prenatal exposure to maternal depression, neonatal methylation of

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[Epigenetics 3:2, 97-106; March/April 2008]; ©2008 Landes Bioscience

Background: In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal (HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression.

Objective: To study this in humans, relationships between prenatal exposure to maternal mood and the methylation status of a CpG-rich region in the promoter and exon 1F of the human GR gene (NR3C1) in newborns and HPA stress reactivity at age three months were examined.

Results: Prenatal exposure to increased third trimester maternal depressed/anxious mood was associated with increased methylation of NR3C1 at a predicted NGFI-A binding site. Increased NR3C1 methylation at this site was also associated with increased salivary cortisol stress responses at 3 months, controlling for prenatal SRI exposure, postnatal age and pre and postnatal maternal mood.

Methods: The methylation status of a CpG-rich region of the NR3C1 gene, including exon 1F, in genomic DNA from cord blood mononuclear cells was quantified by bisulfite pyrosequencing in infants of depressed mothers treated with a serotonin reuptake inhibitor antidepressant (SRI) (n = 33), infants of depressed non treated mothers (n = 13) and infants of non depressed/non treated mothers (n = 36). To study the functional implications of the newborn methylation status of NR3C1 in newborns, HPA func-tion was assessed at three months using salivary cortisol obtained before and following a non noxious stressor and at a late afternoon basal time.

Conclusions: Methylation status of the human NR3C1 gene in newborns is sensitive to prenatal maternal mood and may offer a potential epigenetic process that links antenatal maternal mood and altered HPA stress reactivity during infancy.

Introduction

Early life experience can set a course leading to health or disease, often mediated by an altered capacity to regulate responses to stressful events.1 Among the earliest adverse experience is prenatal exposure to maternal depressed and anxious mood that confers life long risk for behavioral disturbances in childhood and beyond.2 This process of “fetal programming” is thought to be in part mediated by the impact of prenatal experience on the developing hypothalamic- pituitary-adrenal (HPA) axis stress system.3,4 The HPA axis is a dynamic metabolic system that regulates homeostatic mechanisms, such as the ability to respond to stressors, and from early fetal develop-ment it is highly sensitive to the impact of early adverse experience.5 In animal models, early exposure to stress increases corticosterone responses to mild stressors in adulthood6 and is associated with a reduction in hippocampal glucocorticoid receptor (GR) expression.7 In humans, prenatal depressed and anxious/stressed maternal mood is associated with increased rates of preterm delivery and lower birth weights,8-10 altered neonatal neurobehaviors, elevated cortisol11 and lower levels of serotonin12 in early infancy that may persist to at least six months of age.13 Exposure to prenatal stress in animal models and to maternal depression in humans may not constitute similar adverse prenatal experiences; however, they possibly share a final common pathways that affects HPA function in offspring. The mechanisms underlying environmental “programming” of the HPA system in humans following prenatal exposure to maternal depressed mood and other related factors remains unknown.

In sequential studies using a rodent model, Weaver, Meaney and colleagues have shown that variations in early maternal care (at a developmental stage analogous to the human third trimester of gesta-tion) are associated with differences in HPA stress response in the offspring,7 and importantly, associated with epigenetic alterations in gene expression.14 Offspring of mothers exhibiting increased levels of maternal care (licking, grooming, archedback nursing [LG-ABN]) in the early neonatal period are less fearful and show a blunted HPA stress response compared with offspring of Low-LG ABN mothers. Moreover increased HPA stress reactivity in offspring of mothers exhibiting decreased levels of maternal care appears to be medi-ated by decreased hippocampal GR gene (Nr3c1) expression.7,15

Research Paper

Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responsesTim F. Oberlander,1,2,* Joanne Weinberg,2,3 Michael Papsdorf,1 Ruth Grunau,1,2 Shaila Misri4 and Angela M. Devlin1,2

1Department of Pediatrics; Early Human Experience Unit; 2Child and Family Research Institute; 3Department of Cellular and Physiological Sciences; 4Department of Psychiatry; University of British Columbia; Vancouver, British Columbia Canada

Key words: NR3C1 methylation, prenatal maternal depression, hypothalamic pituitary adrenal, (HPA), infant stress response

*Correspondence to: Tim F. Oberlander; Early Human Experience Unit; Centre for Community Child Health Research; Room L408; 4480 Oak St.; Vancouver, British Columbia V6H 3V4 Canada; Tel: +604.875.3570; Fax: +6034.875.3569; Email: [email protected]

Submitted: 02/29/08; Accepted: 04/04/08

Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/6034

www.landesbioscience.com Epigenetics 97

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Neonatal NR3C1 methylation

Conversely, enhanced maternal care is associated with the opposite effects on GR expression and reduced stress responsiveness. Increased hippocampal GR expression has been shown to be mediated by increased central serotonin (5HT) turnover,16 leading to enhanced hippocampal expression of nerve growth factor inducible protein A (NGFI-A) and HPA negative feed back sensitivity.7,17 Using a rat model, McCormick18 identified the alternate first exons and demonstrated their role in controlling hippocampal GR levels via exon 17. Subsequently, crossfostering studies provided an indication that maternal care may be associated with epigenetic phenomena (i.e., a modification of the DNA that influences gene expression without altering nucleotide sequence) conferring a particular stress reactivity capacity across generations.5,15,19 Epigenetic mechanisms, such as DNA methylation and changes in chromatin structure, have been implicated as a means by which environmental factors, such as maternal behavior, can influence gene expression and are thought to produce long-term health consequences.20

Taking this one step further, Weaver et al.,14 showed that partic-ular patterns of maternal care have epigenetic effects that alter the methylation status of the NGFI-A binding site in a region of the Nr3c1 promoter responsible for control of hippocampal GR expres-sion and are critical for these effects.14 Decreased methylation of the NGFI-A consensus binding site in the rat Nr3c1 promoter results in increased expression of exon 17 Nr3c1 hippocampal transcripts and subsequently increased negative HPA feedback stress regulation.14 Methylation status of the 5' CpG within the NGFI-A binding site of the rat Nr3c1 promoter regulates binding of NGFI-A to this region and Nr3c1 transcription,21 offering evidence supporting a role for epigenetic processes in early environmental programming of HPA function.5,14,22 To date, findings supporting this hypothesis are limited to a very few rat studies and has not been investigated in humans.14,21 Studies of the human GR (NR3C1) (hGR) promoter region,23-25 identification of a possible analogous human NGFI-A site25 and methylation status studied in post mortem brains26 have been recently reported, however, the role of methylation status of the human NGFI-A binding site plays in NR3C1 expression remains to be experimentally proven.

Depression is often accompanied by hypersecretion of cortisol and altered HPA axis function27 and similar patterns have been observed in the offspring of depressed mothers.11 Serotonin reuptake inhibitor (SRI) antidepressants, which potentiate central 5HT, are increasingly used to manage maternal mood disorders during preg-nancy28 and are thought to work by “normalizing” dysregulated HPA function, possibly by increasing hippocampal GR expres-sion.29 Moreover, chronic fluoxetine exposure in an animal model, appears to increase NGFI-A mRNA expression in the hippocampus and cerebral cortex,30 which might reflect the adaptive neuronal and neuroendocrine changes, such as HPA axis negative feedback regulation, that follows antidepressant treatment.

We undertook this study to investigate whether prenatal exposure to maternal depressed/anxious mood is associated with methylation status of the promoter and exon1F of the human NR3C1 gene in newborns and whether methylation status of this specific region of NR3C1 at birth is associated with altered HPA function during infancy. Given the importance of the methylation status of the 5' NGFI-A bindings site in the rat Nr3c1 gene14,21 we expected that prenatal exposure to maternal mood would increase methylation at

the human homologue of this region27 and this would be associated with altered HPA stress reactivity during infancy. Moreover, given that SRIs may influence NGFI-A expression via altered 5HT levels, we expected that prenatal SRI-exposure would also effect HPA func-tion mediated by the methylation status of the human NR3C1 gene in neonates of mothers who experienced antenatal depression and were treated with an SRI antidepressant medication.

Results

Maternal characteristics. This study included a cohort of 82 women, including 33 depressed and SRI treated (SRI Treated), 13 depressed and not medicated (Depressed/Non SRI Treated) and 36 not depressed and not SRI treated (Non SRI Treated) mothers prospectively recruited during pregnancy. All were assessed during the second and third trimesters, and at three months postpartum (Table 1). With the exception of mood and medication used, demo-graphic characteristics did not vary significantly between groups (p > 0.05). All mothers took a prenatal vitamin containing the typical prenatal folic acid dose (0.8–1 mg). As maternal mood measures were continuous, they were used as predictors in all regres-sion models. This also allowed us to account for the wide range of depressive symptoms observed among both SRI treated and SRI untreated groups across time, as some mothers in our untreated group became depressed and some crossed over to the SRI treated group. It is noteworthy that the SRI treated group was significantly higher on all of the depressed mood measures at all time points (all p’s < 0.005), and that there was no time by group interaction for any measure (all p’s > 0.05).

Neonatal and infant characteristics. Infant characteristics are presented in Table 2. All infants were full term. SRI-exposed neonates had lower five minute Apgar scores compared with non exposed infants (p = 0.049). At the time of the three month HPA stress study, prenatally SRI-exposed exposed infants were older than infants of non-SRI treated but depressed mothers (p < 0.05).

Maternal and neonatal methylation status. Maternal methyla-tion levels at each of the 13 CpG sites analyzed in exon 1F of NR3C1 during the third trimester are shown in Figure 2A and were compa-rable in magnitude to neonatal levels (Fig. 2A). Group differences were not observed (p > 0.05).

Third trimester maternal mood was not associated with methy-lation status at any of the maternal 13 CpG sites (all p’s > 0.05). There were no significant differences in maternal methylation levels between samples drawn at 33 weeks gestation and delivery (p > 0.05). Maternal methylation was not associated with neonatal status at any site (p < 0.05).

Neonatal methylation levels at each of the 13 CpG sites analyzed in exon 1F of NR3C1 in newborns are shown in Figure 2B.

Differences between the exposure groups and the methylation status of NR3C1 at each of these sites were not significant (all p’s > 0.05).

LINE-1 methylation levels did not vary between SRI exposed and non-exposed infants (p > 0.05) even when accounting for prenatal exposure to maternal depressed mood (Fig. 2C).

LINE-1 methylation levels in newborns were also not associated with baseline or stress cortisol levels in infants at three months (p > 0.05). Methylation status did not vary with mode of delivery.

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Table 1 Maternal demographic, medication and mood characteristics

Maternal characteristics SRI Treated (n = 36) Depressed/Non SRI Treated (n = 13) Non SRI treated (n = 33) (mean (SD)) (mean (SD)) (mean (SD))Maternal age (yrs) 32.0 (4.7) 32.7 (5.2) 32.8 (5.0)First pregnancy (%) 39 58 48Smoking (n) 1 0 1Caesarean section rate % 36 31 33Reported alcohol use (drinks/pregnancy) (%) • 0 74 64 54 • 1–5 19.4 27 26 • 6–10 2.8 0 14 • 11–25 8.3 0 7Prenatal SRI use (days) 222.2 (74) n/a n/aPrenatal SRI medication use (2nd trimester) (n) (median dose, mg) • paroxetine (18) 27.5 n/a n/a • fluoxetine ( 6) 35 n/a n/a • sertraline (5) 100 n/a n/a • venlafaxine (2) 56.3 n/a n/a • citalopram (5) 30 n/a n/aMaternal mood measures2nd trimester maternal mood: (mean 23.8 ± 3.5 wks) • Ham‑A 14.2 (7.9) 17.2 (7.86) 4.38 (3.74)* • Ham‑D 12.6 (6.7) 14.8 (9.32) 3.70 (3.52)* • EPDS 10.3 (6.1) 14.0 (6.32) 3.29 (3.68)*3rd trimester maternal mood (mean 33.7 ± 1.1 wks) • Ham‑A 11.6(7.4) 10.8 (5.2) 4.88 (5.0)* • Ham‑D 9.6 (6.0) 9.4 (6.7) 2.45 (2.4)* • EPDS 8.4 (4.6) 10.7 (5.7) 3.45 (3.4)*Maternal mood 3 months: • Ham‑A 10.4 (7.4) 6.9 (5.5) 3.1 (2.7)* • Ham‑D 7.2 (5.6) 6.8 (5.3) 2.1 (3.1)* • EPDS 7.3 (5.5) 6.5 (4.7) 2.7 (3.1)* • PSI 71.0 (16.3) 73.7 (26.4) 61.1 (16.6)

*p < 0–0.05 for differences between SRI exposed and Depressed/non SRI groups and non‑depresed/non‑treated. Abbreviations: HAM‑A, hamilton rating scale for anxiety; HAM‑D, hamilton rating scale for depression; EPDS, edinburgh postnatal depression scale; PSI, parent stress index; n/a, not applicable; SD, standard deviation.

Table 2 Neonatal and infant characteristics

SRI Exposed Depressed/Non SRI exposed Non SRI Exposed (n = 33) [mean (SD)] (n = 13) (mean SD) (mean SD)Gestational age at birth (weeks) 39.4 (1.5) 40.3 (0.87) 40.0 (1.3)Sex (M:F) 16:20 5:7 16:17Birth Weight (g) 3427 (495) 3756 (574) 3536 (504)Apgar Score 1 minute 7.56 (1.48) 8.33 (0.89) 8.03 (1.49)Apgar Score 5 Minute 8.72 (0.85) 9.17 (0.39) 9.0 (0.50)*Age at 3 month visit (months) 3.37 (0.47) 2.93 (0.32) 3.18 (0.34)

*p < 0–0.05 for differences between SRI exposed and non‑exposed.

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Neonatal methylation status at specific CpG sites and maternal mood. Increased second trimester EPDS symptom scores were associated with increased methylation levels at CpG2 (β = 0.286; t = 2.216; p = 0.031; η′2 = 0.075) in the fragment of the NR3C1 gene analyzed in cord blood mono-nuclear cells (Fig. 1) as shown in Figures 3A, even when controlling for prenatal SRI exposure.

Similarly, second trimester HAM-D scores were positively related to neonatal methylation levels, though this relationship only approached significance (β = 0.253; t = 1.874, p = 0.066, η′2 = 0.056, controlling for SRI exposure). Methylation levels at CpG1 and CpG3 were not related to second trimester maternal mood (depression and anxiety) reports (p > 0.05).

At mid-third trimester, again controlling for SRI use, HAM-A and HAM-D scores were positively associated with methylation status at CpG1 (β = 0.312, t = 2.614, p = 0.011, η′2 = 0.081 and β = 0.317, t = 2.602, p = 0.011, η′2 = 0.081, respectively) (Fig. 3B).

Similarly, EPDS, HAM-D and HAM-A scores (third trimester) were positively associated with CpG2 methylation (β = 0.305, t = 2.651; p = 0.010; η′2 = 0.084; β = 0.371, t = 3.082, p = 0.003; η′2 = .110; and β = 0.363, t = 3.080, p = 0.003; η′2 = 0.110, respectively) (Fig. 3C).

Increased third trimester EPDS and HAM-D scores were also associated with increased neonatal methylation levels at CpG3 (β = 0.244; t = 2.105, p = 0.039, η′2 = 0.054 and β = 0.284, t = 2.314, p = 0.023, η′2 = 0.065, respectively) (Fig. 3D).

Second and third trimester maternal mood measures were not associated with methylation status at any other remaining CpG sites analyzed in exon 1F of NR3C1. Controlling for prenatal SRI expo-sure, gestational age at birth and 5 minute Apgar scores did not alter these relationships.

Neonatal methylation status and infant HPA stress reactivity. Using a regression model, controlling for third trimester maternal mood (EPDS scores), neonatal birth characteristics (five minute Apgar and gestational age at birth), parental stress (PSI), and infant age at the three month visit, HPA reactivity, as reflected by increasing cortisol stress change scores (baseline minus recovery), was predicted by greater NR3C1 methylation at CpG3 in newborns (β = 0.390, t = 2.466, p = 0.018, η′2 = 0.110) (Fig. 4A).

However, as cortisol stress responses varied, increasing in some infants and decreasing in others in response to the stressor (i.e., cortisol change scores had negative and positive values), a direction of change score (i.e., an increased cortisol response vs. a decreased cortisol response) was also tabulated to determine whether there was a relationship between direction of cortisol change and NR3C1 CpG3 methylation status. Infants who responded with increased cortisol levels had significantly higher NR3C1 CpG3 methyla-tion status compared with infants whose cortisol levels declined in response to the three month stress challenge [F(1,50) = 7.430; p = 0.010] (Fig. 4B).

Importantly, prenatal SRI exposure did not mediate any of these relationships and beyond CpG3, these relationships were not observed at any other CpG sites in the fragment of the NR3C1 gene that was analyzed (Fig. 1).

Afternoon-early evening cortisol levels in infants at three months of age were not associated with the methylation status of NR3C1

at birth (p > 0.05), but were significantly lower in infants with prenatal exposure to maternal SRIs (β = 0.355, t = 2.437; p = 0.018, η′2 = 0.100).31

Discussion

Exposure to increased third trimester depressed maternal mood was associated with increased neonatal methylation of CpG3, the 5' CpG in the potential NGIF-A consensus binding site 5' to exon 1F of the NR3C1 gene. Importantly, increased methylation at CpG3 in newborns was the only CpG site in the fragment of NR3C1 analyzed that was associated with increased HPA stress reactivity at three months in response to a visual novel stimuli, even when accounting for neonatal characteristics, maternal pre and post natal maternal mood, and three month maternal stress. Decreased methylation at this site accounted for approximately 11% of the variance associ-ated with decreased salivary cortisol stress responses at three months. Increased second trimester maternal depressed mood was also asso-ciated with increased methylation at the CpG2 site and increased methylation status for CpG and CpG2 were associated with increased maternal third trimester depressed mood. Mean LINE-1 methylation levels, a proxy measure of global methylation, was not associated with maternal mood, did not vary with SRI medication exposure, nor was it associated with three month infant cortisol stress reactivity, suggesting that our findings did not reflect possible other genomic disturbances in methylation (i.e., secondary to disor-dered methyl metabolism). While methylation status at CpG3 in newborns was associated with infant stress cortisol reactivity, it was not associated with afternoon/early evening levels. Contrary to our expectation, prenatal SRI exposure was not itself associated with an altered methylation pattern, though this does not rule out a drug-related methylation effect. Together, these findings suggest that increased third trimester depressed maternal mood is associated with increased infant HPA stress responsiveness, via a potential epigenetic link that involves methylation of the human NR3C1 gene. Prenatal exposure to maternal mood, even at non-clinical levels, remained the key predictor of NR3C1 methylation status in newborns.

Figure 1. Schematic representation of the human NR3C1 gene and the CpG-rich region analyzed by bisulfite pyrosequencing. The diagram is based on the previ-ously characterized 5'-end of the human NR3C1 gene, which contains multiple first exons, with multiple transcriptional start sites and mRNA splice variants.15 The numbering is relative to the translational start site (+1), which is located 13 nucleotides from the start of exon 2. The region analyzed by bisulfite pyrosequenc-ing is illustrated. This region contains 13 CpGs and encompasses exon 1F (capital letters), which is the human homologue of the rat exon 1–7,12 previously shown to be differentially methylated. The potential NGFI-A consensus binding site is underlined.

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Importantly, maternal methylation status during the third trimester was not affected by maternal SRI exposure nor was it associated with maternal third trimester depressed or anxious mood.

Gestational SRI exposure leads to transplacental transfer42,43 and neonatal withdrawal symptoms,44,45 and concerns about behavioral effects following prenatal exposure remain unresolved.46 While SRI exposure in this study was not associated with NR3C1-methylation status, this should not imply that SRIs did not alter brain GR meth-ylation. Yau et al.,47 showed that SRIs had a significant effect on GR mRNA levels in an animal model, thereby raising the possibility, albeit hypothetical at this point, that prenatal exposure to SRIs could also subsequently lead to epigenetically programmed higher hippo-campal GR levels. This remains to be determined in humans.

The methylation status in newborns of key regions of the promoter and exon 1F of the human NR3C1 gene appears to be at some level developmentally sensitive to prenatal exposure to maternal depressed mood. Neonatal CpG2 methylation status was associated with both second and third trimester maternal mood, indicating that methylation status of CpG2 may also be important in regulating NR3C1 expression. Recently in silico searches using MatInspector software48 revealed that this site corresponds to a consensus binding site for neural restrictive silencer factor (NRSF). However, in this study methylation status of the NR3C1 promoter was more sensitive to maternal third trimester mood. Further, CpG3 in the neonate was the only site where increased methylation status was associated with increased third trimester affective symptoms and increased infant cortisol stress reactivity at three months. This is consistent with increased methylation at the 5' NGFI-A binding site, decreased GR expression and increased corticosterone stress responses observed in rodent offspring of LG-ABN mothers.6,13 In a single human study, however, Moser et al.,26 recently reported that neither the 5' nor 3' CpG sites within the NGFI-A consensus binding site in the human NR3C1 promoter for exon 1F from human post mortem hippocampal tissue were methylated (mean age 81 years in individuals with dementia), reflecting possible differences in promoter methylation patterns between humans and animals or within a heterogeneous sample of cells within an individual. In that study no information was available regarding the potential influence of other environmental factors, disease processes or medications on NR3C1 methylation status. The developmental ontogeny of methy-lation of these sites remains to be studied.

This is the first human study to show an association between prenatal exposure to maternal depressed mood and the methylation status of the promoter and exon 1F of the gene for GR (NR3C1) in newborns. Consistent with previous studies using animal models, we observed that the methylation status of CpGs contained within a potential NGFI-A binding site (CpG3) were most sensitive to prenatal exposure to maternal depressed mood. The association between methylation status of CpG3 in the NR3C1 promoter and the three month HPA stress response raises the possibly that neonatal methylation at this site might offer an early epigenetic marker of exposure to late gestational maternal depressed mood and risk for altered HPA function in humans. While methylation of CpG3 was positively correlated with infant cortisol change scores suggesting that methylation of this gene has functional consequences, more direct evidence, such as the relationship between methylation status and GR mRNA levels, is required as a crucial next step needed to link

Figure 2. Methylation levels (%) at each of the 13 CpG sites analyzed in exon 1F of NR3C1 in maternal (Fig. 2A) and newborns (Fig. 2B) (expo-sure group means ± SEM). Prenatal exposure to SRIs did not influence the methylation status of NR3C1 CpG sites (all differences between groups p’s > 0.05). (C) Neonatal LINE-1 methylation (% ± SEM) status and prenatal exposure. LINE-1 methylation levels did not vary between SRI exposed and non-exposed infants (p > 0.05) even when accounting for prenatal maternal mood (Fig. 2C). LINE-1 methylation levels in newborns were not associated with basal or stress cortisol levels in infants at three months (p > 0.05).

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Figure 3. Associations between exposure to prenatal maternal mood as assessed during the second and third trimesters and newborn NR3C1 methylation status. Increased second trimester EPDS symptom scores, controlling for SRI exposure, were associated with increased neonatal methylation levels at CpG2 (β = 0.286; t = 2.216; p = 0.031, η′2 = 0.075) (A). At midthird trimester, again controlling for SRI use, HAM-A and HAM-D scores were positively associated with methylation status at CpG1 (β =.312, t=2.614, p = 0.011, η′2 = 0.081 and β =.317, t = 2.602, p = 0.011, η′2 = 0.081, respectively) (B). Similarly, EPDS, HAM-D and HAM-A scores (third trimester) were positively associated with increased levels of CpG2 methylation (β = 0.305, t = 2.651; p = 0.010; η′2 = 0.084; β = 0.371, t = 3.082, p = 0.003; η′2 = 0.110; and β = 0.363, t = 3.080, p = 0.003; η′2 = 0.110, respectively) (Fig. 3C). Increased third trimester EPDS and HAM-D scores were also associated with increased neonatal methylation levels at CpG3 (β = 0.244; t = 2.105, p = 0.039, η′2 = 0.054 and β = 0.284, t = 2.314, p = 0.023, η′2 = 0.065, respectively) (D).

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methylation status with its functional consequence. Similarly, future studies are also needed to study relationships between GR mRNA and maternal mood.

Importantly, these are correlational findings and an under-standing of direction or mediational role of the methylation status at CpG3 in the NR3C1 gene can not be inferred from this initial study. Studies of the impact of additional environmental factors, maternal diet (i.e., folate), toxin exposure (i.e., prenatal alcohol exposure) and the direct impact of antidepressants on methylation status are also required.49,50 While the fragment of the human NR3C1 promoter and exon 1F was recently identified by others to contain a potential

NGFI-A binding site,35 experimental confirmation is required to determine a role for NGFI-A in transcriptional regulation of the human NR3C1 gene. Relationships between glucocorticoid receptor expression in a hetero-geneous population of venous cord mononuclear cells, hippocampal NR3C1 expression and HPA stress responses in newborns needs to be determined. Further studies are needed to determine links between the methylation status of this gene in these cell types and methylation status in central and peripheral (i.e., adrenal) regions more directly related to the stress response. Studies examining infant HPA reactivity under other provocative challenges (social, emotional, physical), with infants of mothers with a wider range of mood symptoms are also required. Finally, studies of links between methylation status of CpG3, human NR3C1 (i.e. mRNA) and links to subsequent HPA func-tion later in infancy are needed to elucidate the functional implications of how early exposure to maternal depressed and anxious mood epigenetically influences infant and child development.

Methods

Subjects. With approval from the University of British Columbia Research Ethics Board, Children’s and Women’s Health Centre of British Columbia Research Review Committee, and informed parent consent, a cohort (n = 98) of mothers was recruited in their early second trimester as part of a study of the impact of prenatal psychotropic medication exposure on neonatal health. Of the original 98 mothers who completed a second trimester data collection, samples from 16 mothers and infants at delivery were not available for analysis (i.e., mothers with-drew prior to delivery, inadequate DNA yield, infant cord blood sample was not obtained at birth) leaving 36 infant samples in the non medicated non-depressed group, 13 in the depressed non medication group and 33 samples in the SRI treated group. Mothers were included if they took no other serotoninergic medications or other psychotropic medications during their pregnancy. All mothers were taking folate during their pregnancies (1 mg folate/day) either as a component of a prenatal vitamin supplement or on its own.

Salivary cortisol, Infant HPA stress challenge responses and basal cortisol levels. To examine the functional impli-cations related to neonatal methylation status, two separate

studies were undertaken at three months of age as a part of a parallel study of HPA stress reactivity following among infants with prenatal SRI exposure.31 Stress challenge saliva samples were collected just before the start (baseline) of an established infant-controlled habit-uation-information processing task, where visual stimuli presented in a fixed order on a screen facing the infant.32 The stress challenge study took place in the morning (mean 11:53 ± 0.06 hrs). A baseline saliva sample was collected when the infant was in a quiet awake alert state33 period ten minutes before the start of the information processing task (Baseline), then followed by a post stress recovery sample following baseline (Recovery, mean 39.3 ± 13.8 min) at

Figure 4. (A) Cortisol stress change score at three months and neonatal methylation status at CpG3 site. (B) Direction of cortisol change with stress at three months and CpG3 neonatal methylation levels. Increased cortisol change with the stress/challenge was associated with significantly greater NR3C1 methylation at CpG3 in newborns (β = 0.390, t = 2.466, p = 0.018, η′2 = 0.110), controlling for third trimester maternal mood (EPDS scores) and parental stress report at three months (PSI) (A). Infants who responded with increased cortisol levels had significantly higher NR3C1 CpG3 meth-ylation status compared with infants whose cortisol levels declined in response to the three-month stress [F(1,50) = 7.115; p = 0.010] (B).

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the end of the procedure. Cortisol levels at baseline and at recovery were used to calculate a cortisol change score (baseline cortisol levels minus post stress recovery cortisol measure). Infants were last fed 106 ± 51 minutes (mean ± SD) before the procedure. To study basal cortisol levels, on a day separate from the stress reactivity study, a late afternoon/early evening salivary sample (mean 17:45 ± 0.09 hrs) was obtained by the mother at home during a three week period following the laboratory visit.

Bisulfite pyrosequencing DNA methylation analysis. Maternal blood (mid 3rd trimester) and neonatal cord (venous) blood samples were obtained for DNA methylation analysis. Cord blood was collected at delivery and methylation status was determined using two approaches: long interspersed nucleotide elements (LINE-1) were quantified as an indicator of global methylation status34 using the PyroMark LINE-1 kit (Biotage).34 The methylation status of a CpG-rich region of the human NR3C1 gene, including exon 1F 35 (Fig. 1), was quantified by bisulfite pyrosequencing.36

This region contains 13 CpG sites, with CpGs 3 and 4 repre-senting a potential NGFI-A consensus binding site. This region is considered to be homologous to the region of the rat Nr3c1 gene35 shown by Weaver et al.,14 to be differentially methylated in response to differences in maternal care.

Genomic DNA was extracted from maternal and newborn leuko-cytes (a mixture of monocytes, lymphocytes and granulocytes) using the Flexigene DNA Blood Kit (Qiagen, Valencia, CA). Genomic DNA samples (1 μg) were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen) following the manufacturer’s suggested protocol, and stored at -20°C until further analysis. A 410 bp fragment corresponding to a portion of the NR3C1 gene between -3492 and -3082 relative to the translational start site (Fig. 1) was amplified by PCR from bisulfite-treated DNA. The PCR reaction used HotStar Taq DNA Polymerase (Qiagen) and the following primers: PMHumGCCRF, 5'-TTTTTTTTTTGAAGTTTTTTTA-3' and PMHumGCCRR, 5'-CCCCCAACTCCCCAAAAA-3' (IDT Inc., Coralville, IA), with the reverse primer containing a biotin at the 5' end. Cycling condi-tions were 94°C for 15 minutes followed by 50 cycles of 94°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute with a final extension of 10 minutes at 72°C. PCR products were purified and sequenced using a PyroMark MD System (Biotage, Foxboro, MA) following the manufacturer’s suggested protocol and two sequencing primers: PMHumGCCRS2, 5'-GAGTGGGTTTGGAGT-3' and PMHumGCCRS3, 5'-AGAAAAGAAATTGGAGA AATT-3' (IDT). The PMHumGCCRS2 sequencing primer was designed to sequence a 50 bp region of the 410 bp PCR fragment that contains the first five CpG sites (see Fig.) and the PMHumGCCRS3 sequencing primer was designed to sequence a 104 bp region encompassing CpG sites 6–13. The methylation status of LINE-1 repetitive elements was also analyzed as an index of global methylation status,34 and was conducted using the PyroMark LINE-1 kit (Biotage) and following the manufacturers suggested protocol. This method involved incu-bation of genomic DNA with sodium bisulfite, which converts all unmethylated cytosine residues to thymidine. Sequencing of PCR products amplified from LINE-1 and the NR3C1 promoter and exon 1F of bisulfite-treated DNA indicate whether the cytosine residues at each of the CpG sites were methylated. The percent methylation at each CpG site was quantified using the Pyro Q-CpG software, version 1.0.9 (Biotage).

Maternal mood. Prenatal maternal mood was assessed using clinician- (blinded to medication group status) and mother-rated measures at the time of study enrollment (approximately 26 weeks), at 33 weeks gestation and at three months postpartum using three instruments. Measures included the Hamilton Rating Scale for Depression (HAM-D),37 a 21-item clinician administered scale designed to assess the severity of depression. Scores on this scale have a possible range of 0–63, with higher scores being associated with higher levels of depression in the patient. The Hamilton Rating Scale for Anxiety (HAM-A)38 is a 14-item clinician administered scale that measures the severity of anxiety. Total scores on this scale have a possible range of 0–56, with higher scores being associated with higher levels of anxiety in the patient. The Edinburgh Postnatal Depression Scale39 is a ten item, patient-rated instrument used to assess symptoms of depressed mood in both pre and postnatal settings. At three months post partum, the Parenting Stress Index-Short Form (PSI-SF),40 a 36-item questionnaire was used to assess an overall level of parenting stress. Total stress scores on this inventory reflect personal parental stress, stresses associated with caregiver-child interaction, and stresses relating to the child’s behavioral character-istics. Total scores on this inventory can range from 36 to 180, with higher scores being indicative of higher levels of stress, with scores above 90 indicative of clinically significant levels of stress.

Salivary cortisol. The saliva was expressed into a vial and stored at -20°C until assayed using the Salimetrics High Sensitivity Salivary Cortisol Enzyme Immunoassay Kit for quantitative determination of salivary cortisol (Salimetrics LLC, Philadelphia, PA) in Weinberg’s laboratory at the University of British Columbia. Intra and inter assay coefficients of variation were 2.92 and 3.41%, respectively.

Data reduction and statistical analyses. Cortisol values were log transformed to normalize the distribution, and means, SD and cortisol change scores (Baseline-Recovery) were calculated. Group means were compared using univariate analyses of variance (ANOVAs), followed by post hoc tests as appropriate. For display and descriptive purposes methylation outcomes are presented for the three exposure groups (SRI, depressed not medicated, not depressed/not medicated), however, given that maternal mood is a continuous variable, multiple regression models were used to examine all predic-tive models in the study. First, to focus on relationships between antenatal maternal mood and newborn methylation status, we examined methylation status at the 5' and 3' ends of the NGFI-A consensus binding sites (CpGs 3 and 4 sites), in a region encom-passing exon 1F. As a control, we also examined methylation at each of the 11 other possible CpG sites within the promoter and exon 1F region of the NR3C1 gene, controlling for prenatal SRI exposure. Relationships between newborn methylation status, specifically at CpG 3 and 4 sites, and measures of cortisol stress reactivity at three months (means and a cortisol change score [baseline minus post stress, recovery]) were examined, controlling for neonatal charac-teristics, prenatal maternal mood and SRI exposure, and post natal maternal stress report. A similar model was also used to examine associations between newborn methylation status and afternoon basilar (i.e., non challenge) cortisol levels at three months. Based on the work of Weaver et al., indicating that methylation status of the 5' NGFI-A binding site was a key control point to GR expression, our study tested the hypothesis that antenatal maternal depression would be associated with altered neonatal methylation at the 5' and

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3' sites (CpGg 3 and 4). In exploratory analyses, we also looked at the methylation status of 11 other CpG sites in the human NR3C1 gene, including exon 1F. Given exploratory nature of this approach, Bonferroni corrections were not applied due to the heightened risk of Type II errors associated with such corrections; instead, effect sizes for the regressions (squared semi partial correlations or η′2) were computed to allow examination of the magnitude of the predictive relationships.41

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research (CIHR #MOP 54490 and 57837), the March of Dimes Foundation (USA) (#12-FY01-30) and the Early Human Learning Partnership (HELP, UBC). T.O. is a Senior HELP Scholar and is the R. Howard Webster Professor in Early Child Development. A.D. is a New Investigator of the Heart & Stroke Foundation of Canada. R.E.G. is supported by a Distinguished Scholar Award from the Child & Family Research Institute, UBC and a Senior Scholar Award from HELP. J.W. is a Distinguished University Scholar, UBC.

We are grateful to Ursula Brain, Jodi Pawluski and Tracy Weir for their assistance with preparation of this manuscript and to Rachel Wade for her technical assistance with the methylation assays.

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prenatal exposure to maternal depression, neonatal methylation of

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