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Maternal Cortisol Concentrations During Pregnancy and Sex-Specific Associations With Neonatal Amygdala Connectivity and Emerging Internalizing Behaviors

      Abstract

      Background

      Maternal cortisol during pregnancy has the potential to influence rapidly developing fetal brain systems that are commonly altered in neurodevelopmental and psychiatric disorders. Research examining maternal cortisol concentrations across pregnancy and offspring neurodevelopment proximal to birth is needed to advance understanding in this area and lead to insight into the etiology of these disorders.

      Methods

      Participants were 70 adult women recruited during early pregnancy and their infants born after 34 weeks gestation. Maternal cortisol concentrations were assessed serially over 4 days in early, mid, and late gestation. Resting state functional connectivity magnetic resonance imaging of the neonatal amygdala was examined. Mothers reported on children’s internalizing behavior problems at 24 months of age.

      Results

      Maternal cortisol concentrations during pregnancy were significantly associated with neonatal amygdala connectivity in a sex-specific manner. Elevated maternal cortisol was associated with stronger amygdala connectivity to brain regions involved in sensory processing and integration, as well as the default mode network in girls, and with weaker connectivity to these brain regions in boys. Elevated maternal cortisol was associated with higher internalizing symptoms in girls only, and this association was mediated by stronger neonatal amygdala connectivity.

      Conclusions

      Normative variation in maternal cortisol during pregnancy is associated with the coordinated functioning of the amygdala soon after birth in a sex-specific manner. The identified pathway from maternal cortisol to higher internalizing symptoms in girls via alterations in neonatal amygdala connectivity may be relevant for the etiology of sex differences in internalizing psychiatric disorders, which are more prevalent in women.

      Keywords

      Despite advancements in characterizing alterations in brain functioning underlying neurodevelopmental and psychiatric disorders, the disease burden and challenges of successful treatment remain high (
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      ). Several studies in humans suggest that heightened negative emotionality and development of internalizing symptoms is more common in girls (
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      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
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      ). However, only one study has explicitly examined the role of the amygdala in the link between elevated maternal cortisol and risk for internalizing symptoms (
      • Buss C.
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      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
      ). This study identified an association between heightened maternal cortisol during pregnancy and larger right amygdala volume in school-aged girls and a pathway from higher maternal cortisol to greater internalizing in girls via this altered amygdala phenotype (
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      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
      ).
      The scientific literature to date thus suggests a potentially important role for maternal cortisol during pregnancy in influencing offspring brain development with implications for subsequent internalizing behaviors, which may be more pronounced in girls. The current study advances this line of work in several ways. First, prior studies in humans have examined maternal cortisol during pregnancy at a single time point during the day in relation to offspring brain development (
      • Buss C.
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      • Shahbaba B.
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      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
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      Prenatal maternal cortisol concentrations predict neurodevelopment in middle childhood.
      ). In contrast, we employed ambulatory cortisol assessment over multiple days in early, mid, and late gestation, allowing for reliable and comprehensive estimation of overall maternal cortisol output during pregnancy. Second, while prior studies have examined brain development in school-aged children (
      • Buss C.
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      • Shahbaba B.
      • Pruessner J.C.
      • Head K.
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      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
      ,
      • Kim D.-J.
      • Davis E.P.
      • Sandman C.A.
      • Sporns O.
      • O’Donnell B.F.
      • Buss C.
      • Hetrick W.P.
      Prenatal maternal cortisol has sex-specific associations with child brain network properties.
      ,
      • Davis E.P.
      • Head K.
      • Buss C.
      • Sandman C.A.
      Prenatal maternal cortisol concentrations predict neurodevelopment in middle childhood.
      ), the current study focused on the neonatal brain, increasing capacity to differentiate effects of maternal cortisol concentrations during pregnancy from exposure to postnatal environmental stressors as well as the influence of heightened offspring stress reactivity and internalizing over time.
      Third, this study employed resting state functional connectivity magnetic resonance imaging (rs-fcMRI) to examine the coordinated functioning of the neonatal amygdala with other brain regions. The rs-fcMRI reveals information about the functional architecture of the brain beginning during early infancy and is sensitive enough to capture individual differences that relate to emerging behavioral phenotypes relevant for psychiatric disorders (
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      • Demeter D.V.
      • Gilmore J.H.
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      Implications of newborn amygdala connectivity for fear and cognitive development at 6-months-of-age.
      ,
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      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
      ,
      • Graham A.M.
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      Early life stress is associated with default system integrity and emotionality during infancy.
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      Neonatal amygdala functional connectivity at rest in healthy and preterm infants and early internalizing symptoms.
      ). Therefore, we examined maternal cortisol concentrations during pregnancy in relation to neonatal amygdala functional connectivity. Based on prior research examining other stress-related processes during pregnancy (
      • Graham A.M.
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      • Rudolph M.D.
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      • Gilmore J.H.
      • Styner M.
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      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
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      • Broekman B.F.P.
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      Prenatal maternal depression alters amygdala functional connectivity in 6-month-old infants.
      ) and stress exposure later in life (
      • van Marle H.J.F.
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      • Martis B.
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      Altered amygdala resting-state functional connectivity in post-traumatic stress disorder.
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      • Sripada R.K.
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      Altered resting-state amygdala functional connectivity in men with posttraumatic stress disorder.
      ), we expected to see altered amygdala functional connectivity to multiple brain regions involved in different sensory, emotional, and cognitive functions. We tested associations between the identified patterns of neonatal amygdala connectivity and subsequent child internalizing behavior. We hypothesized that elevated maternal cortisol concentrations during pregnancy would be associated with neonatal amygdala connectivity and subsequent internalizing behaviors in a sex-specific manner, such that they confer increased risk for internalizing behaviors in girls.

      Methods and Materials

      Participants

      Mothers and children in this study (n = 70 with neonatal rs-fcMRI data and n = 45 with behavioral data at 24 months of age) were part of an ongoing prospective longitudinal study conducted at the University of California, Irvine, for which mothers were recruited during early pregnancy. Exclusionary criteria were as follows: maternal age <18 years, maternal use of psychotropic medications or systemic corticosteroids during pregnancy, infant birth before 34 weeks gestation, and infant congenital, genetic, or neurologic disorder. Demographic characteristics are presented in Table 1. A very small portion of mothers reported a mental health diagnosis at study entry (n = 2). Behavioral follow-up data were obtained when children were 24 months of age (mean = 24.00 months, SD = 0.866). There were no significant differences in maternal cortisol concentrations or demographics for those lost to follow-up versus the sample with behavioral follow-up data. See Supplemental Table S1 for a comparison of demographics for the full sample versus the follow-up sample. All procedures were approved by the Institutional Review Board at the University of California, Irvine, and written informed consent was obtained from all mothers.
      Table 1Demographics (n = 70)
      CharacteristicValue
      Maternal Age in First Trimester, Years28.30 (5.40)
      Infant Age, Weeks
      Gestational Age at Birth, Weeks39.30 (1.39)
      Age at MRI Data Collection, Years3.65 (1.72)
      Infant Sex
       Male62.5
       Female37.5
      Race/Ethnicity
       Caucasian Non-Hispanic42.6
       African American Non-Hispanic2.13
       Asian Non-Hispanic10.6
       Multiracial Non-Hispanic10.6
       Caucasian Hispanic29.8
       Asian Hispanic2.13
       Multiracial Hispanic2.13
      Highest Level of Maternal Education
       High School or Test Equivalent10.4
       Vocational School or Some College50.0
       Associate Degree4.20
       Bachelor- or Graduate-Level Degree35.5
      Gross Annual Household Income
       <$15,0006.38
       $15,000–$29,99919.1
       $30,000–$49,99929.8
       $50,000–$100,00036.2
       >$100,0008.51
      Values are mean (SD) or %.
      MRI, magnetic resonance imaging.

      Maternal Cortisol Concentrations

      As described in our previous work (
      • Entringer S.
      • Buss C.
      • Rasmussen J.M.
      • Lindsay K.
      • Gillen D.L.
      • Cooper D.M.
      • Wadhwa P.D.
      Maternal cortisol during pregnancy and infant adiposity: A prospective investigation.
      ) and in the Supplement, pregnant women collected saliva samples 5 times a day for 4 consecutive days in early (T1), mid (T2), and late (T3) pregnancy (see Table 2), resulting in 60 samples per woman across pregnancy (Supplemental Figure S1). For each day, total cortisol output was estimated using area under the curve (AUC) with respect to ground. AUC values across days within each time point were significantly correlated (T1, r = .474–.838, p < .001; T2, r = .630–.800, p < .001; T3, r = .370–.700, p < .001) and were averaged to create a reliable indicator of cortisol output at each stage of pregnancy. These average AUC values were base 2 logarithm transformed to bring outliers closer to the mean and normalize the distributions. These values were also highly correlated (T1–T2, r = .664, p < .001; T1–T3, r = .649, p < .001; T2–T3, r = .743, p < .001) and therefore were averaged to create a composite representing overall cortisol output during pregnancy, which is the focus of analyses.
      Table 2Mean (SD) of Maternal Cortisol AUC and Gestational Age at Each Collection
      First TrimesterSecond TrimesterThird Trimester
      Gestational Age, Weeks12.80 (1.77)20.50 (1.40)30.30 (1.26)
      Cortisol AUC2.93 (1.62)3.25 (1.64)4.11 (1.68)
      Cortisol AUC was log transformed prior to analyses.
      AUC, area under the curve.

      MRI and fMRI Data Acquisition and Processing

      Data Acquisition

      Neuroimaging data were collected at approximately 4 weeks of age (mean = 3.65 weeks, SD = 1.72) during natural sleep on a TIM Trio (Siemens Medical System, Erlangen, Germany) 3.0T scanner. High-resolution T2-weighted scans (repetition time = 3200 ms, echo time = 255 ms, resolution = 1 × 1 × 1 mm, 4 hours 18 minutes) and T1-weighted scans (magnetization prepared rapid acquisition gradient-echo repetition time = 2400 ms, inversion time = 1200 ms, echo time = 3.16 ms, flip angle = 8°, resolution = 1 × 1 × 1 mm, 6 hours 18 minutes) were collected. Functional images for rs-fcMRI were obtained using a gradient-echo echo-planar imaging sequence sensitive to blood oxygen level–dependent contrast (repetition time = 2000 ms, echo time = 30 ms, field of view = 220 × 220 × 160 mm, 195 repetition times, 32 interleaved ascending axial slices, 1-mm gap, resolution = 3.4 × 3.4 × 4 mm, flip angle = 77°).

      MRI and fMRI Data Preprocessing

      Processing followed established procedures for neuroimaging with neonates as described in our previous work (
      • Graham A.M.
      • Buss C.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Demeter D.V.
      • Gilmore J.H.
      • et al.
      Implications of newborn amygdala connectivity for fear and cognitive development at 6-months-of-age.
      ,
      • Graham A.M.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Heim C.M.
      • Gilmore J.H.
      • Styner M.
      • et al.
      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
      ) and in the Supplement.

      rs-fcMRI Preprocessing

      Additional preprocessing steps for rs-fcMRI were implemented to account for signal stemming from non-neuronal processes (
      • Fox M.D.
      • Raichle M.E.
      Spontaneous fluctuations in brain activity observed with functional magnetic resonance imaging.
      ,
      • Fair D.A.
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      Distinct neural signatures detected for ADHD subtypes after controlling for micro-movements in resting state functional connectivity MRI data.
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      Functional brain networks develop from a “local to distributed” organization.
      ) as described in the Supplement. Volume censoring was employed to reduce effects of motion determined by framewise displacement (
      • Power J.D.
      • Barnes K.A.
      • Snyder A.Z.
      • Schlaggar B.L.
      • Petersen S.E.
      Spurious but systematic correlations in functional connectivity MRI networks arise from subject motion.
      ) of 0.3 mm. Remaining mean framewise displacement was subsequently examined as a potential confound. As in our prior work (
      • Graham A.M.
      • Buss C.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Demeter D.V.
      • Gilmore J.H.
      • et al.
      Implications of newborn amygdala connectivity for fear and cognitive development at 6-months-of-age.
      ,
      • Graham A.M.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Heim C.M.
      • Gilmore J.H.
      • Styner M.
      • et al.
      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
      ), seed regions for rs-fcMRI analysis were individually segmented amygdala (see Supplement).

      Potential Confounds Relevant for Maternal Cortisol and Neonatal Brain Outcomes

      Potential confounds relevant to maternal cortisol concentrations and infant brain development were examined. These included maternal prepregnancy body mass index, maternal cigarette smoking during pregnancy, obstetric risk, annual household income, and maternal systemic inflammation during pregnancy (as indexed by interleukin-6) (see Supplement).

      Internalizing Behavior at 24 Months of Age

      Mothers reported on children’s internalizing behavior problems at 24 months of age on the Internalizing Behavior scale (α = .966) of the Child Behavior Checklist/1.5–5 (
      • Achenbach T.M.
      • Rescorla L.
      Manual for the ASEBA School-Age Forms & Profiles.
      ).

      Maternal Postnatal Depression

      The 20-item Center for Epidemiological Studies of Depression Scale (
      • Radloff L.S.
      The CES-D scale: A self report depression scale for research in the general population.
      ) was used to assess maternal depression symptoms repeatedly over the first 2 years of life, and a composite score was used in analyses (see Supplement).

      Analyses

      The interaction between mean maternal cortisol AUC and fetal sex was regressed on whole-brain amygdala voxelwise connectivity while adjusting for the main effects of mean maternal cortisol and infant sex as well as infant gestational age at birth and age at scan. This whole-brain approach was deemed appropriate given our expectation that amygdala connectivity with multiple brain regions would be altered, and the lack of prior research examining amygdala connectivity in relation to maternal cortisol during pregnancy. Left and right amygdala connectivities were examined separately owing to evidence for lateralized effects of prenatal influences (
      • Qiu A.
      • Anh T.T.
      • Li Y.
      • Chen H.
      • Rifkin-Graboi A.
      • Broekman B.F.P.
      • et al.
      Prenatal maternal depression alters amygdala functional connectivity in 6-month-old infants.
      ) and asymmetry in relation to psychiatric outcomes (
      • Okada N.
      • Fukunaga M.
      • Yamashita F.
      • Koshiyama D.
      • Yamamori H.
      • Ohi K.
      • et al.
      Abnormal asymmetries in subcortical brain volume in schizophrenia.
      ). Multiple comparisons correction for p < .05 voxel clusters required a threshold of 35 contiguous voxels with a Z value > 2.50 based on Monte Carlo simulation (
      • Forman S.D.
      • Cohen J.D.
      • Fitzgerald M.
      • Eddy W.F.
      • Mintun M.A.
      • Noll D.C.
      Improved assessment of significant activation in functional magnetic resonance imaging (fMRI): Use of a cluster-size threshold.
      ). Connections identified in the whole-brain analyses were extracted (see Supplement for details). To probe the interaction, correlations between maternal cortisol and these connections were examined separately for girls and boys. Next, we tested for potential confounding influences on each extracted connection.
      We then tested associations between the strongest results from the whole-brain analyses (based on Z value) and child internalizing behaviors at 24 months of age. A covariate for maternal postnatal depression was included to account for variation in the postnatal environment and potential bias in maternal report on child internalizing. Next, we examined the direct association between maternal cortisol and child internalizing behaviors, considering infant sex as a moderator and postnatal depression as a covariate. Finally, we planned to examine neonatal amygdala connectivity as a mediator of any identified association between maternal cortisol during pregnancy and child internalizing using a structural equation modeling framework Mplus Version 7 (
      • Muthén L.K.
      • Muthén B.O.
      Mplus User’s Guide, 7th ed.
      ).

      Results

      The Association Between Maternal Cortisol During Pregnancy and Neonatal Amygdala Connectivity Is Moderated by Infant Sex

      The interaction between maternal cortisol during pregnancy and infant sex was significantly associated with neonatal amygdala connectivity. For the right amygdala, the maternal cortisol–infant sex interaction was significantly associated with connectivity to the following regions: left supramarginal gyrus (SMG) and superior temporal gyrus (STG) and right inferior temporal gyrus (ITG) (extending into fusiform gyrus) and dorsolateral prefrontal cortex (DLPFC). Results for the left amygdala were consistent with regard to the left SMG, right ITG, and a left STG region (although more rostral and less extensive). However, findings for the left amygdala also included the right and left precuneus and a more ventral and rostral fusiform gyrus region (Table 3 and Figure 1).
      Table 3The Interaction Between Maternal Cortisol During Pregnancy and Infant Sex Is Prospectively Associated With Neonatal Amygdala Connectivity
      RegionHemxyzZFemale InfantsMale Infants
      Right Amygdala
       SMGL−56−35202.97+
      p < .01.
      p < .10.
      L−51−25262.88+
      p < .10.
      p < .01.
       STGL−48−7−72.85+
      p < .10.
      p < .01.
      L−58−4−42.65+
      p < .10.
      p < .01.
       ITGR52−17−242.84+
      p < .05.
      p < .01.
       DLPFCR384915−2.67
      p < .05.
      +
      p < .05.
      R315024−2.50+
      p < .01.
      Left Amygdala
       ITGR52−7−233.11+
      p < .01.
      p < .05.
       Precuneus/superior parietalR16−54553.05+
      p < .05.
      p < .05.
       SMGL−52−21233.03+
      p < .01.
      p < .05.
       PrecuneusL−8−59322.92+
      p < .10.
      p < .01.
      L−10−49512.79+
      p < .01.
      p < .05.
       STGL−50−2442.69+
      p < .05.
      p < .10.
      L−47−14−12.67+
      p < .01.
      L−52−37152.60+
      p < .05.
      p < .05.
       Fusiform gyrusR40−76−15−2.83
      p < .05.
      +
      p < .05.
      Regions are in descending order based on highest Z value. The Pearson correlation between mean maternal gestational cortisol area under the curve and the extracted connection between the amygdala and each identified region was examined separately for female vs. male infants. The direction (positive or negative) and statistical significance of the correlation is indicated for female and male infants, respectively, in the last two columns.
      DLPFC, dorsolateral prefrontal cortex; Hem, hemisphere; ITG, inferior temporal gyrus; L, left; R, right; SMG, supramarginal gyrus; STG, superior temporal gyrus.
      a p < .10.
      b p < .05.
      c p < .01.
      Figure thumbnail gr1
      Figure 1The interaction between maternal cortisol during pregnancy and infant sex relates to neonatal amygdala connectivity. The association between maternal cortisol during pregnancy and whole brain left (A) and right (C) neonatal amygdala connectivity is moderated by infant sex (n = 70). Panels (A) and (C) show brain regions for which connectivity of the neonatal amygdala is altered in relation to the interaction between maternal cortisol and infant sex. Scatter plots demonstrate an example of probing the maternal cortisol–infant sex interaction for a specific connection (left amygdala–inferior temporal gyrus [ITG]). For female offspring (B), maternal cortisol indicates a positive association with the strength of this connection (n = 31, r = .490, p = .005). For male offspring (D), maternal cortisol indicates a negative association with the strength of this connection (n = 39, r = −.342, p = .033). See for direction and statistical significance of the bivariate correlations between maternal cortisol and each connection for male and female offspring separately. AUC, area under the curve; DLPFC, dorsolateral prefrontal cortex; L, left; R, right; SMG, supramarginal gyrus.

      Probing the Interaction Between Maternal Cortisol and Infant Sex Reveals Distinct Effects for Girls and Boys

      We extracted all significant connections identified in the whole-brain analysis (see Supplement) and examined the Pearson correlations between maternal cortisol and these connections separately for boys and girls. Overall, for girls, higher maternal cortisol was associated with stronger right and left amygdala connectivity to cortical brain regions. In contrast, for boys, higher maternal cortisol was associated with weaker amygdala connectivity to these regions. Right amygdala–DLPFC and left amygdala–fusiform gyrus connectivity were the two exceptions, such that higher maternal cortisol was associated with weaker connectivity in girls and with stronger connectivity in boys (Table 3). These results suggest a crossover interaction, indicating that sex differences were not characterized by a stronger association for one sex compared with the other; rather, the associations between maternal cortisol and neonatal amygdala connectivity were opposite for girls versus boys.

      Potentially Confounding Factors Do Not Affect the Associations Between Maternal Cortisol and Neonatal Amygdala Connectivity

      The interaction between maternal cortisol during pregnancy and infant sex remained significantly associated with all the identified connections (p < .05) after adjusting for all the potential confounds as well as remaining framewise displacement (micromovements during functional data acquisition remaining after frame removal).

      Neonatal Amygdala Phenotypes Associated With Maternal Cortisol During Pregnancy Are Relevant for Child Internalizing Behavior

      The strongest finding for the right amygdala (based on Z value), amygdala–SMG connectivity, was positively associated with child internalizing behavior at 24 months of age (β = .336, p = .017). Infant sex was not a significant moderator (β = .058, p = .737). Of the covariates in the model, higher maternal postnatal depression was significantly associated with greater child internalizing (β = .408, p = .004).
      The strongest finding for the left amygdala, left amygdala–ITG connectivity, was positively associated with child internalizing behavior only at the trend level (β = .252, p = .094). Infant sex was again not a significant moderator (β = −.190, p = .301), and the covariate for maternal depression was associated with child internalizing (β = .349, p = .018). Thus, patterns of neonatal amygdala connectivity associated with maternal cortisol during pregnancy are relevant for later emerging child internalizing symptoms. These associations are not modulated by infant sex, indicating that the moderation effect is specific to the association between maternal cortisol during pregnancy and the neonatal brain phenotypes.

      The Association Between Maternal Cortisol During Pregnancy and Child Internalizing Behavior Is Moderated by Infant Sex

      The interaction between maternal cortisol and infant sex was significantly associated with child internalizing (β = 1.527, p = .026). The covariate for maternal postnatal depression was also significantly associated with internalizing (β = .390, p = .010). Probing the interaction revealed that maternal cortisol was significantly associated with higher internalizing behaviors for girls (β = .533, p = .013) but not for boys (β = −.175, p = .358).

      Neonatal Amygdala–SMG Connectivity Mediates the Association Between Maternal Cortisol During Pregnancy and Subsequent Internalizing Behavior in a Sex-Dependent Manner

      Based on the significant effect of maternal cortisol on child internalizing, and the significant pathways from maternal cortisol to neonatal amygdala connectivity and from amygdala–SMG connectivity to internalizing, we tested for mediation. We used a moderated mediation model to account for the moderating effect of infant sex. For girls, higher maternal cortisol was associated with higher child internalizing via stronger right amygdala–SMG connectivity (indirect effect = 3.14, 95% confidence interval: [0.198, 10.2], based on 5000 bootstrap samples). Interestingly, the indirect path was also significant for boys, such that higher maternal cortisol was associated with lower child internalizing via weaker right amygdala–SMG connectivity (indirect effect = −3.61, 95% confidence interval: [−10.3, −0.645], based on 5000 bootstrap samples). Thus, for girls, the direct effect of maternal cortisol during pregnancy on child internalizing is mediated by higher neonatal amygdala–SMG connectivity. For boys, there was no direct association between maternal cortisol and subsequent internalizing, but the significant mediation result suggests that heightened maternal cortisol during pregnancy is associated with lower internalizing behavior via this specific pathway involving amygdala–SMG connectivity (Figure 2). The lack of a direct effect for boys indicates that other pathways exist through which heightened maternal cortisol has the opposite effect on internalizing behavior in boys.
      Figure thumbnail gr2
      Figure 2Conceptual model representing sex-specific associations between maternal cortisol during pregnancy and offspring internalizing behaviors via neonatal amygdala connectivity. The direct effect of maternal cortisol on child internalizing behavior was significant only for girls. This model is based on the moderated mediation analysis, which identified significant indirect paths for girls and boys. A negative or positive sign indicates the direction of effect for each association. Gray arrows represent the mediation (indirect) effect. For girls, higher maternal cortisol concentrations during pregnancy are associated with higher levels of internalizing behaviors at 24 months of age via stronger neonatal amygdala–supramarginal gyrus connectivity. For boys, higher maternal cortisol during pregnancy is associated with lower levels of internalizing behaviors via weaker neonatal amygdala–supramarginal gyrus connectivity.

      Discussion

      Summary of Findings

      Although extensively studied in animal models, the implications of variability in maternal cortisol concentrations during pregnancy for offspring brain and behavioral development in humans are not well understood. The findings of the current study indicate that normative variations in maternal cortisol concentrations during pregnancy are associated with alterations in neonatal amygdala functional connectivity to multiple cortical brain regions involved in sensory processing and integration, the default mode network (DMN), and emotion regulation. These associations differed by offspring sex, such that elevated maternal cortisol was associated with stronger neonatal amygdala connectivity in girls and with weaker connectivity in boys. The amygdala connections most strongly associated with maternal cortisol in turn predicted internalizing behavior when children were 2 years of age after accounting for maternal postnatal depressive symptoms. A significant direct effect of maternal cortisol during pregnancy on child internalizing was identified in girls only. Stronger neonatal amygdala connectivity mediated the effect of elevated maternal cortisol on higher internalizing in girls. We consider the potential implications of these findings for the etiology of sex differences in internalizing psychiatric disorders, which are more prevalent in women (
      • Bangasser D.A.
      • Valentino R.J.
      Sex differences in stress-related psychiatric disorders: Neurobiological perspectives.
      ,
      • Altemus M.
      • Sarvaiya N.
      • Neill Epperson C.
      Sex differences in anxiety and depression clinical perspectives.
      ,
      • Kessler R.C.
      • Chiu W.T.
      • Demler O.
      • Walters E.E.
      Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication.
      ,
      • McLean C.P.
      • Asnaani A.
      • Litz B.T.
      • Hofmann S.G.
      Gender differences in anxiety disorders: Prevalence, course of illness, comorbidity and burden of illness.
      ).

      Potential Mechanisms Relevant to Observed Sex Differences

      Research to date suggests several potential mechanisms, at the level of the placenta and fetal brain, through which maternal cortisol concentrations during pregnancy may exert sex-specific effects on fetal neurodevelopment and particularly brain regions such as the amygdala, with high potential to be influenced by GCs. The placenta is an organ of fetal origin and therefore has the same X and Y chromosome composition as the fetus (
      • Rossant J.
      • Cross J.C.
      Placental development: Lessons from mouse mutants.
      ). Sex differences in placental gene expression and regulation, including of the placental enzyme 11β-hydroxysteroid dehydrogenase 2 (
      • Cuffe J.
      • O’Sullivan L.
      • Simmons D.
      • Anderson S.
      • Moritz K.
      Maternal corticosterone exposure in the mouse has sex-specific effects on placental growth and mRNA expression.
      ,
      • Stark M.
      • Wright I.
      • Clifton V.
      Sex-specific alterations in placental 11-hydroxysteroid dehydrogenase 2 activity and early postnatal clinical course following antenatal betamethasone.
      ) and placental GC receptor isoforms (
      • Meakin A.S.
      • Saif Z.
      • Jones A.R.
      • Aviles P.F.V.
      • Clifton V.L.
      Review: Placental adaptations to the presence of maternal asthma during pregnancy.
      ,
      • Saif Z.
      • Hodyl N.A.
      • Stark M.J.
      • Fuller P.J.
      • Cole T.
      • Lu N.
      • Clifton V.L.
      Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight.
      ,
      • Saif Z.
      • Hodyl N.A.
      • Hobbs E.
      • Tuck A.R.
      • Butler M.S.
      • Osei-Kumah A.
      • Clifton V.L.
      The human placenta expresses multiple glucocorticoid receptor isoforms that are altered by fetal sex, growth restriction and maternal asthma.
      ,
      • Scott N.
      • Hodyl N.
      • Osei-Kumah A.
      • Stark M.
      • Smith R.
      • Clifton V.
      The presence of maternal asthma during pregnancy suppresses the placental pro-inflammatory response to an immune challenge in vitro.
      ), affect both the passage of active maternal GCs to the fetal compartment and the extent to which maternal GCs stimulate fetal GC production. These mechanisms lead to sex differences in fetal GC exposure and associated phenotypic alterations in the context of elevated maternal GCs (
      • Chan J.C.
      • Nugent B.M.
      • Bale T.L.
      Parental advisory: Maternal and paternal stress can impact offspring neurodevelopment.
      ,
      • Cuffe J.
      • O’Sullivan L.
      • Simmons D.
      • Anderson S.
      • Moritz K.
      Maternal corticosterone exposure in the mouse has sex-specific effects on placental growth and mRNA expression.
      ,
      • Stark M.
      • Wright I.
      • Clifton V.
      Sex-specific alterations in placental 11-hydroxysteroid dehydrogenase 2 activity and early postnatal clinical course following antenatal betamethasone.
      ,
      • Meakin A.S.
      • Saif Z.
      • Jones A.R.
      • Aviles P.F.V.
      • Clifton V.L.
      Review: Placental adaptations to the presence of maternal asthma during pregnancy.
      ,
      • Saif Z.
      • Hodyl N.A.
      • Stark M.J.
      • Fuller P.J.
      • Cole T.
      • Lu N.
      • Clifton V.L.
      Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight.
      ,
      • Saif Z.
      • Hodyl N.A.
      • Hobbs E.
      • Tuck A.R.
      • Butler M.S.
      • Osei-Kumah A.
      • Clifton V.L.
      The human placenta expresses multiple glucocorticoid receptor isoforms that are altered by fetal sex, growth restriction and maternal asthma.
      ,
      • Scott N.
      • Hodyl N.
      • Osei-Kumah A.
      • Stark M.
      • Smith R.
      • Clifton V.
      The presence of maternal asthma during pregnancy suppresses the placental pro-inflammatory response to an immune challenge in vitro.
      ,
      • Bale T.L.
      The placenta and neurodevelopment: Sex differences in prenatal vulnerability.
      ,
      • Nugent B.M.
      • Bale T.L.
      The omniscient placenta: Metabolic and epigenetic regulation of fetal programming.
      ). Sex differences in the timing and pattern of GC receptor expression in the developing fetal brain have also been observed and likely contribute to differences in how GC exposure shapes ongoing neural development (
      • Owen D.
      • Matthews S.G.
      Glucocorticoids and sex-dependent development of brain glucocorticoid and mineralocorticoid receptors.
      ,
      • Liu L.I.
      • Li A.
      • Matthews S.G.
      • Li A.
      • Maternal S.G.M.
      Maternal glucocorticoid treatment programs HPA regulation in adult offspring: Sex-specific effects.
      ,
      • Dean F.
      • Matthews S.G.
      Maternal dexamethasone treatment in late gestation alters glucocorticoid and mineralocorticoid receptor mRNA in the fetal guinea pig brain.
      ).

      Brain Regions and Networks Involved and Relation to Internalizing Symptoms

      Results of the current study reveal a pattern of increased amygdala connectivity for girls in association with elevated maternal cortisol concentrations. In adults, stronger amygdala connectivity has been observed following stress exposure (
      • van Marle H.J.F.
      • Hermans E.J.
      • Qin S.
      • Fernández G.
      Enhanced resting-state connectivity of amygdala in the immediate aftermath of acute psychological stress.
      ,
      • Rabinak C.A.
      • Angstadt M.
      • Welsh R.C.
      • Kenndy A.E.
      • Lyubkin M.
      • Martis B.
      • Phan K.L.
      Altered amygdala resting-state functional connectivity in post-traumatic stress disorder.
      ,
      • Sripada R.K.
      • King A.P.
      • Garfinkel S.N.
      • Wang X.
      • Sripada C.S.
      • Welsh R.C.
      • Liberzon I.
      Altered resting-state amygdala functional connectivity in men with posttraumatic stress disorder.
      ) and in association with higher subclinical and pathological internalizing symptoms (
      • Qin S.
      • Young C.B.
      • Duan X.
      • Chen T.
      • Supekar K.
      • Menon V.
      Amygdala subregional structure and intrinsic functional connectivity predicts individual differences in anxiety during early childhood.
      ,
      • Baur V.
      • Hänggi J.
      • Langer N.
      • Jäncke L.
      Resting-state functional and structural connectivity within an insula-amygdala route specifically index state and trait anxiety.
      ,
      • Admon R.
      • Lubin G.
      • Stern O.
      • Rosenberg K.
      • Sela L.
      • Ben-Ami H.
      • Hendler T.
      Human vulnerability to stress depends on amygdala’s predisposition and hippocampal plasticity.
      ). This finding is also in line with the limited prior research examining infant amygdala functional connectivity in relation to other indicators of prenatal adversity [heightened maternal systemic inflammation (
      • Graham A.M.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Heim C.M.
      • Gilmore J.H.
      • Styner M.
      • et al.
      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
      ) and depressive symptoms (
      • Qiu A.
      • Anh T.T.
      • Li Y.
      • Chen H.
      • Rifkin-Graboi A.
      • Broekman B.F.P.
      • et al.
      Prenatal maternal depression alters amygdala functional connectivity in 6-month-old infants.
      )], suggesting that various forms of psychological stress and biological stress mediators during pregnancy may result in a neural phenotype of increased amygdala integration into early emerging functional brain architecture.
      The findings for girls specifically indicate stronger amygdala connectivity to brain regions involved in sensory processing and integration (SMG and STG) as well as the DMN (precuneus and ITG). Increased coordinated functioning of the amygdala with sensory processing and integration regions may be indicative of heightened sensitivity to sensory information and has been observed in patients with subclinical and clinical anxiety (
      • Qin S.
      • Young C.B.
      • Duan X.
      • Chen T.
      • Supekar K.
      • Menon V.
      Amygdala subregional structure and intrinsic functional connectivity predicts individual differences in anxiety during early childhood.
      ,
      • Li Y.
      • Qin W.
      • Jiang T.
      • Zhang Y.
      • Yu C.
      Sex-dependent correlations between the personality dimension of harm avoidance and the resting-state functional connectivity of amygdala subregions.
      ,
      • Roy A.K.
      • Fudge J.L.
      • Kelly A.M.C.
      • Perry J.S.A.
      • Daniele T.
      • Carlisi C.
      • et al.
      Intrinsic functional connectivity of amygdala-based networks in adolescent generalized anxiety disorder.
      ,
      • Greening S.G.
      • Mitchell D.G.V.
      A network of amygdala connections predict individual differences in trait anxiety.
      ,
      • He Y.
      • Xu T.
      • Zhang W.
      • Zuo X.N.
      Lifespan anxiety is reflected in human amygdala cortical connectivity.
      ) and disorders involving chronic pain (
      • Hadjikhani N.
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      • Boshyan J.
      • Napadow V.
      • Maeda Y.
      • Truini A.
      • et al.
      The missing link: Enhanced functional connectivity between amygdala and visceroceptive cortex in migrain.
      ,
      • Linnman C.
      • Becerra L.
      • Borsook D.
      Inflaming the brain: CRPS a model disease to understand neuroimmune interactions in chronic pain.
      ). Heightened functional connectivity of the amygdala with regions of the DMN has been observed in response to environmental stress (
      • Bluhm R.L.
      • Williamson P.C.
      • Osuch E.A.
      • Frewen P.A.
      • Stevens T.K.
      • Boksman K.
      • et al.
      Alterations in default network connectivity in posttraumatic stress disorder related to early-life trauma.
      ) and in association with internalizing disorders (
      • Lanius R.A.
      • Bluhm R.L.
      • Coupland N.J.
      • Hegadoren K.M.
      • Rowe B.
      • Théberge J.
      • et al.
      Default mode network connectivity as a predictor of post-traumatic stress disorder symptom severity in acutely traumatized subjects.
      ,
      • Zhou Y.
      • Wang Z.
      • Qin L.
      • Wan J.
      • Sun Y.
      • Su S.
      • et al.
      Early altered resting-state functional connectivity predicts the severity of post-traumatic stress disorder symptoms in acutely traumatized subjects.
      ). Interestingly, heightened maternal cortisol was also associated with girls’ showing weaker functional connectivity of amygdala to the DLPFC, a region involved in effectively regulating negative emotions (
      • Etkin A.
      • Büchel C.
      • Gross J.J.
      The neural bases of emotion regulation.
      ). Taken together, the findings suggest a neural phenotype potentially indicative of increased sensitivity and vulnerability to experiencing heightened negative emotionality with decreased capacity for emotion regulation. This interpretation is supported by the association of stronger neonatal amygdala connectivity to the SMG, an important region for multimodal sensory integration (
      • Zhang S.
      • Li C.-S.R.
      Functional clustering of the human inferior parietal lobule by whole-brain connectivity mapping of resting-state functional magnetic resonance imaging signals.
      ,
      • Lewis J.W.
      • Wightman F.L.
      • Brefczynski J.A.
      • Phinney R.E.
      • Binder J.R.
      • DeYoe E.A.
      Human brain regions involved in recognizing environmental sounds.
      ,
      • Kheradmand A.
      • Lasker A.
      • Zee D.S.
      Transcranial magnetic stimulation (TMS) of the supramarginal gyrus: A window to perception of upright.
      ,
      • Balestrini S.
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      • Marino D.
      • et al.
      Multimodal responses induced by cortical stimulation of the parietal lobe: A stereo-electroencephalography study.
      ), with higher subsequent internalizing behavior.
      Boys displayed the opposite pattern, such that amygdala connectivity to these sensory processing and integration regions, as well as the DMN, was weaker and amygdala–DLPFC connectivity was stronger in association with heightened maternal cortisol. This neural phenotype could be indicative of decreased sensitivity to sensory stimuli and increased capacity for emotion regulation. Interestingly, prior research has indicated that alterations in the coordinated functioning of the amygdala associated with exposure to postnatal environmental stress may confer resilience to internalizing symptoms (
      • Herringa R.J.
      • Burghy C.A.
      • Fox M.
      • Davidson R.J.
      • Essex M.J.
      Enhanced fronto-subcortical connectivity following childhood adversity as a protective mechanism against internalizing in adolescence.
      ,
      • Gee D.G.
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      • Goff B.
      • Humphreys K.L.
      • Telzer E.H.
      • et al.
      Early developmental emergence of human amygdala–prefrontal connectivity after maternal deprivation.
      ). This may be the case for boys in the context of exposure to heightened maternal cortisol during pregnancy given that weaker neonatal amygdala–SMG connectivity was associated with lower internalizing symptoms at 2 years of age.
      Stronger neonatal amygdala connectivity mediated the association between elevated maternal cortisol during pregnancy and higher internalizing symptoms in girls. For boys, there was no direct association between maternal cortisol and internalizing symptoms. However, the significant indirect effect of heightened maternal cortisol on internalizing behavior via altered amygdala–SMG connectivity suggests one potential pathway through which heightened cortisol could lead to lower internalizing behaviors in boys. This finding is in line with a recent large study that identified an association between heightened maternal cortisol during pregnancy and lower negative emotionality in male infants (
      • Braithwaite E.C.
      • Pickles A.
      • Sharp H.
      • Glover V.
      • O’Donnell K.J.
      • Tibu F.
      • Hill J.
      Maternal prenatal cortisol predicts infant negative emotionality in a sex-dependent manner.
      ).
      These findings may suggest one early point of divergence in terms of later risk for internalizing psychiatric disorders, which are known to be more prevalent in girls (
      • Altemus M.
      • Sarvaiya N.
      • Neill Epperson C.
      Sex differences in anxiety and depression clinical perspectives.
      ,
      • Kessler R.C.
      • Chiu W.T.
      • Demler O.
      • Walters E.E.
      Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication.
      ,
      • McLean C.P.
      • Asnaani A.
      • Litz B.T.
      • Hofmann S.G.
      Gender differences in anxiety disorders: Prevalence, course of illness, comorbidity and burden of illness.
      ,
      • Rossant J.
      • Cross J.C.
      Placental development: Lessons from mouse mutants.
      ) beginning during puberty (
      • Mendle J.
      Why puberty matters for psychopathology.
      ). Future research will be needed to examine whether these early sex-specific alterations in brain connectivity and behavior increase sensitivity to the hormonal and social changes of puberty or even relate to shifts in pubertal timing, which in turn are associated with heightened internalizing in girls (
      • Mendle J.
      • Leve L.D.
      • Van Ryzin M.J.
      • Natsuaki M.N.
      Linking childhood maltreatment with girls’ internalizing symptoms: Early puberty as a tipping point.
      ). It is important to note that while girls are at greater risk for developing internalizing disorders, boys are at greater risk for developing other psychiatric diagnoses, including autism, substance abuse, and externalizing disorders (
      • Altemus M.
      • Sarvaiya N.
      • Neill Epperson C.
      Sex differences in anxiety and depression clinical perspectives.
      ,
      • McLean C.P.
      • Asnaani A.
      • Litz B.T.
      • Hofmann S.G.
      Gender differences in anxiety disorders: Prevalence, course of illness, comorbidity and burden of illness.
      ,
      • Kim Y.S.
      • Leventhal B.L.
      • Koh Y.-J.
      • Fombonne E.
      • Laska E.
      • Lim E.-C.
      • et al.
      Prevalence of autism spectrum disorders in a total population sample.
      ). Ongoing research following brain and behavioral development across infancy and childhood will be needed to delineate such pathways of risk and resilience for boys and girls.

      Limitations and Alternative Explanations for Findings

      Several limitations of this study should be considered. First, cortisol does not act in isolation to influence the developing fetus. The endocrine system interacts with others, including the immune system, with potential to influence fetal neurodevelopment (
      • Graham A.M.
      • Rasmussen J.M.
      • Rudolph M.D.
      • Heim C.M.
      • Gilmore J.H.
      • Styner M.
      • et al.
      Maternal systemic interleukin-6 during pregnancy is associated with newborn amygdala phenotypes and subsequent behavior at 2 years of age.
      ). While results of the current study remained consistent after adjusting for maternal systemic inflammation, we did not have sufficient statistical power to examine the interactive and cumulative influence of these systems, which will be a critical topic for future studies. With regard to genetic contributions to internalizing behavior, maternal stress during pregnancy has been found to be similarly associated with elevated child anxiety when mothers carry genetically related children (via in vitro fertilization) or unrelated children (via in vitro fertilization with egg donation), suggesting a large environmental influence (
      • Rice F.
      • Harold G.T.
      • Boivin J.
      • van den Bree M.
      • Hay D.
      • Thapar A.
      The links between prenatal stress and offspring development and psychopathology: Disentangling environmental and inherited influences.
      ). Our analyses also adjusted for maternal postnatal depression over a 2-year period, making it unlikely that shared genetic vulnerability to internalizing symptoms explains these findings.
      With regard to the neuroimaging results, although we hypothesized that maternal cortisol concentrations would be associated with altered neonatal amygdala connectivity to multiple brain regions spanning sensory, emotional, and cognitive functions, we did not set forth hypotheses about specific regions, and thus our interpretations regarding these connections are post hoc. Similarly, the lateralized findings, including left SMG and STG and right ITG and DLPFC, are consistent with prior research indicating asymmetry in effects of prenatal stress exposure on neurodevelopment (
      • Buss C.
      • Davis E.P.
      • Shahbaba B.
      • Pruessner J.C.
      • Head K.
      • Sandman C.A.
      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
      ) but were not specifically hypothesized. It should also be noted that neuroimaging with infants, by necessity, occurred during natural sleep. Ongoing investigation into the differences in coordinated brain functioning during infant sleep and wake cycles will likely aid in interpretation of findings moving forward. Lastly, the measure of child internalizing symptoms was based on maternal report and could be improved by the addition of a diagnostic interview and observational assessment.

      Conclusions

      The findings of the current study provide support for the sensitivity of developing fetal limbic brain circuitry to variation in maternal cortisol during pregnancy in a normative sample of pregnant women. This work builds on prior research (
      • Buss C.
      • Davis E.P.
      • Shahbaba B.
      • Pruessner J.C.
      • Head K.
      • Sandman C.A.
      Maternal cortisol over the course of pregnancy and subsequent child amygdala and hippocampus volumes and affective problems.
      ,
      • Kim D.-J.
      • Davis E.P.
      • Sandman C.A.
      • Sporns O.
      • O’Donnell B.F.
      • Buss C.
      • Hetrick W.P.
      Prenatal maternal cortisol has sex-specific associations with child brain network properties.
      ,
      • Braithwaite E.C.
      • Pickles A.
      • Sharp H.
      • Glover V.
      • O’Donnell K.J.
      • Tibu F.
      • Hill J.
      Maternal prenatal cortisol predicts infant negative emotionality in a sex-dependent manner.
      ) and provides novel insight into a potential pathway through which prenatal conditions may lead to increased risk for internalizing symptomatology in girls. More broadly, these findings advance understanding of one key aspect of maternal stress biology during pregnancy in relation to offspring brain and behavioral development. This work can provide a foundation for subsequent investigations, including examination of the multitude of factors that may contribute to elevated maternal cortisol levels during gestation and the longer-term sequelae of the observed alterations in offspring amygdala connectivity and emerging internalizing behaviors.

      Acknowledgments and Disclosures

      Support for this work was provided by the National Institute of Child Health and Human Development (Grant No. R01 HD060628 to PDW [EMA Assessment of Biobehavioral Processes in Human Pregnancy]), National Institute of Mental Health (Grant No. R01 MH091351 to CB and PDW [Fetal Programming of the Newborn and Infant Human Brain], Supplement to Grant No. R01 MH091351 to CB and DAF [Fetal Programming of Brain Functional Connectivity in Neonates and Infants], NIH Office of the Director (Grant No. UG3 OD023349 02 [Pre- and Postnatal Exposure Periods for Child Health: Common Risks and Shared Mechanisms] to CB, Richard K. Miller, Hyagriv N. Simhan, and Pathik D. Wadhwa) and Grant No. K99 MH111805 to AMG [A Targeted Approach to Examine the Influence of Maternal Psychological Stress on Newborn Brain Outcomes]), Gates Foundation Grand Challenges Explorations (to DAF and R. Nardos [Early Markers to Predict Cognition and Brain Development]), and Grant Nos. R00 MH091238 and R01 MH096773 (both to DAF).
      The authors report no biomedical financial interests or potential conflicts of interest.

      Supplementary Material

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