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Alcohol 49 (2015) 79e87

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Alcohol

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Salivary cortisol levels are elevated in the afternoon and at bedtimein children with prenatal alcohol exposure

Kathy Keiver a,*, Chris P. Bertram a, Alison Pritchard Orr a, Sterling Clarren b

aDepartment of Kinesiology and Physical Education, University of the Fraser Valley, 33844 King Road, Abbotsford, British Columbia V2S 7M8, CanadabCentre for Community Child Health Research, Canada Northwest FASD Research Network, Vancouver, British Columbia, Canada

a r t i c l e i n f o

Article history:Received 3 July 2014Received in revised form12 November 2014Accepted 12 November 2014

Keywords:Fetal Alcohol Spectrum DisorderCortisolPrenatal alcohol exposureHPA axisExercisePhysical activity

* Corresponding author. Tel.: þ1 604 504 7441x413E-mail address: kathy.keiver@ufv.ca (K. Keiver).

http://dx.doi.org/10.1016/j.alcohol.2014.11.0040741-8329/� 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

Prenatal alcohol exposure can cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis,which may underlie some of the behavioral and adaptive problems seen in individuals with Fetal AlcoholSpectrum Disorders (FASD). Infants prenatally exposed to alcohol show altered basal and post-stresscortisol levels, but it is unknown if this persists beyond 2 years of age. It is also unknown if cortisollevels can be normalized through intervention programs. In this study, we investigated the effects of aphysical activity program for children with FASD to determine: 1) if HPA dysregulation persists in school-age children with FASD, and 2) the effect of our program on cortisol levels. Twenty six children (ages 6e14 years) with FASD participated in an 8 week motor skill development program. Salivary cortisol levelswere measured in 24 children and compared at 4 time points: before, immediately after, 3 months, and 1year after program completion. Cortisol levels were also compared to 32 control children to evaluate thelong-term effects of prenatal alcohol exposure on HPA regulation. For each time point, saliva wascollected on each of 2 days at 3 times in the diurnal cycle: awakening, after school, and just beforebedtime. Cortisol levels were significantly higher in the afternoon and at bedtime in children with FASDwith confirmed prenatal exposure to high levels of alcohol (alcohol exposure rank 4), compared withControl children or children with FASD with exposure to low or unknown levels of alcohol (alcoholexposure rank 3). The program did not significantly affect cortisol levels in children with FASD as a group.These results provide support for long-term effects of prenatal alcohol exposure on the HPA system inhumans, which could increase vulnerability to mental health issues and diseases later in life.

� 2015 Elsevier Inc. All rights reserved.

Introduction

Fetal Alcohol Spectrum Disorder (FASD) describes a broad rangeof deficits resulting from prenatal alcohol exposure (PAE). In-dividuals with FASD have a variety of physical, cognitive, behavioral,and psychosocial problems, resulting in poor performance inschool, theworkplace, and an impaired ability to function in society.Neuropsychological effects resulting from PAE include impairmentsin cognitive and motor function, hyperactivity, and problems inadaptive function (Mattson, Crocker, & Nguyen, 2011). In addition,individuals with FASD are at high risk for mental health problemssuch as depression and anxiety disorders (Kodituwakku, 2007;O’Connor et al., 2002), and sleep disorders (Jan et al., 2010).

Among the physiological effects of PAE is dysregulation ofthe hypothalamic-pituitary-adrenal (HPA) system (Weinberg,Sliwowska, Lan, & Hellemans, 2008). Developmental changes in

2; fax: þ1 604 855 7558.

the HPA system occur throughout prenatal life and childhood, anddetermine the basal HPA activity (across the diurnal cycle), thecortisol awakening response (CAR, the rise in cortisol during thefirst hour after waking), and reactivity to stress. Studies havedemonstrated that adverse experiences (e.g., prenatal alcoholexposure, maternal/caregiver separation, childhoodmaltreatment)can affect the development of the HPA system and result inlong-term changes in HPA function, and such changes may be acentral mechanism by which early life adversity predisposes anindividual to later development of physical and psychiatric disease(Essex et al., 2011; Tarullo & Gunnar, 2006; Weinberg et al., 2008).In the case of FASD, it is thought that PAE sensitizes the HPA sys-tem, making individuals hyper-responsive to the stresses andchallenges of life, which increases their vulnerability to stress-related physical and mental disorders (Weinberg et al., 2008).Thus, HPA axis dysregulation may underlie, or at least contributeto, some of the cognitive, behavioral, and adaptive problems seenin individuals with FASD, as well as their vulnerability to mentalhealth and sleep disorders.

K. Keiver et al. / Alcohol 49 (2015) 79e8780

Animal models of FASD show increased HPA hyper-responsiveness and delayed or deficient recovery following stress.These effects vary depending on the sex of the animal and the timein the diurnal cycle they are measured, and last into adulthood(Weinberg et al., 2008). The effects of PAE on HPA dysregulationhave not been well studied in humans, but the reaction to acutestress has been investigated in children under 2 years of age.Studies have shown that infants with PAE exhibit increased basal(pre-stress) and/or abnormal post-stress cortisol levels (Haley,Handmaker, & Lowe, 2006; Jacobson, Bihun, & Chiodo, 1999;Oberlander et al., 2010; Ramsay, Bendersky, & Lewis, 1996), indi-cating HPA axis abnormalities. However, the effects of PAE inhumans older than 2 years, or on basal cortisol levels across thediurnal cycle (an index of physiological regulation), are unknown.Moreover, most children in the above studies were tested in a clinicor laboratory rather than their home or usual environment, whichcould potentially be stressful for the child and elevate the basalcortisol level.

Intervention programs have the potential to improve neuro-psychological deficits in children with FASD and thus reduce theburden on affected individuals, families, and society. Althoughseveral interventions have been developed to address specificdeficits (Kodituwakku, 2010), there is an urgent need to increasethe number of effective programs available. One interventionstrategy that appears promising for improving multiple areas of thelives of children with FASD is exercise or physical activity. Asidefrom the general health benefits of exercise (e.g., cardiovascularbenefits, weight control), physical activity appears to be protectiveagainst stress-induced physical and psychological illness and mayattenuate HPA axis dysregulation (Salmon, 2001; Tsatsoulis &Fountoulakis, 2006). Physical activity has been demonstrated toimprove various aspects of cognitive function and behavior,improve sleep, and decrease risk and symptoms of depression andanxiety disorders (Erickson, Gildengers, & Butters, 2013;Ploughman, 2008; Tomporowski, 2003; Tsatsoulis & Fountoulakis,2006). In humans, physical activity appears to decrease sensitivityto stress, which is likely at least partly due to adaptations of the HPAaxis (Tsatsoulis & Fountoulakis, 2006). Animal studies have shownthat physical activity can decrease HPA activation to low-intensitystressors (Campeau et al., 2010; Droste, Chandramohan, Hill,Linthorst, & Reul, 2007) and enhance habituation to repeatedstress (Nyhuis, Masini, Sasse, Day, & Campeau, 2010; Sasse et al.,2008). Moreover, physical activity has been shown to amelioratethe HPA dysregulation and associated depression-like behaviorsinduced by prenatal exposure to excess glucocorticoids (Liu et al.,2012).

Importantly, studies using animal models suggest that increasedphysical activity can attenuate some of the deficits resulting fromPAE (Klintsova, Hamilton, & Boschen, 2013). Voluntary exercise hasbeen shown to ameliorate deficits in spatial learning and memory(Christie et al., 2005; Thomas, Sather, & Whinery, 2008) andattenuate anxiety and depressive-like symptoms (Brocardo et al.,2012; Thomas et al., 2008) in rodent models of FASD. To ourknowledge, the effect of exercise on HPA axis dysregulation has notbeen examined. However, exercise has been shown to increaseplasticity in regions of the brain associated with learning andmemory in rodents with PAE by increasing levels of neurotrophins,cell proliferation, and decreasing oxidative stress (Boehme et al.,2011; Brocardo et al., 2012; Helfer, Goodlett, Greenough, &Klintsova, 2009; Redila et al., 2006).

Although increasing physical activity as an intervention strategyfor individuals with FASD appears promising, this approach has notbeen previously investigated in humans. We have completed astudy developing and evaluating the effects of a physical activityprogram designed to develop motor skills for children with FASD.

We hypothesized that our intervention program may improve HPAaxis dysregulation in children with FASD, and thus may improvesome aspects of neuropsychological function.

Our study measured salivary cortisol levels as an indicator ofHPA activity and compared levels in children with FASD to those ofcontrol children to evaluate the long-term effects of PAE on HPAregulation. We are the first to investigate effects in humans beyond2 years of age, across the diurnal cycle, and in the child’s usualenvironment. In addition, we compared salivary cortisol levels inchildren with FASD before and after the physical activity program.Our hypotheses were that: 1) cortisol levels will differ betweenchildren with FASD and control children, and 2) cortisol levels willbe normalized in children with FASD after completion of the pro-gram. This paper presents the data on cortisol levels. The effects ofour intervention program on cognitive function and motor skillswill be presented separately.

Methods

Participants

The study procedures were reviewed and approved by the Hu-man Research Ethics Board at the University of the Fraser Valley(UFV), and written informed consent was obtained from the legalguardians of all children. Twenty six children (age 6e14 years) with adiagnosis under the FASD umbrella participated in our interventionprogram. Using the 4 digit diagnostic code of Astley and Clarren(2000), criteria for participation were a rank of 3 or 4 on the gesta-tional exposure to alcohol scale (rank 3 ¼ confirmed exposure, levelof exposure unknown or less than high levels; or rank 4¼ confirmedexposure to high levels), and rank 2, 3, or 4 on the central nervoussystem damage or dysfunction scale (rank 2 ¼ evidence ofdysfunction, but less than rank 3; rank 3 ¼ significant dysfunctionacross 3 or more domains; rank 4 ¼ structural or neurologic evi-dence) (Chudley et al., 2005). Each child’s 4 digit code was deter-mined by S. Clarren through examination of the child’s pertinentmedical and diagnostic records.

Of the 26 children recruited into the study, saliva was success-fully collected from 24 children (FASD group). In addition, salivasamples were collected from 32 children without FASD, otherdisability, or condition requiring use of steroid medication (Controlgroup). Control children were of similar age and gender to thechildren with FASD and represented a convenience samplerecruited from the families of UFV employees, students, and theirfriends.

Project design

Twenty six children diagnosed with an FASD participated in an 8week after-school motor skill development program that consistedof two 1.5 h sessions/week. A cross-over design (waitlist controldesign) was used to deliver the program, such that half the childrenparticipated in the program at any one time, with the other halfserving as controls for confounds such as history, maturation, andtesting. Assessments, including collection of saliva samples, weremade prior to, immediately following, and approximately 2.5e3months and 1 year after completion of the program. Due to thecrossover design, assessments prior to the program were madetwice on some children (the waitlisted group), resulting in 5 ratherthan 4 assessment periods for this group. However, saliva sampleswere not obtained for all children at every assessment period. Forchildren with FASD, saliva was collected from 12 children for allassessment periods, 4 children for 3 assessment periods, 3 childrenfor 2 assessment periods, and 5 children for only 1 assessmentperiod. For children in the Control group, saliva was collected from

Table 1Participant characteristics and mean interval between time of awakening andcollection of the awakening saliva sample. p values are for comparisons, by t test orChi square, between children with FASD and Control children.

Children withFASD

Controlchildren

p value(t test orChi square)

Total number 24 32Females (%) 14 (58%) 16 (50%) 0.536a

Males (%) 10 (42%) 16 (50%)Mean age (�SEM) 10.4 � 0.4 y 9.7 � 0.4 y 0.266Mean age females (range) 10.2 � 0.6 y

(6e14 y)9.2 � 0.5 y(6e12 y)

0.180

Mean age males (range) 10.6 � 0.6 y(7e13 y)

10.4 � 0.6 y(6e13 y)

0.783

Mean interval from awakeningto saliva collection

11.4 � 3.0 min(n ¼ 13)

16.6 � 2.3 min(n ¼ 19)

0.173

a For comparison of the sex ratio between children with FASD and Control chil-dren by Chi square.

K. Keiver et al. / Alcohol 49 (2015) 79e87 81

20 children for all assessment periods, 4 children for 3 assessmentperiods, 2 children for 2 assessment periods, and 6 children for only1 assessment period.

Salivary cortisol

During each assessment period, saliva was collected from chil-dren in the FASD and Control conditions to determine cortisol levelson each of 2 days at 3 points in the diurnal cycle: at awakening, inthe afternoon (after school), and just before bedtime. All sampleswere collected prior tomeals or snacks (including beverages, exceptwater) and prior to teeth brushing. Caregivers were asked to avoidcollection on a day the child had a dentist appointment.

Saliva samples were collected with the caregiver’s assistanceusing sorbettes (Salimetrics LLC, State College, PA, USA), placed incapped plastic tubes and stored in the participant’s freezer (atapproximately �15 �C) until collection and transportation on ice toUFV for storage (at �20 �C). Samples were then transported to theUniversity of British Columbia, Vancouver, B.C., for analysis by AxisResearch Solutions. Salivary cortisol levels weremeasured using theSalimetrics High Sensitivity Salivary Cortisol Enzyme Immunoassaykit. The minimum amount of saliva required by this assay is 25 mL.Intra- and inter-assay coefficients of variation were 2.92% and3.41%, respectively. Values obtained at each time point for each ofthe 2 days were averaged for analysis.

Motor skill intervention program (FAST Club)

Our project adopts a strength-based, rather than deficit-based,approach to intervention programming. Strength-based ap-proaches seek to improve on areas that are relative strengths(rather than weaknesses) and thus may be more amenable tomodification. Although motor deficits have been documented inindividuals with FASD (e.g., Adnams et al., 2001; Mattson, Riley,Gramling, Delis, & Jones, 1998), motor skills, especially less com-plex gross motor skills, have been recognized as an area of relativestrength (Adnams et al., 2001; Jirikowic, Kartin, & Olson, 2008).Prior to the start of the intervention program (called FAST Club), amotor skill proficiency test, the BruininkseOseretsky Test of MotorProficiency (BOT 2), was administered to the children. The BOT 2 isdesigned to illuminate both strengths and weaknesses within in-dividual components of the motor domain. The test consists of 14motor skill subtests, which measure 8 determinants of gross andfine motor skills. These include measures of running speed andagility, strength, balance, bilateral coordination, upper-limb coor-dination, manual dexterity, fine motor precision, and fine motorintegration. From this test, the top 3 motor strengths of each childwere determined. In addition, the child identified a motor skill goalof his/her choice to work on. Previous studies on physical activityprograms suggest that interventions that allow children to choosetheir own functional goals increase participation and the develop-ment of social skills (Mandich, Polatajko, & Rodger, 2003). Based ontheir strengths and choices, individualized programs were devel-oped for each child.

Each FAST Club session consisted of a period of warm-up andcool-down, three 15 min periods spent completing activities thatdeveloped each of the child’s top 3 identified areas of strength, anda 20 min period working on the activity chosen by the child. Groupsof 5e11 children participated in the sessions at any one time. Theprogram was run in a school gymnasium, in which 7 stations wereset up that were designed to include the various components ofgross and fine motor skills tested in the BOT 2 (but not the identicaltasks): speed and agility, strength, balance, bilateral coordination,upper-limb coordination, manual dexterity, and fine motor skills.Each station consisted of a variety of activities that allowed for

progression in both intensity and complexity as the child’s skillsdeveloped throughout the program. Each child received individualinstruction throughout the session by a student instructor fromUFV, which provided motivation and positive reinforcement, andhelped to manage challenging behaviors. The students receivedtraining onworking with childrenwith FASD by kinesiology faculty(UFV) and occupational therapists (Fraser Valley Child Develop-ment Centre).

Statistics

Group differences (e.g., FASD vs. Control children, male vs. fe-male, children with an alcohol exposure rank of 3 vs. a rank of 4 vs.Control children) in salivary cortisol levels for between-subjectfactors were determined by t tests or ANOVAs as appropriate. Forthe ANOVAs, significant main effects or interactions were furtherexamined using NewmaneKeuls post hoc test. The effects of within-subjects factors such as assessment time (pre-program 1, pre-program 2, immediately post-program, 3 months post-program, 1year post-program) and time of day (awakening, afternoon,bedtime) were assessed by paired t tests or repeated-measuresANOVAs as appropriate for children with FASD and Control chil-dren separately. Relationships between salivary cortisol levels andthe children’s ages were analyzed by correlation analyses. Corre-lation coefficients are presented as Pearson r values. The proportionof males and females in the group of children with FASD andControl children was compared using Chi square (c2). Level of sig-nificance was p � 0.05; p values between 0.05 and 0.10 wereconsidered trends. Power analysis was performed and effect sizes(Cohen’s d) were determined for comparisons with p valuesconsidered trends. Values presented are the mean � S.E.M.

Results

Participants

Participant characteristics are given in Table 1. There were nosignificant differences between children with FASD and Controlchildren in sex ratio or overall mean age. There were also no sig-nificant differences in the mean age of males or females betweenchildren with FASD and Control children, or between males andfemales within each group. For saliva samples obtained at awak-ening, the actual time interval between awakening and samplecollection is important due to the rapid rise in cortisol that occurs inthe first hour (the CAR). Thus, differences in the time interval couldpotentially influence the awakening values. Although all families

K. Keiver et al. / Alcohol 49 (2015) 79e8782

were asked to record both the time of awakening and samplecollection, this information was obtained for only 13 out of 24children with FASD and 19 out of 32 Control children. For thesechildren, there was no significant difference in mean interval be-tween actual awakening and time of sample collection betweenchildren with FASD and the Control condition (Table 1).

At the time of the program, 11 children with FASD were infoster care (1 in a group home), 6 were adopted, 4 were with agrandparent, and 3 were with their biological mothers. The historyof the child’s caregiver situationwas not obtained. With respect totheir 4 digit diagnostic code, 16 children had an alcohol exposurerank of 3, while 8 children had a rank of 4. Five children had acentral nervous system damage or dysfunction rank of 2, 16 had arank of 3, and 3 had a rank of 4. Using the Canadian diagnosticsystem, 12 children had alcohol-related neurodevelopmentaldisorder (ARND), 9 had partial fetal alcohol syndrome (pFAS), and1 had fetal alcohol syndrome (FAS). Two children had confirmedPAE, but did not meet all of the diagnostic criteria to receive adiagnosis of FASD under Canadian diagnostic guidelines (Chudleyet al., 2005).

Cortisol levels

The changes in mean salivary cortisol levels (for all assessmentperiods combined) for children with FASD and Control childrenacross the day are shown in Fig. 1. Cortisol levels showed the ex-pected diurnal rhythm, with levels at awakening, afternoon, andbedtime all being significantly different from each other (repeated-measures ANOVAs and NewmaneKeuls post hoc tests, p’s < 0.05),regardless if analyzed for each assessment period individually or forthe mean of all assessment periods. All further analyses were per-formed separately for each time of day.

There was no significant effect of the intervention program onsalivary cortisol levels of children with FASD at any time of day.Changes in salivary cortisol levels with assessment period wereanalyzed by paired t tests and repeated-measures ANOVAs for bothFASD and Control groups. Because samples were not obtained fromall children at every assessment period, separate analyses wereperformed for assessment of pre-program 1 vs. pre-program 2, andpre-program and immediate post-program effects (n ¼ 20 FASDchildren), as well as across all assessment periods. In fact, therewere no significant differences found for cortisol levels among any

Fig. 1. Salivary cortisol levels across the day in childrenwith FASD (n ¼ 24) and Controlchildren (n ¼ 32) for all assessment periods combined. Values are means � S.E.M.*Significant difference between children with FASD and Control children (t tests:p ¼ 0.033 for the afternoon and p ¼ 0.026 at bedtime).

of the assessment periods for children with FASD or Control chil-dren. Assessment periods were therefore combined for all furtheranalyses.

Importantly, significant differences were found in cortisol levelsbetween children with FASD and Control children (Fig. 1). Cortisollevels were higher in children with FASD compared to Controlchildren in the afternoon (t [54] ¼ 2.194, p ¼ 0.033) and at bedtime(t [54] ¼ 2.286, p ¼ 0.026). Although levels of cortisol at awakeningappeared to be lower in children with FASD compared to Controlchildren (Fig. 1), this difference was not significant (t [54] ¼ 1.440,p ¼ 0.156).

Cortisol and age, gender, and diagnostic code

The relationships between salivary cortisol levels and age,gender, and the child’s 4 digit diagnostic code (central nervoussystem damage or dysfunction rank, alcohol exposure rank) werealso examined within the FASD and Control groups. Correlationanalysis showed no significant relationship between cortisol levelsand age at awakening, afternoon, or bedtime for FASD or Controlchildren, whether males and females were analyzed together orseparately.

The relationship between cortisol levels and gender is shown inFig. 2. There was a trend within the children with FASD for cortisollevels to be lower in females compared with males at awakening (t[22] ¼ 1.877, p ¼ 0.074), but not in the afternoon or at bedtime.Effect size was mediumehigh (Cohen’s d ¼ 0.764), and poweranalysis indicated that a sample size of 28/group would be neededto achieve significance for the difference between males and fe-males with FASD at awakening. No significant difference in cortisollevels was found between males and females within the Controlgroup for any time of day. Because of the trend for a gender dif-ference in cortisol levels within the FASD group at awakening,differences between children with FASD and Control children werere-examined separately for each gender. For females, there was atrend (t [28] ¼ 1.900, p ¼ 0.068) for cortisol levels to be lower inchildren with FASD compared to Control children at awakening.Again, effect size was mediumehigh (Cohen’s d¼ 0.712) and poweranalysis indicated a sample size of 32/group would be needed toachieve significance for this comparison. No significant differencewas found in cortisol levels in males between children with FASDand Control children at awakening.

Fig. 2. The effect of gender on salivary cortisol levels across the day in children withFASD (n ¼ 14 for females, n ¼ 10 for males) and Control children (n ¼ 16 for females,n ¼ 16 for males). Values are means � S.E.M. There was a trend for a difference be-tween females with FASD and Control females (t test: p ¼ 0.068) at awakening.

Fig. 4. Influence of gender on the effect of the intervention program (or equivalenttime period) on salivary cortisol levels at awakening in children with FASD (n ¼ 11 forfemales, n ¼ 7 for males) and Control children (n ¼ 14 for females, n ¼ 12 for males).Values are means � S.E.M.

K. Keiver et al. / Alcohol 49 (2015) 79e87 83

ANOVA showed no significant differences in cortisol levels withcentral nervous system damage or dysfunction rank at any time ofday. However, as there were only 3 children with a rank of 4, theseresults are inconclusive. Interestingly, a significant relationshipbetween alcohol exposure rank and cortisol levels was found(Fig. 3), which suggests that the elevation in cortisol levels inchildren with FASD compared to Control children was mainly, if notentirely, due to elevated cortisol levels in children exposed tohigher levels of alcohol in utero. One way ANOVAs comparingcortisol levels in children with an alcohol exposure rank of 4, vs. arank of 3, vs. Control children showed significant differences in theafternoon (F[2,53] ¼ 4.385, p ¼ 0.017) and at bedtime (F[2,53] ¼ 17.726, p < 0.001), but not at awakening. Further analysisrevealed that, for both the afternoon and at bedtime, cortisol levelswere higher in children with an alcohol exposure rank of 4compared to an alcohol exposure rank of 3 (NewmaneKeuls: af-ternoon, p 0.033; bedtime, p < 0.001) and compared to children inthe Control group (NewmaneKeuls: afternoon, p ¼ 0.010; bedtime,p < 0.001). There was no significant difference in cortisol levels inthe afternoon or at bedtime between Control children and thosewith an alcohol exposure rank of 3.

Given these results, we speculated that the intervention pro-gram might differentially affect cortisol in children with prior al-terations in cortisol levels (i.e., in females with FASD or children ofboth genders with an alcohol exposure rank of 4). We therefore re-examined the effect of the intervention program on cortisol levelsin females with FASD (Fig. 4) and in all children in relation to theiralcohol exposure rank (Fig. 5).

To explore the influence of gender on the effect of the pro-gram, a two way repeated-measures ANOVA for the factors ofgender and assessment period (pre- or post-program, or equiv-alent times for Control children) was performed for each time ofday and for each group (FASD or Control). At awakening (Fig. 4),there was almost a trend (F[1,16] ¼ 3.006, p ¼ 0.102) for aninteraction between gender and assessment period for childrenwith FASD. As the criterion for a trend was not achieved, furtheranalysis on this interaction was not performed. No significanteffects of program (or equivalent times for Control children) werefound on cortisol levels at awakening for males with FASD, or

Fig. 3. The relationship between alcohol exposure rank and salivary cortisol levelsacross the day in children with FASD (n ¼ 16 for rank 3, n ¼ 8 for rank 4) and Controlchildren (n ¼ 32). Values are means � S.E.M. *Significant difference between childrenwith alcohol exposure rank 4 vs. Control children (NewmaneKeuls: afternoon,p ¼ 0.010; bedtime, p < 0.001) and children with rank 3 (NewmaneKeuls: afternoon,p ¼ 0.033; bedtime, p < 0.001).

Control children of either gender, or for any children in the af-ternoon or at bedtime.

To explore the influence of alcohol exposure rank on the effect ofthe program, a two way repeated-measures ANOVA for the factorsof alcohol exposure (rank 3, rank 4, Control) and assessment period(pre- or post-program, or equivalent times for Control children) wasperformed for each time of day. At awakening (Fig. 5), there was asignificant interaction between alcohol exposure and assessmentperiod (F[2,41] ¼ 4.920, p ¼ 0.012), but no significant main effects.Further analysis indicated that cortisol levels were significantlyhigher (NewmaneKeuls, p ¼ 0.002) after the program in childrenwith an alcohol exposure rank of 4 compared to their levels pre-program. Cortisol was also significantly higher post-program inchildren with an alcohol exposure rank of 4 compared to post-program levels in children with rank 3 (NewmaneKeuls,p¼ 0.002) or Control children (NewmaneKeuls, p¼ 0.020). Cautionmust be used, however, when interpreting these results as pre- and

Fig. 5. Effect of the intervention program (or equivalent time period) on salivarycortisol levels at awakening in children with FASD with different alcohol exposureranks (n ¼ 13 for rank 3, n ¼ 5 for rank 4) and Control children (n ¼ 26). Values aremeans � S.E.M. *Significant difference between pre- and post-program (New-maneKeuls: p ¼ 0.002) in children with an alcohol exposure rank of 4.

K. Keiver et al. / Alcohol 49 (2015) 79e8784

post-programvalues were obtained for only 5 children (3 females, 2males) with an alcohol exposure rank of 4. No significant differ-ences were found between pre- and post-program cortisol levelsfor childrenwith an alcohol exposure rank of 3, or for the equivalenttimes for Control children.

For both the afternoon and bedtime cortisol levels, ANOVAs didnot support an effect of the program for either alcohol exposurelevel, but were consistent with the previous analysis comparingalcohol exposure rank 4 vs. alcohol exposure rank 3 vs. Controlchildren when all assessment periods were combined. Significantmain effects were found for alcohol exposure (afternoon, F[2,41] ¼ 8.521, p < 0.001; bedtime, F[2,41] ¼ 4.338, p ¼ 0.020) only.Further analysis showed that cortisol levels were higher in childrenwith alcohol exposure ranks of 4 compared with ranks of 3 (New-maneKeuls: afternoon, p < 0.001; bedtime, p ¼ 0.005) andcompared to Control children (NewmaneKeuls: afternoon,p < 0.001; bedtime, p ¼ 0.015). No significant differences werefound between Control children and those with alcohol exposureranks of 3.

Discussion

The present study is the first to show alterations in the HPAaxis in school-age children and across the diurnal cycle inhumans with PAE. Effects appeared to vary with severity of PAE,as basal cortisol levels were elevated in the afternoon and atbedtime in children with FASD with confirmed heavy exposurecompared to Control children, but not in children with confirmedexposure at lower or unknown levels. These results suggest PAEat high levels causes dysregulation of the HPA axis across thediurnal cycle such that the change in cortisol levels across theday, i.e., the slope, is flattened.

Previous studies on the effects of PAE in humans have beenlimited to children under 2 years of age, and have looked at pre- andpost-stressor cortisol levels, rather than basal levels across the day.These studies have generally shown that children with moderate-heavy PAE show an abnormal response to stressors (Haley et al.,2006; Oberlander et al., 2010; Ramsay et al., 1996) that is some-times gender-specific (Haley et al., 2006). Studies have had mixedresults, however, onwhether or not PAE affects basal cortisol levels.Children with moderate-heavy PAE had higher basal (pre-stressor)cortisol levels compared to children with low or no PAE at 2(Ramsay et al., 1996) and 13 (Jacobson et al., 1999) months of age,with no apparent difference with gender. Moreover, Jacobson et al.(1999) showed a significant positive relationship between basalcortisol levels and alcohol exposure level. In contrast, no differencein basal (pre-stressor) cortisol levels was found in children withheavy compared to low or no PAE at 3 days (Oberlander et al., 2010)or 5e6 months (Haley et al., 2006; Ramsay et al., 1996) of age.Interestingly, Ouellet-Morin et al. (2011) found lower basal cortisollevels in the morning in 19 month old children with low comparedto no PAE, but only in male children.

The differences among these studies in the effects of PAE onbasal or pre-stressor cortisol levels may be due to a number ofdifferent factors, but probably reflect, at least partly, an effect of age.Although the circadian rhythm begins to develop early in life, theHPA axis shows greater variability during the first 3 years, prior tosettling into an adult-like diurnal rhythm at approximately 4 yearsof age (Jessop & Turner-Cobb, 2008; Tarullo & Gunnar, 2006).Moreover, there is a period of time early in development wheretoddlers and pre-school age children are hypo-responsive tostressors (Tarullo & Gunnar, 2006), such as the exposure to a novelenvironment (e.g., laboratory or clinic), as was the case in most ofthe studies cited above. This hypo-responsiveness would likelyattenuate any differences among groups.

The effect of PAE on HPA axis regulation has been studied muchmore thoroughly in animal models. Studies using rodent modelsshow that PAE results in HPA dysregulation throughout life, withalterations involving both increased HPA drive and deficits in HPAfeedback regulation (Hellemans, Sliwowksa, Verma, & Weinberg,2010; Weinberg et al., 2008). These alterations result in increasedHPA tone, such that the individual is hyper-responsive to manytypes of stressors. Increased stress responsiveness has been shownto occur in non-human primates as well (Schneider, Moore, &Kraemer, 2004). Furthermore, sex differences in HPA axis dysre-gulation occur, but depend on the nature and intensity of thestressor, the time course of testing, and the hormonal endpointmeasured (Hellemans et al., 2010; Weinberg et al., 2008). Incontrast with the present study, rodent models of PAE do nottypically show altered basal corticosterone (the glucocorticoid inrodents) levels (Hellemans et al., 2010). In females, however, al-terations in basal levels may depend on estrogen levels. In adult-hood, female rats with PAE had higher basal corticosterone levels inproestrous (when estrogen levels were high) compared with Con-trol females, but lower levels in estrous (when estrogen levels werelow) (Lan et al., 2009). As cortisol levels were elevated in the af-ternoon and at bedtime in bothmales and females with FASD in ourstudy, it is unlikely that estrogen levels played a role in the elevatedbasal levels found in the children with FASD.

Our saliva samples were collected in the child’s home or usualenvironment. Thus, one might speculate that the increased cortisollevels in the afternoon and at bedtime in the children with FASDreflect a significant difference in stressful events that occurred ondays of saliva collection between children with FASD and Controlchildren. We do not think this is likely, however, because one of thestrengths of our study is that saliva was collected for 2 days at eachassessment period and, for most of the children, over multipleassessment periods. Therefore, the effect of any particular stressfulevent would be minimized. However, it is possible that the elevatedbasal levels reflect increased HPA axis tone, or hyper-responsiveness to the myriad of small events that happenthroughout a normal day e events that would not be significantenough to cause increased cortisol levels in Control children withnormal HPA tone. This could also explain why basal corticosteronelevels seem largely unaffected by PAE in animal models. Animalsare maintained in a controlled laboratory environment wherestressors are absent, or at least minimal. Moreover, in most animalstudies, basal hormone levels are measured prior to stress, andtypically, at the trough of the circadian rhythmwhen corticosteronelevels are low, which would likely minimize differences in levelsamong groups.

Children with FASD are frequently exposed to various forms ofchildhood adversity, including abuse, neglect, and multiple homeplacements (Streissguth et al., 2004). Similar to PAE, childmaltreatment or early caregiving adversity (including maternaldepression) results in HPA dysregulation and increases risk ofphysical and mental health problems (Cicchetti, Rogosch, Toth, &Sturge-Apple, 2011; Essex et al., 2011; Gunnar, Fisher, & The EarlyExperience, Stress, and Prevention Network, 2006). Early lifeadversity can affect basal cortisol levels, with different types ofadversity resulting in effects on the morning peak or eveningtrough of the diurnal rhythm, or the CAR (Cicchetti & Rogosch,2001; Dozier et al., 2006; Tarullo & Gunnar, 2006). However, theexact relationships between different types of adversity and spe-cific effects on the diurnal rhythm are somewhat inconsistent,possibly due to other modifying factors (Chida & Steptoe, 2009).Moreover, the effect on HPA function appears to vary with psychi-atric diagnosis and age of assessment (childhood or later in adult-hood) (Chida & Steptoe, 2009; Tarullo & Gunnar, 2006). We did notobtain information regarding history of abuse or caregiving

K. Keiver et al. / Alcohol 49 (2015) 79e87 85

disruptions/adversity from the families of our children, and therewere likely differences between the childrenwith FASD and Controlchildren in this regard; thus, we cannot be certain that the alter-ations in cortisol levels in the children with FASD resulted solelyfrom PAE. However, our finding that cortisol was elevated aboveControl levels in the afternoon and at bedtime in children withFASD with an alcohol exposure rank of 4, but not a rank of 3, sug-gests that the altered cortisol levels were actually the result of PAErather than differences in childhood adversity. Although we cannotrule out different levels of childhood adversity in children withdifferent alcohol exposure ranks, both groups were likely to have atleast experienced similar caregiving disruptions. Of the 8 childrenwith alcohol exposure rank of 4, 4 (50%) were in foster care and 4were with a grandparent. Of the 16 children with alcohol exposurerank of 3, 7 (44%) were in foster care, 6 were adopted, and 3 werewith their biological parent. Furthermore, although it might bepossible to statistically adjust for childhood adversity factors with alarge enough sample size, it would not be possible to match acontrol group to the childrenwith FASD for such factors. Examiningcortisol levels in children with FASD with no childhood adversitymay possibly be the only way to conclusively determine if alter-ations in cortisol are due solely to PAE in humans.

Interestingly, unpublished work by Dr. Kaitlyn McLachlan, Uni-versity of Alberta, and colleagues (personal communication) showssimilar findings to ours in a completely different cohort of childrenwith PAE. In 85 children and adolescents (age 5e18 years) with PAE,cortisol levels were significantly elevated at bedtime above those ofControl children, and the change in cortisol from morning tobedtime showed a flatter slope (p¼ 0.045). Moreover, similar to ourfindings, morning cortisol levels (within 30 min of awakening)appeared to be lower in children with PAE compared to Controlchildren, but this difference did not reach significance (p ¼ 0.129)(McLachlan, K., personal communication). These morning levelsrepresent cortisol during the morning peak and the CAR. The CAR issuperimposed on the morning peak of the diurnal rhythm and is adiscrete rise in cortisol that is associated with awakening (thesleepewake transition) (Clow, Thorn, Evans, & Hucklebridge, 2004;Pruessner et al., 1997). The CAR peaks at about 20e45 min afterawakening and is believed to be regulated independently from therest of the diurnal cycle (Clow et al., 2004; Pruessner et al., 1997).Because of the CAR, the variability in salivary cortisol levels in themorning is inherently greater than at other times of the day,especially if the time interval between awakening and samplecollection is not constant. This was true in our study and likelycontributed to the lack of statistically significant differences be-tween children with FASD and Control children. These data providefurther support for the idea that PAE causes long-lasting HPA axisdysregulation, and underscore the need for further investigation onthe effect of PAE onmorning cortisol levels with larger sample sizesand tighter control over time of sampling.

The differences in basal cortisol levels between the childrenwith FASD and Control children in this study cannot be explained bygender or age differences among these groups. Gender differencesin HPA axis activity occur in adults, but most studies report nodifferences in cortisol levels between males and females that arepre-pubertal or in early puberty (Jessop & Turner-Cobb, 2008;Netherton, Goodyer, Tamplin, & Herbert, 2004). Age and pubertyare fairly consistently associated with increases in basal cortisollevels in females, and while this has also been reported for males(Törnhage, 2002), the relationship is less consistent (Gunnar,Wewerka, Frenn, Long, & Griggs, 2009; Netherton et al., 2004).We did not record pubertal stage in the present study, but ourchildren were between 6 and 14 years old; thus, we might expectsome, especially females, to be at puberty. In particular, two of ourfemale children with FASD were over 12 years of age, whereas no

Control females were over 12 years. We did not find any relation-ship between age and cortisol levels, however, in children withFASD or Control children (males or females), and mean age did notdiffer between gender or groups. Moreover, dropping the two fe-males over 12 years from the analyses did not substantially changethe results. Cortisol levels also did not vary significantly withgender in Control children or in the children with FASD at any timeof day.

Chronically high or low basal cortisol levels can be harmful tophysical and mental health (Cicchetti et al., 2011; Essex et al., 2011).Although elevated cortisol levels are clearly associated withincreased risk of stress-related disease (e.g., diabetes, hyperten-sion), other specific effects of HPA axis dysregulation are difficult topredict. Rodent models indicate that dysregulation of the HPA axisincreases risk for anxiety and depressive disorders (Liu et al., 2012;Weinberg et al., 2008). Furthermore, the physiological role of theCAR is not clear, and so neither are the effects of alterations in theCAR (Clow, Hucklebridge, Stalder, Evans, & Thorn, 2010).

Intervention programs may be able to attenuate some aspects ofHPA dysregulation associated with early life adversity and sodecrease risk of physical and mental health problems. For example,studies have shown that interventions can change or normalize thealtered basal cortisol levels associated with child maltreatment orcaregiving disruptions (Cicchetti et al., 2011; Dozier, Peloso, Lewis,Laurenceau, & Levine, 2008; Fisher, Stoolmiller, Gunnar, &Burraston, 2007; Gunnar et al., 2006). Evidence is accumulatingto indicate that exercise can play a protective role in dysregulationof the stress system and its potential deleterious effects (Ericksonet al., 2013; Pietropaolo et al., 2008; Tsatsoulis & Fountoulakis,2006). Exercise is thought to affect both the HPA axis and seroto-nergic system (Tsatsoulis & Fountoulakis, 2006). Serotonin modu-lates the HPA axis and, importantly, both the HPA axis andserotonergic function are altered in PAE (Weinberg et al., 2008).Moreover, anxiety and depressive disorders are common in in-dividuals with FASD and are linked to HPA and serotonergic dys-regulation (Hellemans et al., 2010). In humans, female adolescentswith depression showed decreased urinary cortisol levels andimproved symptoms following an exercise program (Nabkasornet al., 2006). In rodent models, exercise can decrease anxiety anddepressive-like behaviors, and normalize changes in glucocorticoidand serotonin receptors induced bymaternal separation (Maniam&Morris, 2010). Furthermore, similar to PAE, prenatal exposure toincreased glucocorticoids results in lifelong alterations in HPA axisfunction, and increased anxiety and depressive-like behavior.Importantly, Liu et al. (2012) have shown that exercise can atten-uate the elevated basal corticosterone levels and depressive-likebehaviors induced by prenatal glucocorticoid exposure in the rat.

We hypothesized that cortisol levels would be normalized inchildren with FASD after completion of our intervention program.We were unable to demonstrate any significant gender specificprogram effects, likely due to power issues. Cortisol levels weresignificantly increased after the program at awakening in children(both genders analyzed together) with alcohol exposure ranks of 4,although to levels above those of Control children. However,because both pre- and post-program saliva samples were obtainedfrom only 5 children (3 females, 2 males) with alcohol exposureranks of 4, decisive conclusions should not be made on this dataalone without further investigation. Unfortunately, the program didnot influence afternoon or bedtime cortisol levels.

Although our intervention program was a physical activityprogram, the focus was on developing motor skills rather thanincreasing fitness. Moreover, it incorporated a mix of activities thatmight be expected to increase both aerobic and anaerobic capacity,depending on the child’s individualized program. Thus, it is possiblethat the overall mean intensity of activity of the children’s

K. Keiver et al. / Alcohol 49 (2015) 79e8786

individualized programs, or the type of activity, was not optimal foreliciting maximal change in cortisol levels. The impact of intensityand type of physical activity has not beenwell studied with respectto effects on the HPA axis. In studies with animal models, mostexercise programs that have affected the HPA axis have involvedvoluntary running in a wheel, which is considered moderate in-tensity aerobic exercise (Greenwood & Fleshner, 2008), or forcedswimming (Liu et al., 2012). In humans, low intensity aerobic ex-ercise was effective in decreasing urinary cortisol and depressivesymptoms (Nabkasorn et al., 2006), and evidence suggests thatphysical activity at light, moderate, and vigorous intensity, and bothresistance and aerobic exercise can decrease symptoms of depres-sion (Dunn, Trivedi, & O’Neal, 2001; Greenwood & Fleshner, 2008).It is also possible that a program of longer duration might have hada greater effect.

Our study had several limitations. First, owing to the relativelylow number of childrenwith FASD, wewere limited in our ability toexamine the effects of variables such as gender, age, and alcoholexposure rank. Second, as we did not obtain information onchildhood adversity or caregiving disruptions from our families, wewere unable to determine the potential role of these factors oncortisol levels. Third, there was a fair amount of variability in thetiming of collection of the saliva samples at awakening, which likelycontributed to the difficulty in observing differences among groupsfor this time point. In addition to reducing variability in the timingof collection, it would also be advantageous to take several samplesat intervals throughout the first hour after awakening to charac-terize any effects of FASD on the CAR itself. These limitations shouldbe addressed in future studies.

In summary, this study supports the hypothesis that PAE at highlevels has long-lasting effects on the HPA axis in humans. Furtherstudy with a larger sample size is needed to determine if PAE haseffects on cortisol levels at awakening, and if exercise-basedintervention programs can modify cortisol levels.

Acknowledgments

We wish to thank Ryan Konarski, Mina Thomas, and the FraserValley Child Development Centre for their invaluable work in thisstudy.We also give our thanks to Dr. JoanneWeinberg, University ofBritish Columbia, for her helpful comments on the manuscript andto Wayne Yu for running the cortisol assays. This study was fundedby the Victoria Foundation (grant $7M 212), the BC ProvincialHealth Services Authority, and the Canada NW FASD ResearchNetwork.

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