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High pregnancy anxiety during mid-gestation is
associated with decreased gray matter density
in 6—9-year-old children
Claudia Bussa, Elysia Poggi Davisa,b, L. Tugan Muftulerc, Kevin Headc,
Curt A. Sandmana,*
aDepartment of Psychiatry and Human Behavior, University of California, Irvine, 333 The City Blvd. W,
Suite 1200, Orange, CA 92868, United States
bDepartment of Pediatrics, University of California, Irvine, CA 92697-5020, United States
cDepartment of Radiological Sciences, University of California, Irvine, CA 92697-5020, United States
Received 30 April 2009; received in revised form 15 July 2009; accepted 16 July 2009
Psychoneuroendocrinology (2010) 35, 141—153
Gray matter volume
vulnerable to environmental insults. There is evidence that maternal stress and anxiety during
pregnancy influences birth outcome but there are no studies that have evaluated the influence of
stress duringhuman pregnancy on brainmorphology.Inthe current prospective longitudinal study
we included 35 women for whom serial data on pregnancy anxiety was available at 19 (?0.83), 25
(?0.9) and 31 (?0.9) weeks gestation. When the offspring from the target pregnancy were
between 6 and 9 years of age, their neurodevelopmental stage was assessed by a structural MRI
scan. With the application of voxel-based morphometry, we found regional reductions in gray
matter density in association with pregnancy anxiety after controlling for total gray matter
volume, age, gestational age at birth, handedness and postpartum perceived stress. Specifically,
independent of postnatal stress, pregnancy anxiety at 19 weeks gestation was associated with
gray matter volume reductions in the prefrontal cortex, the premotor cortex, the medial
temporal lobe, the lateral temporal cortex, the postcentral gyrus as well as the cerebellum
extending to the middle occipital gyrus and the fusiform gyrus. High pregnancy anxiety at 25 and
31 weeks gestation was not significantly associated with local reductions in gray matter
volume.This is the first prospective study to show that a specific temporal pattern of pregnancy
anxiety is related to specific changes in brain morphology. Altered gray matter volume in brain
regions affected by prenatal maternal anxiety may render the developing individual more
vulnerable to neurodevelopmental and psychiatric disorders as well as cognitive and intellectual
Published by Elsevier Ltd.
Because the brain undergoes dramatic changes during fetal development it is
* Corresponding author. Tel.: +1 714 940 1924; fax: +1 714 940 1939.
E-mail address: email@example.com (C.A. Sandman).
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/psyneuen
0306-4530/$ — see front matter. Published by Elsevier Ltd.
Author's personal copy
Programming refers to the action of a factor during a sensi-
tive developmental period affecting the organization and
maturity of specific organs. One key assumption of the
programming hypothesis is that biological systems under-
going rapid developmental changes are especially vulnerable
2003; Seckl and Meaney, 2004). Considering the extended
pre- and postnatal developmental trajectory of the brain, it
is apparent that early life experiences have the potential to
sculpt brain morphology. Such sculpting of the immature
brain is an interactive process between genetic program-
ming, cell function and the environment (Andersen, 2003).
Growing evidence suggests that abnormal development of
the brain during gestation contributes to many neurological
disorders that are manifested throughout the entire lifespan
(Rees et al., 2008).
The immature brain can be considered ‘‘under construc-
tion’’ (Connors et al., 2008) with the development of the
human central nervous system following a protracted, neatly
orchestrated chain of specific ontogenetic events. Although
brain change and adaptation are part of a lifelong process,
the earliest phases of maturation during fetal development
and childhood are perhaps the most dramatic and important
(Toga et al., 2006). Understanding the timing of neurodeve-
lopmental events is essential for determining how particular
environmental disturbances can selectively affect certain
functions. During fetal life, neurons proliferate, migrate,
and aggregate, providing the ‘‘hardware’’ for the developing
brain. Neural proliferation before birth has been estimated
at an average rate of 250,000 cells/min (Cowan, 1979).
Between gestational ages 8 and 16 weeks, migrating neurons
form the subplate zone and await connections from afferent
neurons originating in the thalamus, basal forebrain, and
brainstem (Kostovic et al., 2002). Once neurons reach their
final destination at about the 16th fetal week, they arborize
and branch in an attempt to establish appropriate connec-
tions (Sidman and Rakic, 1973). Axon collaterals connect to
numerous regions in the brain before the neuron finishes
migrating to its target location (Jones et al., 1985). Neuro-
trophins influence the migration or retraction of neurons
(Jones et al., 1993) and ephrins guide neurons further by
establishing a chemical gradient to follow (Knoll and
Drescher, 2002). During development of the human brain,
little synapse formation occurs before the beginning of the
third trimester, when it accelerates to approximately 40,000
specific neurodevelopmental events results in windows of
specific vulnerability for adverse influences. In animal mod-
els, changes in brain morphology have been observed in
offspring of mothers exposed to prenatal stress. In non-
human primates, daily acute prenatal stress is associated
with 10—12% reductions in hippocampal volume and inhibi-
tion of neurogenesis in the dentate gyrus (Coe et al., 2003) as
well as altered size of the corpus callosum (Coe et al., 2002).
the cytoarchitecture of the rat hippocampus is altered as a
consequence of prenatal stress (Hayashi et al., 1998; Gould
and Tanapat, 1999; Lemaire et al., 2000). Impaired neuro-
genesis and associated cognitive impairment have been
repeatedly reported in prenatally stressed animals (Lemaire
et al., 2000, 2006; Fujioka et al., 2006). Furthermore, pre-
natal stress has the potential to alter synaptic plasticity by
impairing long-term potentiation but facilitating long-term
depression (Yaka et al., 2007; Yang et al., 2007). Although
prenatal stress associated changes in the hippocampal for-
mation have received major attention, morphological
changes in other brain regions have been shown as well.
For example significantly expanded dimensions of the lateral
nucleus oftheamygdalawere observedinprenatally stressed
offspring (Salm et al., 2004). Also, reduced spine densities
and significant reduction of dendritic length of pyramidal
neurons in the dorsal anterior cingulate and orbitofrontal
cortex have been reported in offspring of mothers exposed to
stress during pregnancy (Murmu et al., 2006). Since the
rodent brain is less mature at birth than the human brain,
it has been suggested that brain maturation occurring in the
early postnatal period in the rat is analogous to maturational
changes that occur in humans in late gestation (Clancy et al.,
2001). Therefore, it is interesting to note that there is an
impressive body of evidence from rodent studies showing
changes inbrainmorphology inassociation withmanipulation
of the early postnatal environment (e.g. Meaney et al., 1991;
Pham et al., 1999; Helmeke et al., 2001; Huot et al., 2002;
Roceri et al., 2002; Bredy et al., 2003; Ovtscharoff et al.,
2006; Fabricius et al., 2008).
in individuals born prematurely. Thus, low birth weight as well
volumes (e.g. Peterson et al., 2000; Abernethy et al., 2002;
Nosarti et al., 2002; Huizink et al., 2004; Buss et al., 2007;
Beauchamp et al., 2008). Adverse birth outcomes may be
markers of in utero stress exposure (e.g. Wadhwa, 2005) but
the changes in brain morphology may also be due to perinatal
complications that are often associated with premature deliv-
investigated the association betweenprenatal stressexposure
and brain morphology in the offspring.
Pregnancy anxiety has been suggested to be a more
sensitive predictor of birth outcomes than general anxiety
(Wadhwa et al., 1993; DiPietro et al., 2004; Roesch et al.,
2004; Kramer et al., 2009) and has been suggested as a
distinctive syndrome (Huizink et al., 2004). Further evidence
suggests that measures of pregnancy specific stress are bet-
ter than measures of generalized psychological distress for
(DiPietro et al., 2002), infant cognitive and motor develop-
ment (Huizink et al., 2003; Dipietro et al., 2006; Davis and
Sandman, in press) and infant emotional regulation (Dipietro
et al., 2006). The objective of the current study was there-
fore to test in a prospective longitudinal study the associa-
tions between pregnancy anxiety, measured repeatedly over
in their 6—9-year-old offspring.
Pregnant women were recruited for study participation
between 1998 and 2002. Five hundred and fifty seven preg-
nant women, who received prenatal care from the faculty
142C. Buss et al.
Author's personal copy
obstetric practice at the University of California, Irvine
Medical Center or Cedars-Sinai Hospital in Los Angeles, were
recruited by the 15th week of gestation and provided writ-
ten, informed consent. All methods and procedures were
approved by the Institutional Review Board of the participat-
ing institutions. Study participants were English-speaking
adult women (>18 years age) with singleton, intrauterine
pregnancies. Exclusion criteria included tobacco, alcohol, or
other drug use in pregnancy; uterine or cervical abnormal-
dysregulated neuroendocrine function such as endocrine,
hepatic or renal disorders or corticosteroid medication
use. While not an exclusion criterion, none of the women
in this sample were treated for any psychiatric disorders. In
the context of an on-going study on the effects of prenatal
stress exposure on child brain development that started in
2007, women were re-contacted and invited to participate in
a follow-up study of their children. Three hundred and forty
women of the initial sample were located. Fifty-two children
have undergone and MRI scan to date; of those one MRI scan
had to be excluded due to morphological abnormalities and
two MRI scans due to severe motion artifacts. Among the 49
mother—child dyads with usable MRI data, 35 women had
provided complete maternal stress data at three time points
are included in the current report. Among these 35 children
were two siblings; thus one mother was enrolled in the study
with two subsequent pregnancies and consequently two
children for the follow-up study. Women whose children
participated in the follow-up study of brain development
did not differ in sociodemographic characteristics (maternal
age and education, annual household income) from women in
the initial sample (all p > 0.4).
2.2. Assessments in pregnant women
For all pregnant women gestational age was determined by
best obstetric estimate with a combination of last menstrual
period and early uterine size, and was confirmed by obstetric
ultrasonographic biometry before 20 weeks using standard
clinical criteria (O’Brien et al., 1981). Medical risk was
defined as the presence of certain medical conditions in
the index pregnancy or previous pregnancies (e.g. vaginal
bleeding, pregnancy-induced hypertension, preeclampsia,
infection, Hobel, 1982). Risk conditions were determined
through interview and extensive medical chart review. The
sum of medical risk factors was calculated as an indicator of
presence of any current or historical risk conditions. Infor-
mation on birth outcomes was retrieved from medical charts
after delivery. Sociodemographic characteristics and birth
outcomes are summarized in Table 1.
2.3. Pregnancy anxiety
Pregnancy anxiety was assessed over the course of gestation
at 19 (?0.83, SD), 25 (?0.9) and 31 (?0.9) weeks gestation,
and for all 35 children included in the current analyses
complete data was available. A 10-item pregnancy anxiety
scale, which assesses a woman’s feelings about her health
during pregnancy, the health of her baby, and her feelings
about labor and delivery, was administered at all three study
visits. Answers were given on a 4-point scale and included
items such as: ‘‘I am fearful regarding the health of my
baby,’’ ‘‘I am concerned or worried about losing my baby,’’
and ‘‘I am concerned or worried about developing medical
problems during my pregnancy.’’ The final score on this
measure could range from 10 to 40. This reliable measure
(a = 0.75—0.85) was specifically developed for use in preg-
nancy research (Rini et al., 1999; Glynn et al., 2008).
2.4. Pregnancy anxiety and medical risk
To test whether the effects of pregnancy anxiety on the
developing brain could be mediated by the presence of
medical risk, correlation analyses were performed between
At none of the assessments, significant correlations could be
observed(19 weeks:r = ?0.14,
r = ?0.14, p = 0.44, 31 weeks: r = 0.02, p = 0.92).
p = 0.42;26 weeks:
2.5. Pregnancy anxiety, sociodemographic
characteristics and postpartum stress
To test whether the effects of pregnancy anxiety on the
developing brain could be mediated by the quality of the
postnatal environment, correlation analyses were performed
between pregnancy anxiety and sociodemographic charac-
teristics as well as postpartum stress. Generalized stress was
Sociodemographic characteristics and birthoutcomes.
dyads (N = 35)
Maternal age 32.7 ? 6.5
Annual household income
$0 to $30,000
$30,001 to $60,000
$60,001 to $100,000
19 weeks gestation
25 weeks gestation
31 weeks gestation
19.7 ? 6.5
17.7 ? 4.3
17.3 ? 4.4
2.1 ? 0.7
Perceived Stress postpartum
Length of gestation (weeks)38.8 ? 1.83
(11% 34—36.9 weeks)
Birth weight (g) 3527.5 ? 574.2
(0% <2500 g)
Pregnancy anxiety during gestation and brain morphology in 6-9 year-old children143
Author's personal copy
assessed at 8.2 (?2.9) weeks postpartum (range: 5—19
weeks) using a modification of the 10-item version of the
Perceived Stress Scale (Cohen and Williamson, 1988). As
shown in Table 2, pregnancy anxiety was not significantly
associated with maternal sociodemographic characteristics,
whilehighly significant correlations between pregnancy anxi-
ety at all time points during gestation and postpartum per-
ceived stress were observed.
2.6. Assessments in children
All children included in the study had a stable neonatal
course (all Apgar scores >8) and no emotional or physical
conditions were reported in a structured interview using the
MacArthur Health and Behavior Questionnaire (Armstrong
and Goldstein, 2003) at the ages of 6 and 9 years (mean:
7.2 ? 0.86), when they participated in an MRI scan. Child’s
handedness was assessed with a modified version of the
Edinburgh Handedness Inventory (Oldfield, 1971). For the
majority (86%) of children the dominant hand was the right.
2.7. MRI acquisition
Each child underwent an MRI scan conducted on a 3-T Philips
around the head. Ear protection was given to all children. To
fitted with headphones and allowed to watch a movie of their
choice while in the scanner. Following the scanner calibration
and pilot scans, a high resolution T1 anatomical scan was
acquired in the sagittal plane with 1 mm3isotropic voxel
dimensions. An Inversion-Recovery Spoiled Gradient Recalled
was applied: repetition rate (TR) = 11 ms, echo time
(TE) = 3.3 ms, inversion time (TI) = 1100 ms, turbo field echo
factor (TFE) = 192, number of slices: 150, no SENSE accelera-
tion, Flip angle = 188, Shot interval (time from inversion pulse
to the center of acquisition) = 2200 ms. Acquisition time for
this protocol was 7 min. Variations of these parameters were
tested on volunteers to obtain an optimal set that gave us the
while ensuring that there were no discernible artifacts. The
purpose was to keep the total acquisition time at a tolerable
length for children. Automatic brain segmentation software
was tested on these pilot scans to ensure gray-white matter
segmentation with minimal errors.
2.8. Processing of MRI data
Images were visually assessed by a neurologist for normal
anatomic appearance. The structural images were bias field
corrected, and segmented using an integrated generative
model (unified segmentation, Ashburner and Friston, 2005).
Unified segmentation involves alternating between segmen-
tation, bias field correction, and normalization to obtain
local optimal solutions for each process. The pediatric
CCHMC a priori templates (Wilke et al., 2002) were used
to segment and normalize (affine and 16 iteration non-linear
transformations) the children’s images. The resulting images
were modulated to correct voxel signal intensities for the
amount of volume displacement during normalization. The
normalized and segmented images were averaged across the
CSF sample specific a priori templates. The process was then
repeated using the sample specific a priori templates result-
ing in VBM probability maps of 1 mm isotropic voxels. The
normalized, segmented, and modulated images were then
smoothed using a 12 mm kernel to ensure that the data were
normally distributed and to limit the number of false positive
findings (Salmond et al., 2002). Coordinates of clusters (cen-
troids) were converted from original Montreal Neurological
Institute (MNI) coordinates to those of the Talairach brain
atlas (Talairach and Tournoux, 1988) using the mni2tal utility
(Matthew Brett 1999 GPL). Anatomical locations of the sig-
nificant areas are based on the best estimate from the
Talairach atlas using the Tailairach Daemon Client (http://
2.9. Total gray matter analysis
Total gray matter volume for each child was estimated based
on the volumes of the segmented and modulated images. The
sum of the nonzero voxel values in the image was calculated
and multiplied bythe voxel sizetoobtain anestimate oftotal
gray matter volume. Partial correlation analyses were per-
formed to test the association between pregnancy anxiety at
each of the three pregnancy visits and total gray matter
volume controlling for age, sex, gestational age at birth and
2.10. Voxel-based morphometry (VBM) analysis
To examine reductions in regional gray matter volume in
association with pregnancy anxiety, a multiple regression
model was employed with pregnancy anxiety at 19, 25 and
31 weeks gestation as the predictors of interest. Normal-
ization for global differences in gray matter concentration
across subjects was performed by controlling for total gray
matter volume. Consequently, the analysis detected regional
difference rather than overall, large-scale variations in gray
matter concentrations. By controlling for total gray matter
volume, differences in overall brain size were accounted for
Correlations between pregnancy anxiety during pregnancy and sociodemographic characteristics and perceived stress
at 19 weeks GA
at 25 weeks GA
at 31 weeks GA
Maternal education (years of school completed)
Annual household gross income
Perceived stress postpartum
?0.11 (p = 0.54)
0.14 (p = 0.43)
0.17 (p = 0.34)
0.44 (p = 0.01)
?0.17 (p = 0.92)
0.19 (p = 0.27)
0.27 (p = 0.12)
0.36 (p = 0.04)
?0.08 (p = 0.66)
0.18 (p = 0.31)
0.16 (p = 0.35)
0.56 (p = 0.00)
144 C. Buss et al.
Author's personal copy
that varies by sex. We furthermore controlled for child’s age
at assessment. Also, gestational age at birth was controlled
for in these analyses because preterm delivery has been
(e.g. Gimenez et al., 2004). In addition, handedness of the
child was controlled for because structural asymmetries of
the brain may be associated with handedness (Toga and
Thompson, 2003). Because pregnancy anxiety and postpar-
tum stress were highly correlated, we included postpartum
stress as an additional covariate in order to address the
association between prenatal stress and gray matter volume
independent of postnatal stress. Relative threshold masking
(threshold > 0.3) was used to minimize gray-white matter
boundary effects, and implicit masking was used to disregard
voxels with zero values. Analyses for detection of brain
regions that showed significantly reduced gray matter den-
sity in association with high pregnancy anxiety were per-
formed at p < 0.001 uncorrected, but only those voxels
within a cluster of at least 100 voxels that reached a False
Discovery Rate (FDR) threshold of p < 0.05 are reported.
3.1. Pregnancy anxiety over the course of
Table 1 shows mean pregnancy anxiety scores at each preg-
nancy visit and Figure 1 presents the distribution of preg-
nancy anxiety scores over the course of the three pregnancy
visits. With advancing gestational age pregnancy anxiety
scores significantly decreased (F(1.8,
resulting in lower scores at the second and third assessments
56.8)= 5.4, p = 0.01)
as compared to the first. As depicted in Table 3, Spearman’s
rho correlation coefficients suggested significant rank stabi-
lity in pregnancy anxiety scores over the course of gestation.
3.2. Pregnancy anxiety and global reductions in
gray matter volume
Pregnancy anxiety at any of the three time points during
pregnancy was not correlated with total gray matter volume
(T1: r = 0.05, p = 0.81; T2: r = 0.06, p = 0.75; T3: r = ?0.27,
p = 0.15).
3.3. Pregnancy anxiety and regional reductions
in gray matter volume
The VBM analysis revealed lower gray matter density in
several brain areas in association with high pregnancy
anxiety. The significance of the relation between pregnancy
anxiety and gray matter volume reductions varied across
gestation (see Table 4 and Figure 2). High pregnancy anxiety
at 19 weeks gestation was associated with significant,
mostly bilateral, gray matter volume reductions in the
anterior (Brodmann Area (BA) 10), orbitofrontal (BA 11
and BA 47), dorsolateral (BA 46 and BA 9) and ventrolateral
prefrontal cortex (BA 45). Also, lower gray matter density
was observed with high pregnancy anxiety at 19 weeks
gestation in the left precentral gyrus extending to the
middle frontal gyrus (BA 6). Reduced gray matter volume
in association with high pregnancy anxiety at 19 weeks
gestation was furthermore observed in the left medial
temporal lobe, uncus, extending to the entorhinal cortex
(BA 28) and the parahippocampal gyrus (BA 36) as well as in
the left temporal pole (BA 38) and the left inferior temporal
gyrus (BA 20). Bilateral reductions in gray matter volume
were found in children whose mothers reported higher
pregnancy anxiety in the lateral temporal cortex extending
from the superior temporal gyrus (BA 22) to the middle
temporal gyrus (BA 21) and on the right side to the post-
central gyrus. Reduction in gray matter volume in associa-
tion with high pregnancy anxiety at 19 weeks gestation was
also observed in the left postcentral gyrus as well as in the
left supramarginal gyrus (BA 39) and the right angular gyrus
(BA 39). Furthermore, in children of mothers with high
pregnancy anxiety at 19 weeks gestation, pronounced bilat-
eral gray matter volume reduction was found in the cere-
bellum extending to the middle occipital gyrus (BA 19) and
to the fusiform gyrus (BA 37). Clusters of voxels with
reduced gray matter density were found in association with
high pregnancy anxiety at 25 and 31 weeks (see Table 4) but
these did not survive FDR correction and are therefore not
reported here. Excluding either child of the sibling pair did
not significantly change the reported results.
tion. With advancing gestational age, pregnancy anxiety
Pregnancy anxiety scores over the course of gesta-
Rank correlations between pregnancy anxiety scores across pregnancy visits.
at 19 weeks GA
at 25 weeks GA
at 31 weeks GA
Pregnancy anxiety at 19 weeks GA
Pregnancy anxiety at 25 weeks GA
Pregnancy anxiety at 31 weeks GA
0.64 (p < 0.001)
0.65 (p < 0.001)
0.74 (p < 0.001)
Pregnancy anxiety during gestation and brain morphology in 6-9 year-old children145
Author's personal copy
most significant voxel in each of the clusters, and volumes for all clusters in the gray matter SPMs are displayed.
Cluster no Cerebral region Talairach coordinatesCluster
Pregnancy anxiety at 19 weeks gestation
1 Left Superior Frontal Gyrus (BA 10)
Right Superior Frontal Gyrus (BA 10)
2 Left Superior Frontal Gyrus (BA 10)
Left Superior Frontal Gyrus (BA 11)
Left Middle Frontal Gyrus (BA 10)
3 Left Superior Frontal Gyrus (BA 10)
Left Middle Frontal Gyrus (BA 10)
Left Inferior Frontal Gyrus (BA 46)
4 Left Inferior Frontal Gyrus (BA 9)
Left Middle Frontal Gyrus (BA 46)
5Left Inferior Frontal Gyrus (BA 47) 25
6Left Precentral Gyrus (BA 6)
Left Middle Frontal Gyrus (BA 6)
7Right Inferior Frontal Gyrus (BA 46)
Right Middle Frontal Gyrus (BA 46)
Right Inferior Frontal Gyrus (BA 45)
5 3794 4.9
8 Right Superior Frontal Gyrus (BA11)2 55
9Left Uncus (BA 28)
Left Uncus (BA36)
Left Superior Temporal Gyrus (BA 38)
10Left Superior Temporal Gyrus (BA 22)
Left Middle Temporal Gyrus (BA 21)
11Left Inferior Temporal Gyrus (BA 20) 1093.8
12 Right Middle Temporal Gyrus (BA 21)
Right Superior Temporal Gyrus (BA 22)
13 Left Middle Occipital Gyrus (BA 19)
Left Cerebellum (Tuber)
Left Cerebellum (Tuber)
Left Cerebellum (Pyramis)
16Right Cerebellum (Tuber)
Right Middle Occipital Gyrus (BA19)
Fusiform Gyrus (BA37)
Right Cerebellum (Pyramis)
Right Cerebellum (Tuber)
Left Postcentral Gyrus (BA 2)
Right Angular Gyrus (BA 39)
Left Supramarginal Gyrus (BA 39)
Pregnancy anxiety at 25 weeks gestation
22Right Middle Frontal Gyrus (BA 10)
23Left Middle Temporal Gyrus (BA 37)
24Left Lingual Gyrus (BA 17)
Pregnancy anxiety at 31 weeks gestation
25Right Inferior Frontal Gyrus (BA 47)
26 Left Insula (BA13)
27Right Postcentral Gyrus (BA 7)
146C. Buss et al.
Author's personal copy
We present the first evidence in humans that prenatal mater-
nal anxiety is associated with brain morphology in the devel-
oping individual within specific sensitive time periods.
Specifically, pregnancy anxiety at 19 weeks gestation was
associated with gray matter volume reductions in the pre-
frontal cortex, the premotor cortex, the medial temporal
lobe, the lateral temporal cortex, the postcentral gyrus as
well as the cerebellum extending to the middle occipital
gyrus and the fusiform gyrus. These associations with gray
matter density were confined to pregnancy anxiety reported
at 19 weeks gestation, as reports of pregnancy anxiety at 25
and 31weeks gestation were not significantly associated with
gray matter volume. Our findings are consistent with accu-
mulating evidence from animal studies that medial temporal
and prefrontal cortical regions are shaped by early experi-
ence (e.g. Coe et al., 2003; Salm et al., 2004; Fujioka et al.,
2006; Murmu et al., 2006).
Scales measuring pregnancy anxiety have been suggested
to better assess anxieties and worries related specifically to
pregnancy than general scales of stress, depression and
anxiety (Huizink et al., 2004; Dipietro et al., 2006). This is
emphasized by observations of pregnancy anxiety having a
higher predictive quality for birth outcomes and fetal/child
development than general stress scales (Wadhwa et al.,
1993; Huizink et al., 2003; DiPietro et al., 2004; Roesch
et al., 2004; Dipietro et al., 2006; Kramer et al., 2009; Davis
and Sandman, in press) and is furthermore supported by the
highly significant association between self-reported mater-
nal pregnancy anxiety and brain morphology in this study.
The brain regions that we have found to be affected by
pregnancy anxiety are areas specifically associated with
cognitive performance. The prefrontal cortex is sometimes
involved in executive cognitive functions such as reasoning,
planning, attention, working memory, and some aspects of
language (e.g. Connolly et al., 2002). Structures in the
medial temporal lobe, including areas connected to the
hippocampus (entorhinal, perirhinal, parahippocampal cor-
tex), have been proposed to constitute a ‘‘medial temporal
lobe memory system’’ with the primary functions of these
areas related to the storage and recall of facts and events
involved in social and emotional processing including recog-
nition and semantic memory (Nakamura and Kubota, 1996;
Hoistad and Barbas, 2008). A network in the temporal—
parietal cortex consisting of the middle temporal gyrus (BA
21), the superior temporal gyrus (BA 22) and the angular
gyrus (BA 39) has been shown to be important in processes
related to auditory language processing in children (Ahmad
et al., 2003). Also involved in language learning seems to be
another network of brain regions affected by pregnancy
anxiety: the inferior frontal gyrus (BA 45), the middle tem-
poral gyrus (BA 21) and the parahippocampal gyrus (Mestres-
Misse et al., 2008).
Importantly and consistent with the primary functions of
the affected brain regions, a small but growing literature
indicates that prenatal stress influences both cognitive
development as well as temperament. Thus, elevated pre-
ity to attend and with delayed cognitive development
(Brouwers et al., 2001; Huizink et al., 2002; O’Connor
et al., 2002; Buitelaar et al., 2003; Huizink et al., 2003;
Davis and Sandman, in press), lower academic achievement
in school (Niederhofer and Reiter, 2004), higher infant beha-
p < 0.001 (uncorrected) are displayed.
Areas of reduced gray matter volume in association with pregnancy anxiety at 19, 25 and 31 weeks gestation. Voxels with
Pregnancy anxiety during gestation and brain morphology in 6-9 year-old children147
Author's personal copy
vioral reactivity (Davis et al., 2004, 2005, 2007) and emo-
tional/behavioral problems that persisted until adolescence
(Van den Bergh et al., 2005, 2008). Furthermore, offspring of
women who were exposed to a natural disaster during their
pregnancies had poorer general intellectual functioning and
language development (Laplante et al., 2004, 2008) and
maternal exposure to natural disasters, war or stressful life
events have been associated with increased prevalence of
psychopathology in the offspring (van Os and Selten, 1998;
Selten et al., 1999; Watson et al., 1999; Beversdorf et al.,
matter density in the premotor cortex and the cerebellum
delayed motor development in association with prenatal
stress/anxiety (Buitelaar et al., 2003; Huizink et al., 2003).
Interestingly, a recent functional MRI study in humans
found that prefrontal regions, that we found are affected
by high pregnancy anxiety, are involved in the regulation of
stress hormone secretion (Pruessner et al., 2008). These
same brain regions appear to be particularly vulnerable
under conditions of chronic stress due to their high density
of glucocorticoid receptors (Sapolsky et al., 1990). Thus, by
its effect on these brain regions, high maternal prenatal
anxiety may increase the risk for higher stress susceptibility
and reactivity in the developing individuals. This may result
in higher concentrations of stress hormones which could
further delay brain development. These assumptions are
consistent with reports of higher baseline and stress-reactive
cortisol concentrations in children born to mothers with high
anxiety levels during pregnancy (Gutteling et al., 2005;
O’Connor et al., 2005; Van den Bergh et al., 2008).
Reduced gray matter density in the precentral and post-
with evidence for disturbed development of the nociceptive
system and associated behavioral changes in association with
prenatal stress (Smythe et al., 1994; Rokyta et al., 2008).
Occipital—temporal areas (middle occipital gyrus (BA19) and
fusiform gyrus), involved in visual processing, are furthermore
affected by pregnancy anxiety (Brandt et al., 2000).
Limbic structures, especially the hippocampus, have been
shown to be prominent targets for early life stress (e.g. Coe
et al., 2003; Buss et al., 2007). Still, we did not observe a
significant reduction in gray matter density in this region.
Before concluding that this area is not affected by maternal
pregnancy anxiety, alternative, potentially more sensitive,
methods of analyses (e.g. manual segmentation, shape ana-
lyses) are required.
The fetus participates in a dynamic exchange of environ-
mental (intrauterine) information with the maternal host
over the course of gestation. All communication between
the maternal and fetal compartments is mediated via the
placenta, an organ of fetal origin. One of the major placental
signals in pregnant primates is the peptide corticotrophin-
releasing hormone (CRH) which has been shown to be stress-
sensitive in in vitro studies (Petraglia et al., 1987, 1989,
1990). Other in vivo studies have found significant correla-
tions among maternal pituitary—adrenal stress hormones
(ACTH, cortisol) and placental corticotrophin-releasing hor-
mone (pCRH) concentrations (Goland et al., 1992; Chan
et al., 1993; Wadhwa et al., 1997; Hobel et al., 1999). Some
(Hobel et al., 1999; Erickson et al., 2001), but not all studies
(Petraglia et al., 2001), also have reported direct associa-
tions between maternal psychosocial stress and pCRH func-
tion. With the production and release of CRH from the
placenta, regulation of the HPA axis changes dramatically
during pregnancy. Maternal cortisol increases two- to four-
fold over the course of normal gestation (Mastorakos and
Ilias, 2003; Sandman et al., 2006) resulting from pCRH sti-
mulating production of maternal cortisol (Sasaki et al.,
1989). Maternal cortisol passes the placenta with 11b-hydro-
xysteroid dehydrogenase type 2 (11b-HSD2) presenting a
partial barrier (Brown et al., 1996). pCRH furthermore sti-
mulates cortisol secretion from the fetal adrenals directly, as
the CRH 1 receptor is present in human fetal adrenal tissue
from mid-gestation (Smith et al., 1998).
At high concentrations pCRH as well as cortisol may inhibit
growth and differentiation ofthe developing nervous system.
Thus considerable evidence indicates that glucocorticoids
are neurotoxic to hippocampal CA3 pyramidal cells (Sapolsky
to high levels of glucocorticoids produces irreversible
damage to the hippocampus (Uno et al., 1990, 1994). Larger
amounts of exogenously administered CRH increase limbic
neuronal excitation leading to seizures (Ehlers et al., 1983;
Baram et al., 1992, 1997) and may participate in mechanisms
of neuronal injury (Baram and Hatalski, 1998). The potential
mechanisms by which maternal stress and associated
increases in stress-sensitive hormones (pCRH, cortisol) may
produce long-lasting changes in brain function have been
suggested from animal models and may include changes in
neurotransmitter levels (Roceri et al., 2002; Kinnunen et al.,
2003; Pickering et al., 2006), adult neurogenesis (Lemaire
et al., 2000, 2006; Coe et al., 2003; Fujioka et al., 2006;
Odagiri et al., 2008) as well as cell growth and survival
(Roceri et al., 2002; Fumagalli et al., 2004; Van den Hove
et al., 2006; Aisa et al., in press).
Interestingly, pCRH as well as cortisol concentrations
during pregnancy predict fetal and infant development.
Low concentrations of pCRH at the beginning of the second
trimester are associated with precocious maturation of the
human fetus (Class et al., 2008), while elevated concentra-
tions of pCRH during the third trimester of gestation are
associated with impaired fetal learning (Sandman et al.,
1999). The developmental consequences of elevated con-
centrations of pCRH during pregnancy extend into postnatal
life, as higher pCRH concentrations during pregnancy are
associated with delayed neonatal physical and neuromuscu-
lar maturation (Ellman et al., 2008), more fearful tempera-
ments in infants (Davis et al., 2005), and an increase in
central adiposity in 3-year-old children (Gillman et al.,
2006). Endogenous maternal cortisol also plays a role in
shaping human development. Prenatal exposure to elevated
maternal cortisol has been shown to predict increased fussi-
ness,negative behaviorand fearfulnessininfancy(deWeerth
et al., 2003; Davis et al., 2007) and greater cortisol reactivity
in childhood (Gutteling et al., 2005) as well as delays in
mental (Huizink et al., 2003; Davis and Sandman, in press)
and motor development (Huizink et al., 2003).
The results of the current study suggest that earlier in
pregnancy, the effects of pregnancy anxiety on offspring’s
gray matter volume are most pronounced. This effect of
timing may be due to the fact that pregnancy anxiety is
highest at 19 weeks gestation and decreases over the course
of gestation, which is in line with previous observations of
148C. Buss et al.
Author's personal copy
reduced physiological and psychological stress reactivity as
pregnancy advances (Schulte et al., 1990; Glynn et al., 2001,
2004, 2008; de Weerth and Buitelaar, 2005) as well as with a
recent observation that pregnancy anxiety early (around 15
weeks gestation) but not later in gestation predicts mental
The effect of timing may also be related to the fact that
different brain regions have a unique timetable for devel-
This possibility has been supported by observations in rhesus
monkeys, where prenatal exposure to the same stressor had
greater effects on postnatal motor development if it
occurred earlier in gestation, when neuronal migration was
at its peak, than if it occurred in mid- to late gestation, when
synaptogenesis was at its peak (Schneider et al., 1999). The
implication of these findings is that the impact of stress
during pregnancy is not uniform but that stress earlier in
pregnancy may have more pronounced consequences for
brain development than at a later gestational stage.
It important to acknowledge that the observed conse-
quences of prenatal programming not only depend on the
timing of the insult and the brain region of interest but also
on the stage of assessment. Studies on postnatal brain devel-
opmenthaveclearlyshownregionaland temporalpatterns of
dynamic maturational change continuing through childhood
and adolescence. This implies that what we observed and
reported here in children ofthis age range maynot be final. It
is possible that at a later maturational stage, prenatal stress
exposure will confer a different morphological pattern.
Therefore, following-up these children into adolescence
and adulthood will provide valuable information on the
persistence of prenatal stress effects on brain morphology.
It cannot be ruled out that the prenatal stress effects on
brain morphology are moderated by postnatal exposures
(Buss et al., 2007). By controlling for several relevant vari-
ables including postnatal maternal stress and socioeconomic
status, it can be concluded though that the observed effects
of pregnancy anxiety on brain structure were not mediated
by these postnatal factors. Thus, the results suggest that,
independent of postnatal maternal stress prenatal stress has
an impact on the offspring’s brain morphology.
It has to be noted that while there was no indication of
psychiatric disorders and there was no report of treatment
for any disorders in the structured interviews that probed
such issues, the possibility of an undiagnosed disorder cannot
conducted. Pregnancy anxiety may be higher in women with
undiagnosed psychiatric disorders and accompanying endo-
crine alterations could impact on neurodevelopment of the
This is the first study in healthy children to show that
prenatal maternal anxiety is related to distinctive patterns
may increase vulnerability for certain neurodevelopmental
disorders and impair cognitive function. Therefore the results
suggest that addressing mothers’ pregnancy-related concerns
Role of funding source
This research was supported by National Institute of Health
grants NS-41298, HD-51852 AND HD28413 to CAS. The NIH had
interpretation of data, in the writing of this report, or in the
decision to submit this report for publication.
Conflict of interest
All authors acknowledge no conflict of interest.
This research was supported by National Institute of Health
grants NS-41298, HD-51852 and HD28413 to CAS. We grate-
fully acknowledge contributions made by staff of our
Research Laboratory for data collection, especially Christina
Canino and Cheryl Crippen. We also thank the mothers and
children who participated.
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