Prenatal stress and limbic-prefrontal white matter microstructure in children aged 6 9 years: a preliminary diffusion tensor imaging study

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1 The World Journal of Biological Psychiatry, 2014; 15: BRIEF REPORT Prenatal stress and limbic-prefrontal white matter microstructure in children aged 6 9 years: a preliminary diffusion tensor imaging study World J Biol Psychiatry Downloaded from informahealthcare.com by King's College London on 06/26/14 SAGARI SARKAR 1,6 *, MICHAEL C. CRAIG 1,2,3.7 *, FLAVIO DELL ACQUA 1,2,3, THOMAS G. O CONNOR 4, MARCO CATANI 1,2,3, QUINTON DEELEY 1,3, VIVETTE GLOVER 5 * * & DECLAN G.M. MURPHY 1,3 * * 1 Department of Forensic and Neurodevelopmental Science, King s College London, Institute of Psychiatry, London, UK, 2 Natbrainlab, Department of Forensic and Neurodevelopmental Science, King s College London, Institute of Psychiatry, London, UK, 3 NIHR Biomedical Research Centre for Mental Health at South London and Maudsley NHS Foundation Trust and Institute of Psychiatry, Kings College London. London, UK, 4 Department of Psychiatry, University of Rochester Medical Center, Rochester, New York, USA, 5 Institute of Reproductive and Developmental Biology, Imperial College London, London, UK, 6 Department of Neuroimaging, King s College London, Institute of Psychiatry, London, UK, and 7 National Autism Unit, Bethlem Royal Hospital, South London and Maudsley NHS Foundation Trust Abstract Objectives. Maternal prenatal stress is associated with elevated risk of adverse behavioural outcomes in offspring. This association may involve developmental disruption to limbic-prefrontal white matter circuitry, of which the uncinate fasciculus is the major tract. One potential candidate for modulating brain development is maternal prenatal stress. We provide the first prospective study of prenatal stress and white matter microstructure in children. Methods. A total of 22 healthy children (mean age 7 years) of mothers recruited in pregnancy underwent diffusion tensor magnetic resonance imaging. We examined correlations between prenatal stressful life events and white matter microstructural organisation indices (fractional anisotropy (FA) and perpendicular diffusivity ( )) of the uncinate fasciculus and a control tract. Results. Maternal prenatal stressful life events were correlated positively with right uncinate fasciculus FA, and negatively with right uncinate fasciculus in their child, with a similar trend with left uncinate fasciculus. Prenatal stress was not associated with control tract properties; sociodemographic/obstetric variables were not associated with FA/ of either tract. Conclusions. Variation in maternal prenatal stress may be associated with differences in the development of white matter within brain networks underlying child social behaviour. Key words: DT-MRI, prenatal stress, neuroimaging, uncinate fasciculus, white matter Introduction There is increasing evidence that a mother s emotional state during pregnancy can affect her child s behavioural development. For example, many studies have reported that, after controlling for postnatal and other potentially confounding factors, prenatal maternal stress, anxiety and depression are associated with increased rates of developmental difficulties in their offspring. These include conduct and temperament problems (Huizink et al. 2002; O Connor et al. 2002, 2003; Gutteling et al. 2005; Barker and Maughan 2009; Hay et al. 2010; Rice et al. 2010), Autistic Spectrum Disorders (Beversdorf et al. 2005; Kinney et al. 2008), Attention Deficit Hyperactivity Disorder (O Connor et al. 2003; Rodriguez and Bohlin 2005; Huizink et al. 2007), and symptoms of depression (Van den Bergh et al. 2007), and anxiety (O Connor et al. 2003; Van den Bergh and Marcoen 2004). Together these studies suggest an association between high levels of prenatal maternal stress and/or depression and long-term behavioural effects in their offspring. * The first authors have contributed equally to the production of this manuscript. * * The last authors have contributed equally to the production of this manuscript. Correspondence: Michael C. Craig. Tel: Fax: michael.c.craig@kcl.ac.uk (Received 27 August 2013 ; accepted 27 February 2014 ) ISSN print/issn online 2014 Informa Healthcare DOI: /

2 It is unclear what drives the association between maternal prenatal factors and child behaviour. Stress hormones (e.g., glucocorticoids) may play a key role by modulating the development of paralimbic brain regions underlying emotional and social behaviour. For instance, animal studies have reported that exposure to prenatal maternal stress and/or glucocorticoids are associated with changes in the developmental trajectory of the hippocampus and amygdala (Uno et al. 1994; Salm et al. 2004; Kraszpulski et al. 2006; Tamura et al. 2011). In particular, compared to controls, animals exposed to prenatal stress exhibited initially reduced, and subsequently increased, amygdala volume (Salm et al. 2004; Kraszpulski et al. 2006). These findings of an altered neurodevelopmental trajectory are supported by human studies, which have also reported an association between increased maternal prenatal cortisol levels and increased amygdala volume in later childhood (Buss et al. 2010; Buss et al. 2012) and reduced volume in prefrontal cortical brain regions, including orbitofrontal cortex (Buss et al. 2010). However, brain regions do not function in isolation they form networks connected by white matter tracts. Two key paralimbic brain regions that are affected by prenatal maternal stress, the amygdala and prefrontal cortex, are connected by the uncinate fasciculus (UF), whose volume and integrity can be analysed in vivo using diffusion tensor magnetic resonance imaging (DT-MRI) tractography. Using this technique white matter organisation can be quantified through two measures of diffusion properties: fractional anisotropy (FA) and perpendicular diffusivity ( ). FA denotes longitudinal diffusivity of water molecules along the axonal length, whereas reflects diffusion occurring radially, across axons. Increased FA together with decreased reflects greater microstructural organisation, which is underpinned by degree of myelination, and inter- and intra-axonal factors (e.g., axon diameter and fibre number) (Beaulieu, 2009; Paus, 2010). Previous DT-MRI tractography studies have reported abnormal microstructural organisation of the UF associated with behavioural developmental and behavioural disorders, including specific behavioural outcomes associated with prenatal stress, including antisocial behaviour (Craig et al. 2009; Sarkar et al. 2012) and autistic spectrum conditions (Wolff et al., 2012). For example, compared to control subjects, these conditions were associated with initially increased (in childhood), and subsequently reduced (in adulthood), fractional anisotropy (FA) of the UF (Craig et al. 2009; Sarkar et al. 2012; Wolff et al. 2012). In the current study we provide the first test of the hypothesis that prenatal stress predicts microstructural organisation of the UF. DTI prenatal stress 347 Using DT-MRI (diffusion tensor magnetic resonance imaging) tractography we carried out a preliminary investigation in children to test the hypothesis that prenatal exposure to maternal stress is associated with variation in microstructural organisation of the UF limbic white matter tract. We assessed microstructural diffusion properties within the UF in typically developing 6 9-year-old children who had been followed prospectively from the prenatal period. We examined the association between UF FA and and number of prenatal stressful life events. We also analysed a control tract, the inferior longitudinal fasciculus (ILF) tract. The ILF was chosen because it shares a termination point with the UF (temporal lobe), and it has not been reported to be involved in social and emotional behaviour (Ashtari 2011). Associations between DT- MRI indices and postnatal maternal mood (depression and postnatal stressful life events), and several socio-demographic and obstetric variables were also examined. Materials and methods This study was approved by the Brent Research Ethics Committee (H/0717/09). Participants The participants were 22 healthy children, aged between 6 and 9 years old, whose mothers had initially been recruited from an amniocentesis clinic for a study of prenatal stress and prenatal hormone exposure at Queen Charlotte s Maternity Hospital, London. Mothers with full-term ( 37 weeks), healthy singleton infants, with known birth outcomes, were invited to return to the hospital s paediatric clinic for a detailed developmental assessment when their child was ~ 17 months old ( n 123). For full details please refer to Bergman et al. (2007). The current study, which included a further postnatal visit for DT-MRI at the Institute of Psychiatry, is a further phase of the study that took place when the children were between 6 and 9 years of age. The participants recruited into the current study did not differ significantly from the pool from which they were drawn on any prenatal or obstetric measures. Eligibility criteria for the current study included the child being at least 6 years of age and the absence of contraindications to undergoing MRI. Measures Stressful life events. The primary predictor of prenatal stress was based on maternal report on the 26-item

3 348 S. Sarkar et al. Stressful Life Events (SLE) Questionnaire adapted from the Inventory of Ranked Life Events for Primiparous and Multiparous Women, which is a widely used index of stress in pregnancy (Barnett et al. 1983). The inventory, which was completed at the first postnatal assessment at 17 months, contains the most common items indexing major stresses, including illness, financial loss and partner cruelty; mothers completed the inventory twice: once concerning events that occurred in pregnancy and again for events that had occurred since the child s birth. We used the sum of SLE as the index of prenatal stress because we had no a priori hypotheses about the type of stress (for full details see Bergman et al. 2007). In addition, postnatal depression was assessed using the Edinburgh Postnatal Depression Scale; EPDS (Cox et al. 1987) at the first postnatal assessment. Postnatal depression was considered as a possible confounding influence in analyses of DT-MRI measures. Obstetric data and covariates. Obstetric data and information on prenatal and postnatal covariates were collected at the prenatal and first postnatal assessment and from medical records. Maternal smoking and alcohol use were uncommon and were therefore categorized as any/no use in pregnancy (Table I). Neonatal data collected at birth from medical records included birth weight and gestational age at birth; see Table I). Table I. Mother and child characteristics. N 22 Mean (SD)/ n (%) Child age (years) 7 (1) Ethnicity White 17/77% Black/African-Caribbean 2/9% Other 3/14% Annual income 25,000 43,000 8/35% Above 43,000 9/40% Prefer not to say 5/25% Maternal highest level of education GCSEs 2/9% A levels/third level 20/91% Maternal age (years) 36 (4) Birth weight (kilograms) 3.5 (0.4) Any cigarette smoking in pregnancy 3/14% Any alcohol intake during pregnancy 8/36% Female sex 10/45% Handedness of child Right 14/64% Mixed 6/27% Left 15% SD, standard deviation. DT-MRI data collected between 6 and 9 years. DT-MRI acquisition. DT-MRI images were acquired using a GE Signa HDx 3.0T MR scanner (General Electric, Waukshua, WI, USA), with actively shielded magnetic field gradients (maximum amplitude 40 mt m 1 ). The body coil was used for RF transmission, and an eight-channel head coil for signal reception, allowing a parallel imaging (ASSET) speed up factor of two. Head movement was minimised by fitting extra padding beside participants heads. Each volume was acquired using a multi-slice peripherally-gated doubly refocused spin echo EPI sequence, optimised for precise measurement of the diffusion tensor in parenchyma, from 60 contiguous near-axial slice locations with a voxel size of mm. The echo time was ms while the effective repetition time varied between subjects in the range 12 and 20 RR intervals. Based on the recommendations of Jones et al. (2002), the maximum diffusion weighting was 1300 s mm 2, and at each slice location, four images were acquired with no diffusion gradients applied, together with 32 diffusion-weighted images in which gradient directions were uniformly distributed in space. The sequence ran for approximately 12 min. DT-MRI data pre-processing. Data were first converted to NIFTI format and then each raw diffusion dataset underwent a full quality control check where all B 0 s and Diffusion Weighted volumes were visually inspected for image corruption, motion artefacts and signal drop-out effects using the light-box function available inside fslview (fmrib Software Library, Datasets showing 2 motion artefacts in different volumes on the same slice were removed from the study. Datasets showing significant head movements ( 1 cm) were removed. No participant data acquired in this study required removal due to motion artefacts. Data were eddy current and motion corrected using Explore-DTI (Leemans et al. 2009). The diffusion tensor was estimated following removal of outlier data (RESTORE function, Chang et al. 2005), and whole brain tractography was performed on the data. Whole brain tractography parameters selected as seed voxels all those with FA 0.2. Streamlines were propagated using an Euler integration applying a b-spline interpolation of the diffusion tensor field (Basser et al. 2000), and the tractography algorithm step size of 0.5 mm. Where FA 0.2 or when the angle between two consecutive tractography steps was larger than 30 degrees, tractography stopped. Finally diffusion tensor maps (fractional anisotropy; FA, mean diffusivity; MD, FA-colour, parallel diffusivity; D parr, perpendicular diffusivity;, mean diffusion weighted image; meandwi) were estimated and exported to

4 TrackVis (Wang and Weeden 2007). Full details are given elsewhere (Jones et al. 2002). DTI tractography. TrackVis software was used to hand dissect in native space. The tract of interest (UF), then the control tract (inferior longitudinal fasciculus, ILF) were dissected (in the same order) for all data by a blinded, trained and reliable operator (SS). Dissection of both tracts was performed one hemisphere at a time using the region of interest (ROI) approach described elsewhere (Catani et al. 2002; Catani and Thiebaut de Schotten 2008) (see Figure 1). The UF was defined with the first ROI placed on the axial slice at the level of the medial temporal lobe, and the second placed coronally slightly posterior to the external capsule. Short fibres that did not enter either termination of the tract were excluded, as were long fibres extending to regions outside the frontal and temporal lobes. These fibres were omitted through placement of exclusion ROI. Statistical analysis Bivariate analysis using SPSS software (SPSS Inc., Chicago, IL, USA) examined correlations between two measures of white matter microstructure of the UF and control tract (namely, FA and ) and prenatal SLE. Postnatal depression and postnatal SLE were also correlated with UF DT-MRI indices to assess any confounding influence of postnatal factors on UF microstructure. In addition, correlations between DT-MRI indices and other possible confounders (child age, maternal age, birth weight corrected for gestational age, smoking in pregnancy and SES) were examined. Given the small sample size and related distribution concerns we report both Pearson and Spearman rank-order correlations. Results Prenatal stress and DT-MRI measures DTI prenatal stress 349 Prenatal SLE were correlated positively with their offspring s right UF FA ( r 0.48; P 0.024), and negatively with right UF ( r 0.45; P 0.037). There was a similar trend with left UF ( r 0.40; P 0.065). Pearson correlations are robust to non-normal data; nonetheless, we re-ran analyses using Spearman rank-order correlations. Findings were substantively identical. For example, the associations with prenatal stress were 0.37 ( P 0.09) for right UF FA and 0.44 ( P 0.04) for right UF. In contrast, no significant associations were found between maternal prenatal stress and the ILF control tract (Table II, Figure 2). Additionally, sociodemographic or obstetric factors were not significantly correlated with DT-MRI outcome variables (Table III). Associations between maternal postnatal depression or postnatal SLE and UF DT-MRI indices were weaker and not significant (Table II). Additional exploratory analyses indicated that there was a trend for a stronger effect of prenatal stress in females than males for right UF FA, but the difference according to the Fisher r to z transformation was not significant ( z 1.63, P 0.10); similarly, a stronger effect of prenatal stress in females than males for right was not significant ( z 0.69, P 0.10). Further, child age was not associated with prenatal stress ( r 0.13, P 0.57). Discussion In this study we report, for the first time, a significant association between self-reported prenatal maternal stress exposure and proxy measures of white matter microstructural organisation (reduced FA and increased ) of the right UF, in children at age Figure 1. The uncinate fasciculus and inferior longitudinal fasciculus.

5 350 S. Sarkar et al. Table II. Correlations between prenatal stress, postnatal depression, and DT-MRI indices. Prenatal stressful life events Maternal postnatal depression Maternal postnatal stressful life events Tract Value Hemisphere r P R P r P Uncinate fasciculus FA Left Right * Left Right * Inferior longitudinal fasciculus FA Left Right Left Right World J Biol Psychiatry Downloaded from informahealthcare.com by King's College London on 06/26/14 FA, fractional anisotropy;, perpendicular diffusivity; r, correlation coefficient; P, significance level; * P 0.05 (two-tailed). 6 9 years. These findings were significant only for prenatal life events; associations with sociodemographic and obstetric risk, as well as postnatal risk, were weaker and non-significant. Furthermore, prenatal stress was not significantly associated with microstructural organisation in a control tract, suggesting that effects of prenatal stress were tract specific. These results can be viewed as extending findings from earlier animal and human studies: in addition to prenatal stress modifying the neurodevelopment of the amygdala and prefrontal cortex, it also modifies the white matter that connects these regions (i.e., the UF). The biological mechanisms modulating the putative effect of prenatal maternal stress cannot be elucidated by diffusion techniques but probably include proliferation of fibre number and diameter (Lebel et al. 2008; Beaulieu 2009) and increased myelination, a key determinant of FA and values. Myelination begins during foetal life and continues until early adulthood (Marsh et al. 2008). A previous rat study reported that maternal restraint stress applied during the late stages of gestation was associated with hypermyelination in the brain of their offspring (Wiggins and Gottesfeld 1986). Consistent with that study, the increased FA and reduced reported in our study of prenatally stressed children, is likely to be significantly influenced by increased myelination. Our results were also consistent with previous findings that conduct disorder, which occurs more commonly in children born to prenatally stressed mothers, is associated with similar UF changes relative to control children (Sarkar et al. 2012). However, in addition to reflecting myelination, FA and also reflect differences in other variables, such as tract branching (Allin 2010). The methodology in the current study did not permit us to measure myelination per se. However, future studies could incorporate assessment of myelination using new myelin mapping techniques to investigate whether myelination underlies these findings (Deoni et al. 2011). Figure 2. Significant correlations shown are within the uncinate fasciculus: prenatal stressful life events and right hemisphere fractional anisotropy (FA; left); and perpendicular diffusivity ( ; right ).

6 Table III. Correlations between sociodemographic and obstetric factors and DT-MRI indices. DTI prenatal stress 351 Maternal age Birth weight Gestational age at birth Maternal education level Uncinate fasciculus r P r P r P r P FA Left Right Left Right World J Biol Psychiatry Downloaded from informahealthcare.com by King's College London on 06/26/14 Limitations It is important to outline the limitations with this study. First, this preliminary study analysed the effects of prenatal maternal stress in a relatively small sample size. Although our sample was small, our findings did not seem to be affected by outliers (e.g., see Figure 2); we did not adjust for multiple testing given the preliminary nature of the study and because we had a priori hypotheses based on previous work. Replication on a larger sample size would increase the reliability of the findings and provide leverage for assessing potential moderators (e.g., sex of child, quality of postnatal care). The lack of association with maternal (e.g., age), obstetric and postnatal covariates indicated that they did not confound the prenatal prediction. Additionally, we did not have data directly connecting the UF findings with specific behavioural phenotypes, and so the clinical applications of the findings are unclear. A further potential caveat is that correlation analysis with the prenatal SLE questionnaire data may have suffered from a limited range, given that we did not sample clinical cases. Nonetheless our findings provide preliminary evidence that stress during pregnancy may be associated with differences in foetal brain development. Also, it should be noted that prenatal SLE were measured retrospectively. Significantly, however, the prenatal prediction was significant whereas the postnatal prediction was not. That implies that the prenatal prediction was not confounded by postnatal stress, a pattern that has been found for behavioural phenotypes in prior work (Sarkar et al. 2006; Bergman et al. 2007, 2008). Finally, this was an association study in which causality cannot be established. For example, data were not available that pertained to childhood trauma, negative family experiences, illnesses and other events/neurochemical mechanisms that may influence brain development throughout infancy and childhood. While further research is needed to establish the mechanism underlying this association, this is the first human study in which the relationship between prenatal stress and white matter has been investigated. Conclusion This preliminary study revealed a significant relationship between maternal prenatal stress and limbicprefrontal white matter microstructural organisation in their offspring in middle childhood. Larger studies are now required to study these putative effects longitudinally, and to assess brain development (e.g., myelin content) more directly. Acknowledgements This work was funded by the March of Dimes foundation, Baily Thomas Charitable Fund, The Academy of Medical Sciences, the NIHR Biomedical Research Centre for Mental Health at King s College London, Institute of Psychiatry and South London & Maudsley NHS Foundation Trust, and a generous donation from a private individual. We would also like to thank all the children and their parents for taking part. We would like to acknowledge Fiona Rose-Clarke for her assistance with recruitment and data collection. Statement of Interest None to declare. References Allin M Diffusion tensor imaging findings in preterm and low birth weight populations. In: Nosarti C, Murray RM, Hack M, eds. Neurodevelopmental outcomes of preterm birth: from childhood to adult life. Cambridge: Cambridge University Press. p Ashtari M Anatomy and functional role of the inferior longitudinal fasciculus: a search that has just begun. Dev Med Child Neurol 54 : 6 7. Barker ED, Maughan B Differentiating early-onset persistent versus childhood-limited conduct problem youth. Am J Psychiatry 168 : Barnett BE, Hanna B, Parker G Life event scales for obstetric groups. J Psychosom Res 27 : Basser PJ, Pjevic S, Pierpaoli C, Duda J, Aldoubi A In vivo fiber tractography using DT-MRI data. Magn Reson Med 44 :

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