ORIGINAL INVESTIGATION

Prenatal alcohol exposure and cortisol activityin 19-month-old toddlers: an investigationof the moderating effects of sex and testosterone

Isabelle Ouellet-Morin & Ginette Dionne &

Sonia J. Lupien & Gina Muckle & Sylvana Côté &

Daniel Pérusse & Richard E. Tremblay & Michel Boivin

Received: 17 March 2010 /Accepted: 8 July 2010 /Published online: 18 August 2010# Springer-Verlag 2010

AbstractRationale Early exposure to stress and teratogenic sub-stances have an impact on brain structures involved incognition and mental health. While moderate-to-high levelsof prenatal alcohol exposure (PAE) have repeatedly beenassociated with long-term neurodevelopmental deficits, noconsensus has yet been reached on the detrimental effectsof low-to-moderate PAE on the children’s functioning,including the limbic–hypothalamic–pituitary–adrenal axis.Objectives The study aims to examine the associationbetween low PAE and cortisol response to unfamiliarsituations in 19-month-old children and to determine

whether this association was moderated by sex andtestosterone levels.Methods Information regarding PAE, cortisol response tounfamiliar situations, and testosterone activity was avail-able in a total of 130 children participating to the QuébecNewborn Twin Study (Montréal, QC, Canada). Motheralcohol consumption during pregnancy was assessed via asemistructured interview conducted when the children were6 months of age. The contribution of prenatal and postnatalconfounds were examined.Results Disrupted patterns of cortisol activity were ob-served only in PAE males. Testosterone tended to be

Electronic supplementary material The online version of this article(doi:10.1007/s00213-010-1955-z) contains supplementary material,which is available to authorized users.

I. Ouellet-MorinMRC Social, Genetic, and Developmental Psychiatry Centre,Institute of Psychiatry, King’s College London,De Crespigny Park,London SE5 8AF, UK

G. Dionne :G. Muckle :M. Boivin (*)School of Psychology, Université Laval,2325 rue des Bibliothèques, Pavillon Félix-Antoine-Savard,Québec, QC, Canada G1V 0A6e-mail: michel.boivin@psy.ulaval.ca

S. J. LupienCenter for Human Stress Research, Fernand-Seguin ResearchCenter, Université de Montréal,7331 rue Hochelaga,Montréal, QC, Canada H1N 3V2

S. Côté : R. E. TremblayInternational Laboratory for Child and Adolescent Mental HealthDevelopment, INSERM U669,97 Boulevard de Port Royal,75679 Paris cedex 14, France

S. CôtéDepartment of Social and Preventive Medicine, Faculté deMédecine, Université de Montréal,C.P. 6128, succursale Centre-ville,Montréal, QC, Canada H3C 3J7

D. PérusseDepartment of Anthropology, Université de Montréal,C.P. 6128, succursale Centre-ville,Montréal, QC, Canada H3C 3J7

R. E. TremblayDepartment of Pediatrics, Psychiatry and Psychology,Université de Montréal,3175 chemin Côte Sainte-Catherine,Montréal, QC, Canada H3T 1C5

R. E. TremblaySchool of Public Health and Population Science,University College Dublin,Belfield,Dublin 4, Ireland

Psychopharmacology (2011) 214:297–307DOI 10.1007/s00213-010-1955-z

negatively associated with the cortisol response, but not forPAE males, suggesting an altered sensitivity to theinhibitory effects of testosterone in these participants.Conclusions Low levels of PAE were associated withdisrupted cortisol activity, and males may be at higher risk.These findings challenge the existence of a “safe level” ofalcohol consumption during pregnancy and have publichealth implications.

Keywords Prenatal alcohol exposure . Stress . Teratogens .

Adversity . Cortisol . LHPA axis . Sex differences .

Testosterone . Children . Alcohol . Androgen .

Development . Drinking . Environmental . Glucocorticoid .

Prenatal . Sex differences

Introduction

Since the initial description of fetal alcohol syndrome(FAS) in the early 1970s (Jones and Smith 1973), robustevidence from preclinical animal models and human studiessupports a teratogenic effect of prenatal alcohol exposure(PAE) on fetal development (Riley and McGee 2005;Wacha and Obrzut 2007). FAS is characterized by a uniquepattern of facial anomalies, growth retardation, and neuro-developmental deficits of the central nervous system in thecontext of documented PAE (Warren and Foudin 2001).However, not all children exposed to alcohol during fetaldevelopment receive a diagnostic of FAS. A more inclusiveterm, fetal alcohol spectrum disorders, is thus increasinglyused to describe the wider range of physical, neuroanatom-ic, neuropsychological, and sociobehavioral deficits associ-ated with PAE (Mattson et al. 2001; Streissguth andO’Malley 2000).

In humans, moderate-to-high PAE (≥1 standard unit perday) is recurrently associated with long-term neurodeve-lopmental deficits, including lower IQ scores (Jacobson etal. 2004; Mattson et al. 1997; Streissguth et al. 1990),greater problems in attention and executive functions(Burden et al. 2005; Coles et al. 1997; Kable and Coles2004; Kodituwakku 2007; Mattson et al. 1999), andbehavioral problems (Jacobson and Jacobson 1994; Kellyet al. 2000; Mattson and Riley 2000). PAE children alsotend to be smaller in weight, length, and head circumfer-ence at 3 years of age (Day et al. 1991). Conversely, noconsensus has yet been reached on possible detrimentaleffects of low-to-moderate PAE (<1 standard unit per day;Henderson et al. 2007; Nathanson et al. 2007; O’Brien2007; Sayal 2007). The lack of clear guidelines regardinglow-to-moderate maternal alcohol consumption duringpregnancy is troubling considering the dose–responseassociations documented between PAE and higher behav-ioral problems in childhood, greater problems in executive

functions in adolescence, and higher prevalence of antiso-cial personality disorder and alcohol-related problems inearly adulthood (Kelly et al. 2000; Sayal et al. 2007; Soodet al. 2001; Streissguth 2007).

The inconclusive findings regarding the adverse effect oflow-to-moderate PAE may lie in the fact that anatomicalstructures, biological systems, and areas of functioning arenot equally vulnerable to PAE. For example, there ismuch consensus that low PAE has greater and moreenduring effects on neurobehavioral outcomes than onphysical morphology (Streissguth 2007). Accordingly,biomarkers regulated by brain regions sensitive to PAE(frontal cortex, hippocampus, and cerebellum) should beexplored further (Riley and McGee 2005). Specifically, thelimbic–hypothalamic–pituitary–adrenal (LHPA) axis under-goes a period of rapid maturation during fetal develop-ment and early childhood characterized by an increasedvulnerability to environmental influences such as PAE(Gunnar and Vazquez 2006; Sanchez 2006). Becausedisrupted LHPA axis activity and its end-product cortisolhave been associated with mood disorders (Goodyer et al.2001), antisocial behaviors (Susman 2006), and cognitivedifficulties (Blair et al. 2005), investigating the associationbetween low-to-moderate PAE and LHPA axis activitymay reveal subtle biochemical differences and helpidentify mechanisms whereby PAE jeopardize normativedevelopment.

Animal studies indicate that PAE offspring showheightened LHPA axis activity in comparison to nonex-posed animals (Schneider et al. 2002, 2004; Weinberg et al.2008; Zhang et al. 2005). In nonhuman primates, moderatelevels of PAE were associated with higher adrenocortico-tropic hormone and cortisol responses to maternal separa-tion (Schneider et al. 2004). Sexual dimorphic associationsare often reported between PAE and HPA axis activity.Higher glucocorticoid responses to acute/short durationstressors (e.g., restraint) are more often reported in PAEfemales, although PAE males show higher responses toprolonged or repeated stressors (for a review, see Weinberget al. 2008). For example, postnatal handling was associ-ated with higher basal hypothalamic CRH mRNA in PAEmales, but not in PAE females (Gabriel et al. 2005).

Sexual dimorphic effects of PAE have been associatedwith PAE-induced dampened postnatal testosterone secre-tion, which would reduce the inhibiting action oftestosterone on the LHPA axis. Alternatively, PAE mayalter the interactions between the LHPA and limbic–hypothalamic–pituitary–gonadal (LHPG) axes in exposedmales, leading to distinct associations between PAE andcortisol secretion in males versus females. In both cases,PAE would increase LHPA axis activity in a sex-specificmanner, possibly through the role of sexual hormonessuch as testosterone.

298 Psychopharmacology (2011) 214:297–307

Only a few studies have investigated the associationsbetween PAE and cortisol secretion in humans. Ramsay etal. (1996) reported higher pretest levels in 2-month-oldinfants exposed to alcohol and cigarettes during pregnancyand blunted responses to inoculation. Lower cortisolresponses to another noxious medical procedure (heel lane)were found in PAE neonates (Oberlander et al. 2010).Conversely, heavily PAE was related to elevated basal andcortisol responses to physical examination in 13-month-oldtoddlers (Jacobson et al. 1999). Higher cortisol reactivitywas also reported in 5- to 7-month-old PAE infants inresponse to a “still face” procedure, but only in boys bornfrom high-frequency drinkers versus those born from low-frequency drinkers (Haley et al. 2006).

The aim of the present study was to examine theassociation between PAE and cortisol secretion in 19-month-old toddlers. We examined cortisol response tounfamiliar situations, a laboratory paradigm known to bemoderately stressful at this age (Goldberg et al. 2003;Mullen et al. 1993; van Bakel and Riksen-Walraven 2004).More specifically, we hypothesized that PAE toddlerswould show enhanced pretest and cortisol responses tounfamiliar situations compared to nonexposed children.Based on previous rodent studies (Gabriel et al. 2005;Weinberg et al. 2008), we further hypothesized that theassociations between PAE and cortisol responses to ourstressful paradigm would be stronger for boys than for girls.Finally, we explored the possibility that testosteronemoderated the association between PAE and cortisolactivity.

Method

Sample

Participants were families of twins from the QuébecNewborn Twin Study recruited between April 1995 andDecember 1998 in the greater Montréal area. A total of 989families were initially contacted, of which 672 agreed toparticipate (68%). The majority of the families wereCaucasian (92.0%). Twins were first seen at 6 months ofage and assessed yearly on a variety of child and familycharacteristics. Informed written consent was obtained fromthe parents annually. Zygosity was determined through theZygosity Questionnaire for Young Twins when they were 6and 19 months of age (Goldsmith 1991). Independent ratersassessed zygosity through live observation of the twins’physical similarities. DNA-based zygosity was determinedfor 31% randomly selected same-sex twin pairs using eightto ten highly polymorphic microsatellite markers. Acomparison of the two methods indicated a concordanceof 93.8% (Forget-Dubois et al. 2003).

Procedures and measures

The stressful context: exposure to unfamiliar situations

At 19 months of age (mean (SD)=18.85 (0.74)), eachparticipating twin was successively exposed to two unfa-miliar situations known to be moderately stressful at thisage (Goldberg et al. 2003; Mullen et al. 1993; van Bakeland Riksen-Walraven 2004). In the first situation, one twinand the mother were alone in a corner of a room when awoman dressed up as a clown entered the room, went to theopposite corner, and invited the child to approach byoffering a set of familiar toys. In the second situation, anoisy, odd-looking, moving toy robot was placed on aplatform in the opposite corner of the room. Each sessionlasted 280 s separated by 5 min of mother–child free play.For the first 140 s, the mother was asked to remain passiveand not to interact with her child, responding only to child-initiated talk with very brief statements (e.g., “It’s okay”,“It’s a clown”). For the last 140 s, the mother was allowedto do whatever she felt was needed to help her child be atease with the stimulus.

Neuroendocrine measures

We collected saliva samples prior to (pretest; ≈15 min uponarrival to the laboratory) and 20 min after (posttest) theunfamiliar situations to capture the peak cortisol response(Ramsay and Lewis 2003). Saliva samples were collectedusing salivettes (Sarstedt, Canada) and were immediatelyfrozen at −80°C until assay. Although saliva sampling tookplace in the morning for all participants, the time ofcollection varied from 0805 to 1215 hours. Mothers wereinstructed not to give the child anything to eat or drink20 min before each collection. Salivary samples wereanalyzed for cortisol and testosterone using radioimmuno-assay (Diagnostic Systems Laboratories Inc., Webster, TX,USA). Intra-assay variability was less than 10%.

Saliva samples were collected for testosterone andcortisol assays in 212 children at pretest and 200 childrenat posttest. A total of 166 children had enough saliva toallow for reliable cortisol determination at both pre- andposttest. In addition to the inherent difficulty of collectingsufficient volume of saliva at this age, appropriate controlfor the nonindependence of the data (e.g., two children perfamily) required the inclusion of complete twin pairs in thestatistical analyses. PAE information was available for 154children at pretest (72.6%) and 148 children at posttest(74.0%). Cortisol levels did not differ as a function of theavailable PAE information (pretest: t(210)=−0.63, p=0.53and posttest: t(198)=−0.33, p=0.75). We thus consideredthe samples of 154 children at pretest and 148 children atposttest to be representative of the larger sample.

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Two indices of cortisol were considered: the pretestsample and a reactivity ratio. The reactivity ratio was usedto take into account the influence of the initial (i.e., pretest)value on the magnitude of cortisol change between the pre-and posttest (i.e., the law of initial value; Lacey 1956).Accordingly, the reactivity ratio was computed by dividingthe change score between the pre- and posttest by thepretest score (Ouellet-Morin et al. 2008; Ramsay and Lewis2003). A positive reactivity ratio score refers thus to anincreased cortisol response to the unfamiliar situations. Thecortisol indices were positively skewed and were normal-ized using log10 transformation and then standardized(Tabachnick and Fidell 1996). The final samples consideredfor statistical analyses were composed of 154 children atpretest and 130 children for the reactivity ratio.

Prenatal alcohol exposure

Information about maternal alcohol consumption duringpregnancy was collected retrospectively using a computerassisted face-to-face semistructured interview conducted athome when the children were 6 months of age. Theinterviews took place shortly after the families’ visit tothe laboratory. The interview was based on that used in theNational Longitudinal Survey of Children and Youth(Statistics Canada 1995) and included several aspects ofparenting in addition to the alcohol-related items. Theinterviewers were all undergraduate and graduate femalestudents in psychology who received intensive trainingfrom a senior research coordinator and supervision prior toand during data collection. The items included: “Did youdrink alcohol during the pregnancy”, “How many drinkwere you usually drinking per drinking occasion”, “Did youdrink alcohol during the (first/second/third) trimester”, and“Did you drink alcohol through pregnancy”. Responsecategories were: “never”, “less than once per month”, “1–3times per month”, “once a week”, “2–3 times per week”,“4–6 times per week”, or “everyday”. The response choicesfor the number of drinks taken per drinking occasion were:“none”, “1 or 2”, “3 or 4”, and “5 and more”. The motherresponded to the remaining items as “yes” or “no”.

According to the mother’s report, most children were notPAE (61.8%, n=110). Among PAE children (38.2%, n=68), most mothers reported to have drunk only one or twoglasses per drinking occasion (97.1%, n=66), thus prevent-ing the investigation of a dose–response between PAE andcortisol secretion. As would be expected in a population-based study, frequent exposures to alcohol were rare.Indeed, few mothers reported to have drunk ≥1 glass perweek (n=4 mothers or 4.5%), which prevented us fromapplying the same criteria used in a previous population-based study (≥1 glass per week; Sayal et al. 2007). In PAEchildren, 50.0% (n=34) were exposed to alcohol during one

trimester, 8.8% (n=6) were exposed during two trimesters,and 41.2% (n=28) were exposed to alcohol throughoutpregnancy. Considering the pattern of PAE in thispopulation-based sample (infrequent PAE to relative lowamount of alcohol), we opted to examine the associationbetween cortisol secretion and alcohol exposure throughoutthe entire pregnancy. The total sample of children was thussplit into two groups: (1) uninterrupted prenatal alcoholexposure (UPAE), which included children exposed toalcohol throughout pregnancy (15.7% of the sample) and(2) sporadic and not exposed (S/NE), which combinednonexposed and children exposed to alcohol only duringone or two trimesters (84.3% of the sample). The frequencyof maternal alcohol consumption during pregnancy did notdiffer between UPAE and S/NE groups (χ2(4, 68)=7.17,p=0.13), suggesting that the proposed groups captured themost of the variance in PAE duration.

Confounders

Several sociodemographic, maternal, and child risk factorspreviously associated with PAE or LHPA activity wereexamined: prenatal nicotine exposure, birth weight, lowfamily income, young motherhood, single motherhood, andthe children’s sex (Gunnar and Quevedo 2007; Ouellet-Morin et al. 2008; Sanchez 2006). The children’ birthweight and gestational weeks were retrieved from medicalrecords (birth weight: mean (SD)=2.50 kg (0.51); gesta-tional weeks: mean (SD)=36.51 weeks (2.31)). Interviewsconducted with the mother when the twins were 6 monthsof age were used to ascertain the low family income (lessthan CDN $20,000; 15.2%), single parenthood (12.0%), themother’s age when she first gave birth (20-years-old or less;2.7%), and prenatal cigarette exposure (during all pregnancy;20.3%).

Statistical analysis

Correlations and t tests were performed to identifycontinuous and dichotomous confounders of neuroendo-crine measures. Risk factors were considered to be apotential confounder when p<0.10. The associationsbetween UPAE, sex, testosterone, and cortisol were testedusing multilevel modeling (MlwiN Version 1.10.0007;Rasbash et al. 2000) combined with Hubert–White sand-wich estimators to produce robust standard errors (Maasand Howx 2004). Multilevel analyses take into account thedependency of the observations present in a twin design byanalyzing the data hierarchically. At its simplest form (nullmodel; block 1), the models consisted in a lower level ofobservations (within family variance) nested within ahigher level of observations (between family variance).The twins’ familial similarities were thus taken into

300 Psychopharmacology (2011) 214:297–307

account, allowing for unbiased tests of associations betweenUPAE and cortisol measures. Models of increasing complex-ity were conducted to test for the main hypotheses. Eachmodel was compared to the previous one using the change inthe −2 log likelihood estimates to determine whether theinclusion of the variables significantly improved the fit of themodel. Three series of models were performed. First, wetested whether UPAE was associated with each neuroendo-crine measure by successively including to the null model (i.e.,within family variance; block 1), the covariables (block 2), andthe UPAE (block 3). Second, we tested if the sex of the childmoderated the association between UPAE and the neuroen-docrine measures by including successively the covariables(block 2), the main effects of UPAE and sex (block 3), and theinteraction term (UPAE×sex; block 4). Finally, we extendedthe previous analyses to test if testosterone moderated theassociations between UPAE, sex, and cortisol (UPAE×sex×testosterone, block 5).

Results

Identification of covariables

Cortisol and testosterone levels were not associated withcigarette exposure during pregnancy, low income, singleheaded family, and young motherhood (Table 1). No signif-icant associations were detected between the neuroendocrinemeasures and birth weight and the number of gestationalweeks (|rs|<0.07, ps>0.42). Thus, these risk factors were notincluded in the subsequent statistical analyses.

Time of saliva collection was not associated with pretestand posttest cortisol levels (r=−0.09, p=0.22 and r=0.05, p=0.47, respectively). Mothers were asked not to give theirchildren anything to eat or drink 20 min before salivacollection, although noncompliance was documented. Chil-dren who had eaten or drank did not differ on cortisolmeasures compared to those who did not (pretest: t(210)=−1.04, p=0.35; posttest: t(198)=0.59, p=0.56). Nine childrenwere taking steroid medication occasionally. As theircortisol levels did not differ from the rest of the sample(pretest: t(210)=1.19, p=0.24; posttest: t(198)=−0.64, p=0.53), they were retained in the main analyses. No overallsignificant cortisol increase was detected between the pretestand the posttest measures (mean (SD)=0.04 (0.40)μg/dL),although large individual variation was noted (post- andpretest difference varied from −2.11 to 1.78 μg/dL).

UPAE and neuroendocrine measures

Table 2 presents the descriptive statistics for cortisol(pretest and reactive) and testosterone as a function ofUPAE. The analyses revealed no significant associations

between UPAE and cortisol reactivity or testosterone (β=0.56, SE=0.37, z=1.48, p=0.14 and β=0.10, SE=0.30, z=0.32, p=0.75, respectively). In contrast, UPAE children hadlower pretest cortisol levels than S/NE toddlers (β=−0.55,SE=0.17, z=−3.24, p=0.001).

Sex differences

Significant interactions between UPAE and sex wererevealed for the cortisol measures (pretest cortisol: β=0.86, SE=0.36, z=2.40, p=0.02; cortisol reactivity: β=−1.25, SE=0.40, z=3.15, p=0.002), but not for testosterone(β=−0.43, SE=0.26, z=−1.66, p=0.10). As depicted inFig. 1a, UPAE boys showed higher cortisol reactivitycompared to S/NE boys (β=1.14, SE=0.38, z=3.00, p=0.003), whereas this association was not present for girls(cortisol reactivity: β=−0.11, SE=0.36, z=−0.30, p=0.76).A distinct pattern of results emerged for pretest cortisol.UPAE boys had lower, rather than higher, cortisol levelsthan S/NE boys (Fig. 1b; β=−1.89, SE=0.51, z=−3.73, p=0.0002). Again, no significant association was detected forgirls (β=−0.08, SE=0.29, z=0.27, p=0.77; for completestatistics, see Online resource 1).

Moderating effect of testosterone

No interaction between UPAE, sex, and testosterone wasdetected for pretest cortisol (β=0.48, SE=0.32, z=1.48, p=0.14). However, testosterone levels moderated the associa-tion between UPAE, sex, and cortisol reactivity (tripleinteraction: z=−2.45, p=0.01). As illustrated in Fig. 2,higher testosterone levels were associated with an increas-ing difference in cortisol reactivity according to UPEA, butonly for boys: UPEA boys showed high cortisol reactivityirrespective of their testosterone levels (β=0.17, SE=0.31,testosterone=0.55, p=0.59), whereas S/NE boys cortisolreactivity was negatively associated with their testosteronelevels (β=−0.37, SE=0.10, p=0.0005). This downwardtrend in cortisol reactivity as a function of testosteronelevels was found for both UPAE girls (β=−0.53, SE=0.26,p=0.04) and S/NE girls (β=−0.29, SE=0.19, p=0.12),although not reaching significance in the latter.

Discussion

The present study examined the association between UPAEand cortisol secretion in a population-based sample of 19-month-old toddlers and investigated whether the children’ssex and testosterone levels moderated these associations.We showed sexual dimorphic patterns of associationsbetween UPAE and cortisol secretion. Higher cortisolresponses to unfamiliar situations and lower pretest cortisol

Psychopharmacology (2011) 214:297–307 301

levels were detected only for UPAE boys, not for UPAEgirls, in comparison to their same-sex S/NE counterparts.Testosterone further moderated the association betweenUPAE and cortisol reactivity, but again only for boys.These findings are noteworthy considering the low levels ofPAE reported in this population-based sample (≤1–3standard units per month) and the investigated effects ofpotential prenatal and postnatal confounds.

These results are in line with a growing body of evidencesuggesting that early-life stress disrupt the developing brain,particularly the regions involved in stress regulation (Gunnarand Vazquez 2006; Meaney 2001; Sanchez 2006). Thefinding that UPAE boys had higher cortisol responses tounfamiliar situations than S/NE boys is consistent with theincreased LHPA axis reactivity to stress reported in severalhuman and preclinical animal studies (Jacobson et al. 1999;Schneider et al. 2002, 2004; Weinberg et al. 2008).

The limitation of the association between PAE and cortisolreactivity to boys is a salient feature of the present study. Asimilar finding (i.e., limited to boys) has been reported in astudy examining the cortisol responses to a social–emotionalstressful task among 5- to 7-month-old children born eitherfrom high-frequency drinkers (>2 standard units per week) orfrom low-frequency drinkers (≤2 standard units per week;Haley et al. 2006). Sex-specific patterns of associationsbetween PAE and social behaviors, brain structures, andneuroendocrine activity are commonly reported in animalstudies (Kelly et al. 2000). Higher responses to unfamiliarsituations shown in UPAE males, but not in UPAE females,are in agreement with previous findings reporting higherLHPA responses to prolonged/repeated stressors in PAEmale rodents (Gabriel et al. 2005; Weinberg et al. 2008).Notably, previous studies investigating the effects of PAE onstress-related systems does not suggest pervasive increases inall neuroendocrine markers of stress, but rather indicatechanges in specific components of the stress response, whichmay affect differently males and females (Kelly andDillingham 1994). For example, acute exposure to stressreduced cortical norepinephrine (NE) and increased hippo-campal NE in both PAE males and females, but it wasassociated with lower hypothalamic NE in PAE females only(Rudeen and Weinberg 1993). Furthermore, the contexts inwhich stress responses are elicited may lead to sexualdimorphic associations between PAE and LHPA axisactivity. Exposure to novelty in the presence of the motheris a situation commonly experienced and moderatelystressful among young children (Goldberg et al. 2003; vanBakel and Riksen-Walraven 2004). Analog repeated andmoderate stressful laboratory challenges have been reportedto elicit higher corticosterone levels in PAE male rodents(Weinberg et al. 2008). Alternatively, more acute stress mayhave resulted in higher responses among girls (Weinberg etal. 2008).T

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0.44

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0.46

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0.45

(0.18)

0.21

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0.47

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T.10

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11.04(0.58)

10.97(0.88)

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9.94

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302 Psychopharmacology (2011) 214:297–307

One potential mechanism whereby PAE could influenceLHPA axis activity in boys, but not in girls, involvessteroid-dependent processes. We showed that testosteronelevels moderated the association between UPAE, sex, andcortisol response to unfamiliar situations. In contrast to S/NE boys for whom testosterone levels were negativelyassociated to cortisol reactivity, no significant associationwas detected between cortisol and testosterone levels inUPAE boys. Increased corticosterone responses to noxious

stress (noise) have been reported in male rodents deprivedof testosterone during pre- and postnatal developmentcompared to controls. The lack of association betweencortisol and testosterone levels in UPAE boys may thussignal disrupted neonatal androgenization of the brain(Seale et al. 2005; Weinberg et al. 2008). Moreover,hypothalamic GR expression, sensitive to early exposureto androgens, may alter the inhibition of the LHPA axis andthus change the LHPA axis reactivity to stress (Seale et al.

0

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S/NE Girls (n=52)

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UPAE Boys S/NE Girls UPAE Girls S/NE Boys UPAE Boys

S/NE Girls UPAE Girls S/NE Boys UPAE Boys

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Sex x UPAE (p = .02) Sex x UPAE (p = .002)

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a

c

b

Fig. 1 Means and standard error measures of a cortisol reactivity, b cortisol pretest, and c testosterone as a function UPAE and sex. S/NE sporadicand not exposed prenatally to alcohol, UPAE uninterrupted prenatal exposure to alcohol. **p<0.01 and ***p<0.001

Table 2 Means (SEM) of cortisol and testosterone as a function of UPAE

Measures UPAE S/NE p value (ΔAdj R2)

Mean (SEM) n Mean (SEM) n

Pretest cortisol, μg/dL 0.25 (0.03) 22 0.39 (0.03) 132 0.001 (0.044)

Cortisol reactivity, μg/dL 1.48 (1.00) 16 0.30 (0.14) 114 0.14 (0.033)

Testosterone, pg/mL 11.16 (1.12) 28 10.55 (0.43) 150 0.75 (0.008)

Neuroendocrine values were reconverted from log10 to linear scale

SEM standard error of the mean, S/NE sporadic and not exposed prenatally to alcohol, UPAE uninterrupted prenatal exposure to alcohol

Psychopharmacology (2011) 214:297–307 303

2005). The hypothesis of an altered LHPA–LHPG axesinteraction in PAE male rodents is consistent with ourfindings and others (Lan et al. 2009; Zhang et al. 2005).Longitudinal population-based studies including more PAEchildren are needed to replicate this new finding in humans.

A striking and unexpected finding was detected for thepretest cortisol measure. UPAE was associated with lower,rather than higher, pretest cortisol levels (i.e., following thearrival to the laboratory) in UPAE males in comparison toUPAE females and S/NE children. This finding differs fromthe higher pretest cortisol levels noted in 2-month-oldinfants exposed prenatally to alcohol and cigarettes(Ramsay et al. 1996). However, in that same study, thesedifferences in cortisol were not found at 6 months of age(Ramsay et al. 1996). Lower pretest cortisol levels mayemerge with age in PAE children (Ashman et al. 2002;Kelly 1996; Oberlander et al. 2010), possibly because of adownregulation of baseline cortisol secretion resulting fromrepeated activations of the LHPA axis (Miller et al. 2007).Lower pretest cortisol levels found in UPAE boys are alsoconsistent with higher levels of GR messenger RNA detectedin the paraventricular nucleus of the hypothalamus in PAEmale rodents under basal conditions (Halasz et al. 1993),which may underline enhanced inhibition of LHPA axisbasal activity or compensatory responses to deficits inregulatory processes elsewhere in the LHPA axis (Glavas etal. 2001). Lower pretest cortisol levels in UPAE boys mayalso reflect the activation and subsequent retroactive inhibi-tion of the LHPA axis in response to stress experienced priorto the arrival to the laboratory (e.g., leaving home, transport).This explanation would be consistent with the absence ofdifferences noted between nonexposed and PAE rodents inbasal cortical NE levels while lower NE levels were detected

in PAE animals 1 h poststress (recovery; Rudeen andWeinberg 1993). Longitudinal studies are required to betterunderstand the temporal sequence of the influence of PAE onLHPA axis basal activity and reactivity.

The study has limitations that might have constrained thefindings. First and foremost, because few children wereexposed to alcohol during pregnancy, we were unable toinvestigate further the role of the timing (Kelly 1996; Lee etal. 1990; Schneider et al. 2005), dose (Haley et al. 2006;Jacobson and Jacobson 1999), and frequency (Jacobson andJacobson 1999) of PAE on neuroendocrine activity.Additionally, whether the findings generalize to the farend of the spectrum of PAE is open to question. Theevidence of significant direct and indirect effects of UPAEon cortisol secretion suggests, however, that the associa-tions are robust. Further replications are neverthelesswarranted. Second, we relied on retrospective assessmentsof the maternal alcohol consumption during pregnancy.Although mothers tend to report higher alcohol consump-tion retrospectively than prospectively (during the antenatalperiod), the latter tend to be more accurate and correlatemore closely to the children’ neurobehavioral outcomes(Jacobson et al. 2002). More generally, face-to-face inter-views lead to higher reports of alcohol intake duringpregnancy in comparison to self-report questionnaires,although prospectively collected 2 weeks diary may bemore reliable (Kesmodel and Olsen 2001). We collectedinformation regarding the maternal alcohol consumption ateach trimester and offered different choices regarding thefrequency of consumption. These procedures may havecontributed to reduce the error of measurement and under-reporting (Pereira et al. 1993). In future studies, additionalmeasures of biomarkers associated with PAE should be

-1.5

-1

-0.5

0

0.5

1

1.5

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Testosterone (z scores)

Cor

tisol

rea

ctiv

ity (

z sc

ores

)

S/NE Boys

UPAE Boys

S/NE Girls

UPAE Girls

Fig. 2 Slopes illustrating the associations between testosterone and cortisol reactivity as a function UPAE and sex. S/NE sporadic and notexposed prenatally to alcohol, UPAE uninterrupted prenatal exposure to alcohol

304 Psychopharmacology (2011) 214:297–307

considered (e.g., fatty acid ethyl esters in the meconium ormaternal and newborn’s hair; Bearer et al. 2003; Bearer etal. 2004/2005; Klein et al. 2002). Third, the cortisolreactivity measure was based on single pretest and posttestsaliva samples. In most toddlers, cortisol response peaks20 min after stress induction, but marked individualvariability exists around that point (Ramsay and Lewis2003). Multiple pretest and posttest samples may haveyielded more reliable assessments of cortisol reactivity.Fourth, for ethical reasons, a rather mild yet developmen-tally relevant stressful situation was used, resulting in anabsence of increase in cortisol overall. This lack of increaseis consistent with previous studies that used similarprocedures with this age group (Gunnar et al. 2009). Manyfactors may have accounted for this general lack of increasein cortisol, including the calming presence of the motherduring the stress paradigm (Gunnar and Quevedo 2007).Finally, we have not directly asked the mothers whether thechildren were teething the day of or the week preceding thevisit to the laboratory. It is possible that small bloodleakages due to teething have affected the cortisol values,although effects of blood contamination on salivary cortisolare thought to be small (Granger et al. 2007).

In conclusion, the present study reported that lowexposure to alcohol throughout pregnancy was associatedwith disrupted cortisol secretion in a population-basedsample of 19 months of age toddlers. Our findings suggestthat low levels of PAE may be associated with disruptedpatterns of LHPA axis activity and that males may be athigher risk. Our findings challenge the existence of a “safelevel” of alcohol consumption during pregnancy and havepublic health implications.

Acknowledgments This research was supported by grants from theNational Health Research Development Program, the Social Sciencesand Humanities Research Council of Canada, the Québec Ministry ofHealth and Social Services, the Canadian Institutes of HealthResearch, the Canada Research Chair program, the Fonds Québécoisde la Recherche sur la Société et la Culture, and the Fonds de laRecherche en Santé du Québec. Isabelle Ouellet-Morin was supportedby fellowships from the Canadian Institutes of Health Research andthe CIHR Training Grant in Genes, Environment, and Health. Thefunding agencies have no further role in the study design, in thecollection, analysis, interpretation of data, and in the writing ofthe manuscript. We are grateful to the parents and twins of theparticipating families. We thank Jocelyn Malo for coordinating thedata collection and Helene Beaumont, Helene Paradis, Bei Feng,Bernadette Simoneau, and Jacqueline Langlois for the assistancein data management and statistical analyses (H.P. and B.F.).

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