistical power within the Ice Storm group.
We also found that, compared to controls, the Ice Storm youth had larger GMV of the PCC and precuneus, part of the defaultmode network (DMN), and also had higher precuneus-temporal functional connectivity.Given our novel approach to the effects of disaster-related PNMS on offspring brain structure and function, no study, to date, has examined this issue in the same way.Nevertheless, outside of the pregnancy context, it has been shown that, in human individuals, prolonged stress exposure is associated with morphological changes within the DMN (Soares et al., 2017), and stressed participants display greater DMN activation than controls (Soares et al., 2013).One study also reported that the precuneus and temporal regions displayed increased neural activity during the formation of subsequent stressful memory (Henckens et al., 2009).
The ACC and hippocampus express high levels of glucocorticoid receptors (Herman et al., 2005) which may explain why their structure and function are most frequently altered in response to stress.We found that Ice Storm participants exhibited larger ACC volume and higher ACC-occipital functional connectivity compared to controls.
As part of prefrontal regions, the ACC is involved in the "top-down" emotion generation process.Occipital areas are implicated in the "bottom-up" emotion generation process (e.g., detecting inherently aversive images) (Vandekerckhove, 2020).The strengthened ACCoccipital connection revealed the possible modulation of cognitive reappraisal in "bottom-up" processing of environmental stimuli, possibly as a result of fetal programming for those exposed to PNMS.
Likewise, we found that the left occipital pole, showing a GMV increase, also displayed increased functional connectivity with the paracingulate, part of the prefrontal cortex (Jahn et al., 2016).A possible explanation lies in the amygdala linking the prefrontal and occipital regions: the amygdala connects both "bottom-up" and "topdown" emotion generation processes (Ochsner et al., 2009).Briefly, the basolateral complex of the amygdala receives visual stimuli from the occipital cortex that functions as the early stage of emotion processing (Gläscher et al., 2007;Ressler, 2010), and projects to the prefrontal regions that regulate emotional circuits of the amygdala and modulate learning and memory (Sah et al., 2003).In addition, larger left postcentral volume was observed in Ice Storm participants than that in controls.The postcentral gyrus, known as the primary somatosensory area, participates in stress-regulation circuits by projecting sensory stimuli to the amygdala (Ressler, 2010).
Acute psychosocial stress has generally been found to deactivate the hippocampus (Dedovic et al., 2009).Our study observed larger hippocampal GMV and higher hippocampal-postcentral functional connectivity in Ice Storm participants compared to controls.A possible explanation is that hippocampal regulation (i.e., inhibitory or excitatory) of the hypothalamus-pituitaryadrenal axis may be stressor-specific (Herman et al., 2005).For instance, a previous study reported that no association was observed between postnatal maternal dysregulated mood and hippocampal volume in young adult offspring while greater postnatal maternal anxiety was associated with smaller hippocampal volume (Marečková et al., 2018).
TA B L E 4 Clusters of significant (uncorrected voxel-level p < .001,cluster-level p-FDR < .05)differences in resting-state functional connectivity between Ice Storm participants and controls.we were unable to explain the higher precuneus-temporal connectivity detected for between-group comparison: the higher connectivity was, unexpectedly, associated with lower levels of objective hardship level.This effect, however, failed to survive the correction for multiple comparisons.
A valuable aspect of Project Ice Storm is that the acute onset of the 1998 Quebec ice storm allows us to study timing-specific effects of PNMS in pregnancy accurately.We found that greater maternal subjective distress experienced during preconception was associated with thicker left occipital pole, pointing to an important vulnerability window in the 3 months prior to conception.This is in line with a review summarizing the impact of preconception stress on offspring brain development (Keenan et al., 2018); for example, rat pups exposed to maternal preconception stress showed a decrease in the agranular insular dorsal cortex thickness relative to the control group (Jenkins et al., 2018).Given that periods of accelerated growth of different cerebral cortical compartments take place at different gestational ages, there may be more than one sensitive exposure period (Rakic, 1988).For example, after 25 weeks of gestation in humans, the subventricular zone is the principal source of cortical neurons and continues to increase the thickness (Zecevic et al., 2005).
The cortical plate and subplate also contribute to the thickness of the occipital lobes, and the thickness of the cortical subplate increases between 25 and 30 weeks of gestation (Vasung et al., 2016).Our study found that when ice storm exposure occurred during the 2nd and 3rd trimesters, higher maternal subjective distress levels were associated with thinner occipital pole, which is possibly attributed to the disruption in the activities of the subventricular zone, cortical plate and subplate compartments.However, this potential timing effect needs to be replicated in future studies with larger sample sizes.
Although it is interesting to observe associations between PNMS exposure and severity with brain structure and function, These exploratory analyses showed a positive tendency within the Ice Storm youth for higher functional connectivity from the ACC to the right occipital pole to be associated with higher full-scale IQ score (which ranged between 90 and 137) (r = .354,p = .031,Figure S4).This result indicates the possible relevance of cognitive functioning with strengthened neural activity of the prefrontal and occipital cortices.One previous study in healthy young adults (Narr et al., 2007) reported positive associations between full-scale IQ score (which ranged between 74 and 139) and cortical thickness in left and right prefrontal cortices and the right occipital lobe.Future research is needed to replicate the correlation of IQ with prefrontaloccipital connectivity.In contrast, our exploratory analyses with behavioral outcomes showed that, within the Ice Storm youth, there were no associations of the overall severity of autistic-like behaviors with the larger GMV and CT and the higher functional connectivity of the brain regions that were examined in this study.We speculate that the total score on autistic-like behaviors may have correlations with other brain regions that were not examined in this study; for example, the amygdala plays a central role in emotion processing (Ochsner et al., 2009) and social cognition (Emery et al., 2001), and thus is an important brain structure of the neuropathology behind social deficits, the core feature seen in autism (Gotts et al., 2012).
Our future research aims to determine whether volume and functional connectivity of other brain regions (e.g., amygdala) mediate the association between different aspects of PNMS and autistic-like traits at age 19.
There are some limitations to this study.First, our sample was small, limiting the statistical power to control for other possible maternal confounders (e.g., socioeconomic status, marital status, living status, and age at childbirth).Second, MRI data for Ice Storm and control groups were collected from different scanning sites; this Abbreviations: F, female; FD, framewise displacement; IQ, intelligence quotient; L, left; M, male; R, right; SD, standard deviation.an = 55, there are 10 missing values, but the IQs of the 10 participants are ≥80.
tion, and smoothing based on the Statistic Parameter Map package (SPM12 http://www.fil.ion.ucl.ac.uk/spm).First, all native T1w images were segmented into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF).A diffeomorphic non-linear registration algorithm was used for spatially normalizing GM and WM images into the Montreal Neurological Institute (MNI) space.Further modulation was applied to convert the voxel values of tissue concentration (density) to measures of volume.Finally, the segmented, normalized, and modulated GM maps were smoothed with an isotropic Gaussian kernel (full width at half maximum (FWHM) = 8 mm).The quality of preprocessed GM probabilistic images was inspected visually by X.L. and F.L., and any discrepancies were decided by an experienced researcher (M.N.I.).Using R RMINC package (https://github.com/Mouse -Imagi ng-Centr e/RMINC), a voxel-by-voxel analysis of covariance (ANCOVA) controlling for T1w repetition time (TR), echo time (TE) and offspring sex was applied to compare GMV differences between Ice Storm participants and controls.Multiple comparisons were corrected based on Random Field Theory (Worsley

2. 8 |
Seed-based rs-FC analysisSeeds were defined as the brain regions showing between-group differences in GMV.At the individual level, Pearson's correlation coefficients were calculated between average BOLD time courses extracted from each of the seeds and the time courses of all voxels across the brain, and were then converted to normally distributed z-values.At the group level, ANCOVAs (controlling for rs-fMRI TR, TE, in-scanner eye status and offspring sex) were applied to compare differences in the mean z-values between Ice Storm participants and controls.Thresholds of significance were set to uncorrected voxellevel p < .001,cluster-level FDR-corrected p < .05.Finally, the AN-COVA results were Bonferroni-corrected for the number of seeds.All rs-FC analyses were conducted using CONN functional connectivity toolbox 19b (Whitfield-Gabrieli & Nieto-Castanon, 2012).
with timing of exposure, objective hardship, and timing of exposure × objective hardship as predictor variables and offspring sex as the covariate.The same moderated regression models, controlling for offspring sex, were rerun for timing of exposure × subjective distress, and timing of exposure × cognitive appraisal.The results were Bonferronicorrected for the number of regions showing between-group difference and the three aspects of PNMS. 3 | RE SULTS 3.1 | Between-group differences in GMV (VBM) Ice Storm participants had significantly larger GMV compared to controls in the following nine regions: the anterior cingulate cortex (ACC), the posterior cingulate cortex (PCC), the left F I G U R E 1 GMV differences between Ice Storm participants and controls.Clusters displaying larger GMV in Ice Storm participants than controls are depicted in red and yellow colors.ACC, anterior cingulate cortex; GMV, gray matter volume; L, left; LOG, lateral occipital gyrus; OP, occipital pole; PCC, posterior cingulate cortex; PostCG, postcentral gyrus; R, right.
findings.First, relative to controls, young adults exposed in-utero to the ice storm showed enlarged left thalamus, PCC, precuneus, ACC, bilateral hippocampus, left occipital pole, left postcentral gyrus, and right lateral occipital gyrus, and thickened left occipital pole.Second, the enlarged precuneus, ACC, left occipital pole, and right hippocampus were also accompanied by increased functional connectivity in the Ice Storm youth compared to controls.Finally, within the Ice Storm group, maternal subjective distress was associated with left occipital pole thickness in a timing-specific manner; effects from objective hardship and cognitive appraisal were not moderated by timing.
E 3 Resting-state functional connectivity differences between Ice Storm participants and controls.(a) Using the ACC as the seed, clusters displaying higher ACC-right OP connectivity in Ice Storm participants than controls are depicted in yellow colors.(b) Using the left OP as the seed, clusters displaying higher left OP-left PaCiG connectivity in Ice Storm participants than controls are depicted in yellow colors.(c) Using the precuneus as the seed, clusters displaying higher precuneus-left MTG connectivity in Ice Storm participants than controls are depicted in yellow colors.(d) Using the right hippocampus as the seed, clusters displaying higher right hippocampus-right PostCG connectivity in Ice Storm participants than controls are depicted in yellow colors.ACC, anterior cingulate cortex; L, left; MTG, middle temporal gyrus; OP, occipital pole; PaCiG, paracingulate gyrus; PostCG, postcentral gyrus; R, right.
The Research Ethics Board of the Douglas Mental Health University Institute approved all phases of the study at each time point.All the participants provided written informed consent.

Table 3
describes between-group differences with effect sizes in GMV, SA, and CT.Compared to controls, Ice Storm participants TA B L E 2 Larger GMV in Ice Storm participants compared to controls.
(Rimol et al., 2012)with asterisks indicate significant results (Ice Storm > controls) that survived the Bonferroni correction for nine seeds (.05/9 = .006).We observed that Ice Storm participants had larger GMV of the left occipital pole.Our exploratory analyses further showed that Ice Storm participants had larger CT, but not SA, of the left occipital pole compared to controls.Given that a previous study reported that cortical GMV was closely associated with CT in schizophrenia(Rimol et al., 2012), our exploratory result might support a tentative (Cao-Lei, Dancause, et al., 2016;Cao-Lei et al., 2018); LOG, lateral occipital gyrus; MNI, Montreal Neurological Institute; MTG, middle temporal gyrus; OFusG, occipital fusiform gyrus; OP, occipital pole; PaCiG, paracingulate gyrus; PaHC, parahippocampal gyrus; PostCG, postcentral gyrus; R, right; SFG, superior frontal gyrus.aPositiveTscoresdenotethat Ice Storm participants had higher functional connectivity compared to controls; negative T scores denote that Ice Storm participants had lower functional connectivity compared to controls.|1859et al.growth in their offspring.Previous Project Ice Storm analyses have found that maternal negative cognitive appraisal was associated with atypical biological outcomes at age 13 including lower BMI, central adiposity and higher C-peptide secretion(Cao-Lei, Dancause, et al., 2016;Cao-Lei et al., 2018), and thus we propose that maternal negative cognitive appraisal might act as a potential factor of offspring brain development.In addition, within the Ice Storm group, these findings would have greater relevance if they were shown to explain variance in behavioral outcomes.For example, our previous Project Ice Storm findings have shown that the three aspects of PNMS play distinct roles in offspring development: maternal objective hardship predicted altered cytokine production at age 13½ via DNA methylation (Cao-Lei, Veru, et al., 2016); maternal subjective distress predicted externalizing behaviors at age 11½ via increased amygdala volume (Jones et al., 2019); and maternal cognitive appraisal was associated with BMI, central adiposity and C-peptide secretion at age 13½ via DNA methylation (Cao-Lei, Dancause, et al., 2016; Cao-Lei et al., 2018).To test the relevance of the current findings to behavioral outcomes in the Ice Storm group, we correlated the atypical brain structure and function reported above with two outcomes assessed at age 19 (shown in Supporting Information).