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High pregnancy anxiety during mid-gestation isassociated with decreased gray matter densityin 6—9-year-old children

Claudia Buss a, Elysia Poggi Davis a,b, L. Tugan Muftuler c, Kevin Head c,Curt A. Sandman a,*

aDepartment of Psychiatry and Human Behavior, University of California, Irvine, 333 The City Blvd. W,Suite 1200, Orange, CA 92868, United StatesbDepartment of Pediatrics, University of California, Irvine, CA 92697-5020, United StatescDepartment 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

KEYWORDSPregnancy anxiety;Pregnancy;Prenatal stress;Longitudinal;MRI;Gray matter volume

Summary Because the brain undergoes dramatic changes during fetal development it isvulnerable to environmental insults. There is evidence that maternal stress and anxiety duringpregnancy influences birth outcome but there are no studies that have evaluated the influence ofstress during human pregnancy on brain morphology. In the current prospective longitudinal studywe 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 werebetween 6 and 9 years of age, their neurodevelopmental stage was assessed by a structural MRIscan. With the application of voxel-based morphometry, we found regional reductions in graymatter density in association with pregnancy anxiety after controlling for total gray mattervolume, age, gestational age at birth, handedness and postpartum perceived stress. Specifically,independent of postnatal stress, pregnancy anxiety at 19 weeks gestation was associated withgray matter volume reductions in the prefrontal cortex, the premotor cortex, the medialtemporal lobe, the lateral temporal cortex, the postcentral gyrus as well as the cerebellumextending to the middle occipital gyrus and the fusiform gyrus. High pregnancy anxiety at 25 and31 weeks gestation was not significantly associated with local reductions in gray mattervolume.This is the first prospective study to show that a specific temporal pattern of pregnancyanxiety is related to specific changes in brain morphology. Altered gray matter volume in brainregions affected by prenatal maternal anxiety may render the developing individual morevulnerable to neurodevelopmental and psychiatric disorders as well as cognitive and intellectualimpairment.Published by Elsevier Ltd.

* Corresponding author. Tel.: +1 714 940 1924; fax: +1 714 940 1939.E-mail address: [email protected] (C.A. Sandman).

ava i lab le at www.sc ienced i rect .com

journa l homepage: www.e l sev ie r.com/locate/psyneuen

0306-4530/$ — see front matter. Published by Elsevier Ltd.doi:10.1016/j.psyneuen.2009.07.010


1. Introduction

Programming refers to the action of a factor during a sensi-tive developmental period affecting the organization andmaturity of specific organs. One key assumption of theprogramming hypothesis is that biological systems under-going rapid developmental changes are especially vulnerableto organizing and disorganizing influences (Nathanielsz et al.,2003; Seckl and Meaney, 2004). Considering the extendedpre- and postnatal developmental trajectory of the brain, itis apparent that early life experiences have the potential tosculpt brain morphology. Such sculpting of the immaturebrain is an interactive process between genetic program-ming, cell function and the environment (Andersen, 2003).Growing evidence suggests that abnormal development ofthe brain during gestation contributes to many neurologicaldisorders 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 thehuman central nervous system following a protracted, neatlyorchestrated chain of specific ontogenetic events. Althoughbrain change and adaptation are part of a lifelong process,the earliest phases of maturation during fetal developmentand childhood are perhaps the most dramatic and important(Toga et al., 2006). Understanding the timing of neurodeve-lopmental events is essential for determining how particularenvironmental disturbances can selectively affect certainfunctions. During fetal life, neurons proliferate, migrate,and aggregate, providing the ‘‘hardware’’ for the developingbrain. Neural proliferation before birth has been estimatedat an average rate of 250,000 cells/min (Cowan, 1979).Between gestational ages 8 and 16 weeks, migrating neuronsform the subplate zone and await connections from afferentneurons originating in the thalamus, basal forebrain, andbrainstem (Kostovic et al., 2002). Once neurons reach theirfinal destination at about the 16th fetal week, they arborizeand branch in an attempt to establish appropriate connec-tions (Sidman and Rakic, 1973). Axon collaterals connect tonumerous regions in the brain before the neuron finishesmigrating 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 byestablishing a chemical gradient to follow (Knoll andDrescher, 2002). During development of the human brain,little synapse formation occurs before the beginning of thethird trimester, when it accelerates to approximately 40,000synapses/min (Bourgeois, 1997).The presence of periods withspecific neurodevelopmental events results in windows ofspecific vulnerability for adverse influences. In animal mod-els, changes in brain morphology have been observed inoffspring of mothers exposed to prenatal stress. In non-human primates, daily acute prenatal stress is associatedwith 10—12% reductions in hippocampal volume and inhibi-tion of neurogenesis in the dentate gyrus (Coe et al., 2003) aswell as altered size of the corpus callosum (Coe et al., 2002).Several lines of evidence from studies in rodents indicate thatthe cytoarchitecture of the rat hippocampus is altered as aconsequence of prenatal stress (Hayashi et al., 1998; Gouldand Tanapat, 1999; Lemaire et al., 2000). Impaired neuro-genesis and associated cognitive impairment have beenrepeatedly 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 byimpairing long-term potentiation but facilitating long-termdepression (Yaka et al., 2007; Yang et al., 2007). Althoughprenatal stress associated changes in the hippocampal for-mation have received major attention, morphologicalchanges in other brain regions have been shown as well.For example significantly expanded dimensions of the lateralnucleus of the amygdala were observed in prenatally stressedoffspring (Salm et al., 2004). Also, reduced spine densitiesand significant reduction of dendritic length of pyramidalneurons in the dorsal anterior cingulate and orbitofrontalcortex have been reported in offspring of mothers exposed tostress during pregnancy (Murmu et al., 2006). Since therodent brain is less mature at birth than the human brain,it has been suggested that brain maturation occurring in theearly postnatal period in the rat is analogous to maturationalchanges that occur in humans in late gestation (Clancy et al.,2001). Therefore, it is interesting to note that there is animpressive body of evidence from rodent studies showingchanges in brain morphology in association withmanipulationof 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).

Inhumans, changes inbrainmorphology havebeen reportedin individuals born prematurely. Thus, low birth weight as wellas pretermbirth have been related to changes in regional brainvolumes (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 bemarkers of in utero stress exposure (e.g. Wadhwa, 2005) butthe changes in brain morphology may also be due to perinatalcomplications that are often associated with premature deliv-ery. To thebestofourknowledgenostudy todate inhumanshasinvestigated the association between prenatal stress exposureand brain morphology in the offspring.

Pregnancy anxiety has been suggested to be a moresensitive 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 adistinctive syndrome (Huizink et al., 2004). Further evidencesuggests that measures of pregnancy specific stress are bet-ter than measures of generalized psychological distress forpredicting developmental outcomes including, fetal behavior(DiPietro et al., 2002), infant cognitive and motor develop-ment (Huizink et al., 2003; Dipietro et al., 2006; Davis andSandman, in press) and infant emotional regulation (Dipietroet 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 overthe course of gestation, and reductions in graymatter volumein their 6—9-year-old offspring.

2. Methods

2.1. Participants

Pregnant women were recruited for study participationbetween 1998 and 2002. Five hundred and fifty seven preg-nant women, who received prenatal care from the faculty

142 C. Buss et al.


obstetric practice at the University of California, IrvineMedical Center or Cedars-Sinai Hospital in Los Angeles, wererecruited by the 15th week of gestation and provided writ-ten, informed consent. All methods and procedures wereapproved by the Institutional Review Board of the participat-ing institutions. Study participants were English-speakingadult women (>18 years age) with singleton, intrauterinepregnancies. Exclusion criteria included tobacco, alcohol, orother drug use in pregnancy; uterine or cervical abnormal-ities; or presence of any condition potentially associated withdysregulated neuroendocrine function such as endocrine,hepatic or renal disorders or corticosteroid medicationuse. While not an exclusion criterion, none of the womenin this sample were treated for any psychiatric disorders. Inthe context of an on-going study on the effects of prenatalstress exposure on child brain development that started in2007, women were re-contacted and invited to participate ina follow-up study of their children. Three hundred and fortywomen of the initial sample were located. Fifty-two childrenhave undergone and MRI scan to date; of those one MRI scanhad to be excluded due to morphological abnormalities andtwo MRI scans due to severe motion artifacts. Among the 49mother—child dyads with usable MRI data, 35 women hadprovided complete maternal stress data at three time pointsbetween 19 and 31 weeks gestation as well as postpartum andare included in the current report. Among these 35 childrenwere two siblings; thus one mother was enrolled in the studywith two subsequent pregnancies and consequently twochildren for the follow-up study. Women whose childrenparticipated in the follow-up study of brain developmentdid not differ in sociodemographic characteristics (maternalage and education, annual household income) fromwomen inthe initial sample (all p > 0.4).

2.2. Assessments in pregnant women

For all pregnant women gestational age was determined bybest obstetric estimate with a combination of last menstrualperiod and early uterine size, and was confirmed by obstetricultrasonographic biometry before 20 weeks using standardclinical criteria (O’Brien et al., 1981). Medical risk wasdefined as the presence of certain medical conditions inthe index pregnancy or previous pregnancies (e.g. vaginalbleeding, pregnancy-induced hypertension, preeclampsia,infection, Hobel, 1982). Risk conditions were determinedthrough interview and extensive medical chart review. Thesum of medical risk factors was calculated as an indicator ofpresence of any current or historical risk conditions. Infor-mation on birth outcomes was retrieved from medical chartsafter delivery. Sociodemographic characteristics and birthoutcomes are summarized in Table 1.

2.3. Pregnancy anxiety

Pregnancy anxiety was assessed over the course of gestationat 19 (�0.83, SD), 25 (�0.9) and 31 (�0.9) weeks gestation,and for all 35 children included in the current analysescomplete data was available. A 10-item pregnancy anxietyscale, which assesses a woman’s feelings about her healthduring pregnancy, the health of her baby, and her feelingsabout labor and delivery, was administered at all three study

visits. Answers were given on a 4-point scale and includeditems such as: ‘‘I am fearful regarding the health of mybaby,’’ ‘‘I am concerned or worried about losing my baby,’’and ‘‘I am concerned or worried about developing medicalproblems during my pregnancy.’’ The final score on thismeasure 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 thedeveloping brain could be mediated by the presence ofmedical risk, correlation analyses were performed betweenpregnancy anxiety and the number ofmedical risk conditions.At none of the assessments, significant correlations could beobserved (19 weeks: r = �0.14, p = 0.42; 26 weeks:r = �0.14, p = 0.44, 31 weeks: r = 0.02, p = 0.92).

2.5. Pregnancy anxiety, sociodemographiccharacteristics and postpartum stress

To test whether the effects of pregnancy anxiety on thedeveloping brain could be mediated by the quality of thepostnatal environment, correlation analyses were performedbetween pregnancy anxiety and sociodemographic charac-teristics as well as postpartum stress. Generalized stress was

Table 1 Sociodemographic characteristics and birth outcomes.

Mother—childdyads (N = 35)

Maternal age 32.7 � 6.5

Race/ethnicityNon-Hispanic White 37.1%Hispanic White 20.0%African American 8.6%Asian 31.4%Other 2.9%

Annual household income$0 to $30,000 32.4%$30,001 to $60,000 11.8%$60,001 to $100,000 26.4%Over $100,000 29.4%

Pregnancy anxiety19 weeks gestation 19.7 � 6.525 weeks gestation 17.7 � 4.331 weeks gestation 17.3 � 4.4

Perceived Stress postpartum 2.1 � 0.7

Primiparous 44.1%

Child sexMale 51.4%Female 48.6%

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 children 143


assessed at 8.2 (�2.9) weeks postpartum (range: 5—19weeks) using a modification of the 10-item version of thePerceived Stress Scale (Cohen and Williamson, 1988). Asshown in Table 2, pregnancy anxiety was not significantlyassociated with maternal sociodemographic characteristics,while highly 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 neonatalcourse (all Apgar scores >8) and no emotional or physicalconditions were reported in a structured interview using theMacArthur Health and Behavior Questionnaire (Armstrongand Goldstein, 2003) at the ages of 6 and 9 years (mean:7.2 � 0.86), when they participated in an MRI scan. Child’shandedness was assessed with a modified version of theEdinburgh Handedness Inventory (Oldfield, 1971). For themajority (86%) of children the dominant hand was the right.

2.7. MRI acquisition

Each child underwent an MRI scan conducted on a 3-T PhilipsAchieva system. Tominimize headmotion, paddingwas placedaround the head. Ear protection was given to all children. Tofurther increase complianceand reducemotion, childrenwerefitted with headphones and allowed to watch a movie of theirchoice while in the scanner. Following the scanner calibrationand pilot scans, a high resolution T1 anatomical scan wasacquired in the sagittal plane with 1 mm3 isotropic voxeldimensions. An Inversion-Recovery Spoiled Gradient RecalledAcquisition (IR-SPGR) sequence with the following parameterswas applied: repetition rate (TR) = 11 ms, echo time(TE) = 3.3 ms, inversion time (TI) = 1100 ms, turbo field echofactor (TFE) = 192, number of slices: 150, no SENSE accelera-tion, Flip angle = 188, Shot interval (time from inversion pulseto the center of acquisition) = 2200 ms. Acquisition time forthis protocol was 7 min. Variations of these parameters weretested on volunteers to obtain an optimal set that gave us thebest gray-whitematter contrast, sharpness andhigh resolutionwhile ensuring that there were no discernible artifacts. Thepurpose was to keep the total acquisition time at a tolerablelength for children. Automatic brain segmentation softwarewas tested on these pilot scans to ensure gray-white mattersegmentation with minimal errors.

2.8. Processing of MRI data

Images were visually assessed by a neurologist for normalanatomic appearance. The structural images were bias field

corrected, and segmented using an integrated generativemodel (unified segmentation, Ashburner and Friston, 2005).Unified segmentation involves alternating between segmen-tation, bias field correction, and normalization to obtainlocal optimal solutions for each process. The pediatricCCHMC a priori templates (Wilke et al., 2002) were usedto segment and normalize (affine and 16 iteration non-lineartransformations) the children’s images. The resulting imageswere modulated to correct voxel signal intensities for theamount of volume displacement during normalization. Thenormalized and segmented images were averaged across thechildren’s datasets to produce graymatter, whitematter, andCSF sample specific a priori templates. The process was thenrepeated using the sample specific a priori templates result-ing in VBM probability maps of 1 mm isotropic voxels. Thenormalized, segmented, and modulated images were thensmoothed using a 12 mm kernel to ensure that the data werenormally distributed and to limit the number of false positivefindings (Salmond et al., 2002). Coordinates of clusters (cen-troids) were converted from original Montreal NeurologicalInstitute (MNI) coordinates to those of the Talairach brainatlas (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 theTalairach atlas using the Tailairach Daemon Client (http://www.talairach.org/client.html).

2.9. Total gray matter analysis

Total gray matter volume for each child was estimated basedon the volumes of the segmented and modulated images. Thesum of the nonzero voxel values in the image was calculatedandmultiplied by the voxel size to obtain an estimate of totalgray matter volume. Partial correlation analyses were per-formed to test the association between pregnancy anxiety ateach of the three pregnancy visits and total gray mattervolume controlling for age, sex, gestational age at birth andpostpartum stress.

2.10. Voxel-based morphometry (VBM) analysis

To examine reductions in regional gray matter volume inassociation with pregnancy anxiety, a multiple regressionmodel was employed with pregnancy anxiety at 19, 25 and31 weeks gestation as the predictors of interest. Normal-ization for global differences in gray matter concentrationacross subjects was performed by controlling for total graymatter volume. Consequently, the analysis detected regionaldifference rather than overall, large-scale variations in graymatter concentrations. By controlling for total gray mattervolume, differences in overall brain size were accounted for

Table 2 Correlations between pregnancy anxiety during pregnancy and sociodemographic characteristics and perceived stresspostpartum.

Pregnancy anxietyat 19 weeks GA



Maternal age �0.11 (p = 0.54) �0.17 (p = 0.92) �0.08 (p = 0.66)Maternal education (years of school completed) 0.14 ( p = 0.43) 0.19 (p = 0.27) 0.18 (p = 0.31)Annual household gross income 0.17 (p = 0.34) 0.27 (p = 0.12) 0.16 (p = 0.35)Perceived stress postpartum 0.44 (p = 0.01) 0.36 (p = 0.04) 0.56 (p = 0.00)

144 C. Buss et al.


that varies by sex. We furthermore controlled for child’s ageat assessment. Also, gestational age at birth was controlledfor in these analyses because preterm delivery has beenshown to be associatedwith reductions in graymatter volume(e.g. Gimenez et al., 2004). In addition, handedness of thechild was controlled for because structural asymmetries ofthe brain may be associated with handedness (Toga andThompson, 2003). Because pregnancy anxiety and postpar-tum stress were highly correlated, we included postpartumstress as an additional covariate in order to address theassociation between prenatal stress and gray matter volumeindependent of postnatal stress. Relative threshold masking(threshold > 0.3) was used to minimize gray-white matterboundary effects, and implicit masking was used to disregardvoxels with zero values. Analyses for detection of brainregions 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 voxelswithin a cluster of at least 100 voxels that reached a FalseDiscovery Rate (FDR) threshold of p < 0.05 are reported.

3. Results

3.1. Pregnancy anxiety over the course ofgestation

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 pregnancyvisits. With advancing gestational age pregnancy anxietyscores significantly decreased (F(1.8, 56.8) = 5.4, p = 0.01)resulting in lower scores at the second and third assessments

as compared to the first. As depicted in Table 3, Spearman’srho correlation coefficients suggested significant rank stabi-lity in pregnancy anxiety scores over the course of gestation.

3.2. Pregnancy anxiety and global reductions ingray matter volume

Pregnancy anxiety at any of the three time points duringpregnancy 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 reductionsin gray matter volume

The VBM analysis revealed lower gray matter density inseveral brain areas in association with high pregnancyanxiety. The significance of the relation between pregnancyanxiety and gray matter volume reductions varied acrossgestation (see Table 4 and Figure 2). High pregnancy anxietyat 19 weeks gestation was associated with significant,mostly bilateral, gray matter volume reductions in theanterior (Brodmann Area (BA) 10), orbitofrontal (BA 11and BA 47), dorsolateral (BA 46 and BA 9) and ventrolateralprefrontal cortex (BA 45). Also, lower gray matter densitywas observed with high pregnancy anxiety at 19 weeksgestation in the left precentral gyrus extending to themiddle frontal gyrus (BA 6). Reduced gray matter volumein association with high pregnancy anxiety at 19 weeksgestation was furthermore observed in the left medialtemporal lobe, uncus, extending to the entorhinal cortex(BA 28) and the parahippocampal gyrus (BA 36) as well as inthe left temporal pole (BA 38) and the left inferior temporalgyrus (BA 20). Bilateral reductions in gray matter volumewere found in children whose mothers reported higherpregnancy anxiety in the lateral temporal cortex extendingfrom the superior temporal gyrus (BA 22) to the middletemporal 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 wasalso observed in the left postcentral gyrus as well as in theleft supramarginal gyrus (BA 39) and the right angular gyrus(BA 39). Furthermore, in children of mothers with highpregnancy 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) andto the fusiform gyrus (BA 37). Clusters of voxels withreduced gray matter density were found in association withhigh pregnancy anxiety at 25 and 31 weeks (see Table 4) butthese did not survive FDR correction and are therefore notreported here. Excluding either child of the sibling pair didnot significantly change the reported results.

Figure 1 Pregnancy anxiety scores over the course of gesta-tion. With advancing gestational age, pregnancy anxietydecreases.

Table 3 Rank correlations between pregnancy anxiety scores across pregnancy visits.




Pregnancy anxiety at 19 weeks GA — 0.64 ( p < 0.001) 0.65 ( p < 0.001)Pregnancy anxiety at 25 weeks GA — — 0.74 ( p < 0.001)Pregnancy anxiety at 31 weeks GA — — —



Table 4 Local reductions in graymatter density in association with high pregnancy anxiety: Talairach coordinates and z-scores for themost significant voxel in each of the clusters, and volumes for all clusters in the gray matter SPMs are displayed.

Cluster no Cerebral region Talairach coordinates Clustersize

t-Value Voxel p(uncor)

Voxel p(FDR-cor)

x y z

Pregnancy anxiety at 19 weeks gestation1 Left Superior Frontal Gyrus (BA 10) 0 67 25 5523 7.0 <0.001 0.02

Right Superior Frontal Gyrus (BA 10) 34 67 �4 4.8 <0.001 0.02

2 Left Superior Frontal Gyrus (BA 10) �28 69 �9 3582 5.5 <0.001 0.02Left Superior Frontal Gyrus (BA 11) �23 53 �19 5.4 <0.001 0.02Left Middle Frontal Gyrus (BA 10) �39 62 �11 4.8 <0.001 0.02

3 Left Superior Frontal Gyrus (BA 10) �37 64 19 734 4.4 <0.001 0.02Left Middle Frontal Gyrus (BA 10) �50 57 4 3.9 <0.001 0.03Left Inferior Frontal Gyrus (BA 46) �54 51 5 3.7 <0.001 0.03

4 Left Inferior Frontal Gyrus (BA 9) �62 23 30 442 3.9 <0.001 0.03Left Middle Frontal Gyrus (BA 46) �61 31 26 3.5 0.001 0.04

5 Left Inferior Frontal Gyrus (BA 47) �55 25 �9 236 4.2 <0.001 0.03

6 Left Precentral Gyrus (BA 6) �66 10 40 2854 4.3 <0.001 0.02Left Middle Frontal Gyrus (BA 6) �51 6 52 4.3 <0.001 0.03

7 Right Inferior Frontal Gyrus (BA 46) 53 50 5 3794 4.9 <0.001 0.02Right Middle Frontal Gyrus (BA 46) 57 32 28 4.3 <0.001 0.03Right Inferior Frontal Gyrus (BA 45) 61 25 9 4.2 <0.001 0.03

8 Right Superior Frontal Gyrus (BA11) 2 55 �21 1543 4.7 <0.001 0.03

9 Left Uncus (BA 28) �13 3 �25 3398 5.3 <0.001 0.02Left Uncus (BA36) �25 10 �37 5.1 <0.001 0.02Left Superior Temporal Gyrus (BA 38) �20 13 �28 4.7 <0.001 0.02

10 Left Superior Temporal Gyrus (BA 22) �73 �13 0 1375 4.3 <0.001 0.03Left Middle Temporal Gyrus (BA 21) �76 �26 �5 4.2 <0.001 0.03

11 Left Inferior Temporal Gyrus (BA 20) �49 �12 �37 109 3.8 <0.001 0.03

12 Right Middle Temporal Gyrus (BA 21) 75 �33 �8 6241 5.3 <0.001 0.02Right Superior Temporal Gyrus (BA 22) 74 �35 14 4.9 <0.001 0.02

13 Left Middle Occipital Gyrus (BA 19) �58 �71 �12 3969 5.9 <0.001 0.02Left Cerebellum (Tuber) �57 �52 �23 4.0 <0.001 0.03

14 Left Cerebellum (Tuber) �32 �93 �28 4644 5.1 <0.001 0.0315 Left Cerebellum (Pyramis) �54 �69 �32 181 3.6 0.001 0.04

16 Right Cerebellum (Tuber) 61 �64 �19 2742 5.7 <0.001 0.02Right Middle Occipital Gyrus (BA19) 62 �72 �6 3.9 <0.001 0.03Fusiform Gyrus (BA37) 62 �75 1 3.8 <0.001 0.03

17 Right Cerebellum (Pyramis) 53 �72 �32 2747 5.3 <0.001 0.0218 Right Cerebellum (Tuber) 43 �88 �24 827 4.2 <0.001 0.0319 Left Postcentral Gyrus (BA 2) �56 �28 59 348 4.4 <0.001 0.0220 Right Angular Gyrus (BA 39) 60 �68 40 399 4.2 <0.001 0.0221 Left Supramarginal Gyrus (BA 39) �70 �56 24 331 4.1 <0.001 0.02

Pregnancy anxiety at 25 weeks gestation22 Right Middle Frontal Gyrus (BA 10) 33 37 12 115 3.8 <0.001 0.9423 Left Middle Temporal Gyrus (BA 37) �42 �58 �2 106 4.0 <0.001 0.9424 Left Lingual Gyrus (BA 17) �11 �93 �9 148 3.9 <0.001 0.94

Pregnancy anxiety at 31 weeks gestation25 Right Inferior Frontal Gyrus (BA 47) 32 25 2 617 4.7 <0.001 0.9526 Left Insula (BA13) �38 6 17 200 3.9 <0.001 0.9527 Right Postcentral Gyrus (BA 7) 18 �53 65 121 3.9 <0.001 0.95

146 C. Buss et al.


4. Discussion

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 wasassociated with gray matter volume reductions in the pre-frontal cortex, the premotor cortex, the medial temporallobe, the lateral temporal cortex, the postcentral gyrus aswell as the cerebellum extending to the middle occipitalgyrus and the fusiform gyrus. These associations with graymatter density were confined to pregnancy anxiety reportedat 19 weeks gestation, as reports of pregnancy anxiety at 25and 31 weeks gestation were not significantly associated withgray matter volume. Our findings are consistent with accu-mulating evidence from animal studies that medial temporaland 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 suggestedto better assess anxieties and worries related specifically topregnancy than general scales of stress, depression andanxiety (Huizink et al., 2004; Dipietro et al., 2006). This isemphasized by observations of pregnancy anxiety having ahigher predictive quality for birth outcomes and fetal/childdevelopment than general stress scales (Wadhwa et al.,1993; Huizink et al., 2003; DiPietro et al., 2004; Roeschet al., 2004; Dipietro et al., 2006; Kramer et al., 2009; Davisand Sandman, in press) and is furthermore supported by thehighly 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 bypregnancy anxiety are areas specifically associated with

cognitive performance. The prefrontal cortex is sometimesdescribed as the ‘‘highest’’ structure of the brain because it isinvolved in executive cognitive functions such as reasoning,planning, attention, working memory, and some aspects oflanguage (e.g. Connolly et al., 2002). Structures in themedial temporal lobe, including areas connected to thehippocampus (entorhinal, perirhinal, parahippocampal cor-tex), have been proposed to constitute a ‘‘medial temporallobe memory system’’ with the primary functions of theseareas related to the storage and recall of facts and events(Squire et al., 2004). The temporal polar cortex appears to beinvolved 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 (BA21), the superior temporal gyrus (BA 22) and the angulargyrus (BA 39) has been shown to be important in processesrelated to auditory language processing in children (Ahmadet al., 2003). Also involved in language learning seems to beanother network of brain regions affected by pregnancyanxiety: 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 ofthe affected brain regions, a small but growing literatureindicates that prenatal stress influences both cognitivedevelopment as well as temperament. Thus, elevated pre-natal maternal stress/anxiety is associated with infant inabil-ity to attend and with delayed cognitive development(Brouwers et al., 2001; Huizink et al., 2002; O’Connoret al., 2002; Buitelaar et al., 2003; Huizink et al., 2003;Davis and Sandman, in press), lower academic achievementin school (Niederhofer and Reiter, 2004), higher infant beha-

Figure 2 Areas of reduced gray matter volume in association with pregnancy anxiety at 19, 25 and 31 weeks gestation. Voxels withp < 0.001 (uncorrected) are displayed.



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 ofwomen who were exposed to a natural disaster during theirpregnancies had poorer general intellectual functioning andlanguage development (Laplante et al., 2004, 2008) andmaternal exposure to natural disasters, war or stressful lifeevents have been associated with increased prevalence ofpsychopathology in the offspring (van Os and Selten, 1998;Selten et al., 1999; Watson et al., 1999; Beversdorf et al.,2005; Khashan et al., 2008). Our observations of reduced graymatter density in the premotor cortex and the cerebellummay provide the anatomical basis for previous observations ofdelayed motor development in association with prenatalstress/anxiety (Buitelaar et al., 2003; Huizink et al., 2003).

Interestingly, a recent functional MRI study in humansfound that prefrontal regions, that we found are affectedby high pregnancy anxiety, are involved in the regulation ofstress hormone secretion (Pruessner et al., 2008). Thesesame brain regions appear to be particularly vulnerableunder conditions of chronic stress due to their high densityof glucocorticoid receptors (Sapolsky et al., 1990). Thus, byits effect on these brain regions, high maternal prenatalanxiety may increase the risk for higher stress susceptibilityand reactivity in the developing individuals. This may resultin higher concentrations of stress hormones which couldfurther delay brain development. These assumptions areconsistent with reports of higher baseline and stress-reactivecortisol concentrations in children born to mothers with highanxiety 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-centralgyrus inassociationwithpregnancyanxiety isconsistentwith evidence for disturbed development of the nociceptivesystem and associated behavioral changes in association withprenatal stress (Smythe et al., 1994; Rokyta et al., 2008).Occipital—temporal areas (middle occipital gyrus (BA19) andfusiform gyrus), involved in visual processing, are furthermoreaffected by pregnancy anxiety (Brandt et al., 2000).

Limbic structures, especially the hippocampus, have beenshown to be prominent targets for early life stress (e.g. Coeet al., 2003; Buss et al., 2007). Still, we did not observe asignificant reduction in gray matter density in this region.Before concluding that this area is not affected by maternalpregnancy 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 hostover the course of gestation. All communication betweenthe maternal and fetal compartments is mediated via theplacenta, an organ of fetal origin. One of the major placentalsignals 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; Chanet 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 theplacenta, regulation of the HPA axis changes dramaticallyduring pregnancy. Maternal cortisol increases two- to four-fold over the course of normal gestation (Mastorakos andIlias, 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 apartial barrier (Brown et al., 1996). pCRH furthermore sti-mulates cortisol secretion from the fetal adrenals directly, asthe CRH 1 receptor is present in human fetal adrenal tissuefrom mid-gestation (Smith et al., 1998).

At high concentrations pCRH as well as cortisol may inhibitgrowth and differentiation of the developing nervous system.Thus considerable evidence indicates that glucocorticoidsare neurotoxic to hippocampal CA3 pyramidal cells (Sapolskyet al., 1985, 1990; Magarinos et al., 1996), and fetal exposureto high levels of glucocorticoids produces irreversibledamage to the hippocampus (Uno et al., 1990, 1994). Largeramounts of exogenously administered CRH increase limbicneuronal excitation leading to seizures (Ehlers et al., 1983;Baram et al., 1992, 1997) and may participate in mechanismsof neuronal injury (Baram and Hatalski, 1998). The potentialmechanisms by which maternal stress and associatedincreases in stress-sensitive hormones (pCRH, cortisol) mayproduce long-lasting changes in brain function have beensuggested from animal models and may include changes inneurotransmitter levels (Roceri et al., 2002; Kinnunen et al.,2003; Pickering et al., 2006), adult neurogenesis (Lemaireet 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 Hoveet al., 2006; Aisa et al., in press).

Interestingly, pCRH as well as cortisol concentrationsduring pregnancy predict fetal and infant development.Low concentrations of pCRH at the beginning of the secondtrimester are associated with precocious maturation of thehuman fetus (Class et al., 2008), while elevated concentra-tions of pCRH during the third trimester of gestation areassociated with impaired fetal learning (Sandman et al.,1999). The developmental consequences of elevated con-centrations of pCRH during pregnancy extend into postnatallife, as higher pCRH concentrations during pregnancy areassociated 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 incentral adiposity in 3-year-old children (Gillman et al.,2006). Endogenous maternal cortisol also plays a role inshaping human development. Prenatal exposure to elevatedmaternal cortisol has been shown to predict increased fussi-ness, negative behavior and fearfulness in infancy (deWeerthet al., 2003; Davis et al., 2007) and greater cortisol reactivityin childhood (Gutteling et al., 2005) as well as delays inmental (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 inpregnancy, the effects of pregnancy anxiety on offspring’sgray matter volume are most pronounced. This effect oftiming may be due to the fact that pregnancy anxiety ishighest at 19 weeks gestation and decreases over the courseof gestation, which is in line with previous observations of

148 C. Buss et al.


reduced physiological and psychological stress reactivity aspregnancy advances (Schulte et al., 1990; Glynn et al., 2001,2004, 2008; de Weerth and Buitelaar, 2005) as well as with arecent observation that pregnancy anxiety early (around 15weeks gestation) but not later in gestation predicts mentaldevelopment at 12months age (Davis and Sandman, in press).The effect of timing may also be related to the fact thatdifferent brain regions have a unique timetable for devel-opment and therefore specific periods of neural vulnerability.This possibility has been supported by observations in rhesusmonkeys, where prenatal exposure to the same stressor hadgreater effects on postnatal motor development if itoccurred earlier in gestation, when neuronal migration wasat its peak, than if it occurred in mid- to late gestation, whensynaptogenesis was at its peak (Schneider et al., 1999). Theimplication of these findings is that the impact of stressduring pregnancy is not uniform but that stress earlier inpregnancy may have more pronounced consequences forbrain development than at a later gestational stage.

It important to acknowledge that the observed conse-quences of prenatal programming not only depend on thetiming of the insult and the brain region of interest but alsoon the stage of assessment. Studies on postnatal brain devel-opment have clearly shown regional and temporal patterns ofdynamic maturational change continuing through childhoodand adolescence. This implies that what we observed andreported here in children of this age range may not be final. Itis possible that at a later maturational stage, prenatal stressexposure will confer a different morphological pattern.Therefore, following-up these children into adolescenceand adulthood will provide valuable information on thepersistence of prenatal stress effects on brain morphology.

It cannot be ruled out that the prenatal stress effects onbrain morphology are moderated by postnatal exposures(Buss et al., 2007). By controlling for several relevant vari-ables including postnatal maternal stress and socioeconomicstatus, it can be concluded though that the observed effectsof pregnancy anxiety on brain structure were not mediatedby these postnatal factors. Thus, the results suggest that,independent of postnatal maternal stress prenatal stress hasan impact on the offspring’s brain morphology.

It has to be noted that while there was no indication ofpsychiatric disorders and there was no report of treatmentfor any disorders in the structured interviews that probedsuch issues, the possibility of an undiagnosed disorder cannotbe ruled out because clinical diagnostic assessments were notconducted. Pregnancy anxiety may be higher in women withundiagnosed psychiatric disorders and accompanying endo-crine alterations could impact on neurodevelopment of theoffspring.

This is the first study in healthy children to show thatprenatal maternal anxiety is related to distinctive patternsof structuralbraindevelopment.Thesemorphologicalpatternsmay increase vulnerability for certain neurodevelopmentaldisorders and impair cognitive function. Therefore the resultssuggest that addressing mothers’ pregnancy-related concernsandanxietyshouldbeamajorfocusforpublichealth initiatives.

Role of funding source

This research was supported by National Institute of Healthgrants NS-41298, HD-51852 AND HD28413 to CAS. The NIH had

no further role in study design, in the collection, analysis, andinterpretation of data, in the writing of this report, or in thedecision to submit this report for publication.

Conflict of interest

All authors acknowledge no conflict of interest.

Acknowledgments

This research was supported by National Institute of Healthgrants NS-41298, HD-51852 and HD28413 to CAS. We grate-fully acknowledge contributions made by staff of ourResearch Laboratory for data collection, especially ChristinaCanino and Cheryl Crippen. We also thank the mothers andchildren who participated.

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