Parsing the relations between SES and stress reactivity: Examining individual differences in neonatal stress response

Infant Behavior & Development 30 (2007) 134–145

Parsing the relations between SES and stress reactivity: Examining individual differences in neonatal stress response Kate Keenan ∗ , Dana Gunthorpe, Desia Grace Department of Psychiatry (MC 3077), University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637-1470, United States Received 13 February 2006; received in revised form 21 July 2006; accepted 9 August 2006

Abstract In an effort to further delineate the reported relations between socioeconomic status (SES) and stress reactivity in children, associations between three domains of perinatal risk: socio-demographic, obstetrical complications, and maternal psychological factors during the perinatal period, and cortisol and behavioral reactivity were examined in 100 healthy African American neonates whose families resided in low-income environments. Behavioral and cortisol response to a heel stick and the Neonatal Behavioral Assessment Scale (NBAS) was measured within the first 2 days of life. Significant associations were found between socio-demographic risk, obstetrical complications, and maternal psychological factors and neonatal behavior and cortisol in the context of the NBAS; few significant associations were found in the context of the heel stick. Greater magnitude of perinatal risk was associated with both higher and lower than average neonatal stress reactivity. The results provide preliminary data on the types of perinatal experiences that may have significant effects on stress reactivity in humans, especially in the context of families living in poverty. Application of these data to the hypothesis that prenatal stress results in programming of the fetal/neonatal stress response system is discussed. © 2006 Elsevier Inc. All rights reserved. Keywords: Perinatal; Neonates; African American; Stress reactivity; Poverty

A variety of literatures converge to support the hypothesis that exposure to stressors common to environments marked by poverty leads to individual differences in patterns of stress reactivity. The amount or quality of stress that a child is exposed to, even prenatally, may lead to an individual difference in how a child responds to stress early in life. There are four areas of investigation that we draw on as preliminary support for this hypothesis. First, in the U.S., higher levels of acute and chronic stress are found among families living in low-income environments than among families living in other income environments. Neighborhood disorder and lack of safety and exposure to violence are all significantly higher in areas with lower per capita income in rural and urban environments (Evans, 2003; Ewart & Suchday, 2002). Much of the work in this area has been focused on cumulative risk (Atzaba-Poria, Pike, & Deater-Deckard, 2004) and allostatic load (McEwen, 2000), with the idea that there may be a threshold at which exposure to stress leads to less efficiency in the physical and psychological regulation of stress (Evans, 2003). ∗

Corresponding author. Tel.: +1 773 702 4449; fax: +1 773 834 9929. E-mail address: [email protected] (K. Keenan).

0163-6383/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.infbeh.2006.08.001

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Second, in a few studies SES has been associated with cortisol secretion, however the pattern has not been consistent. Evans (2003) found a small but significant positive association between overnight urinary cortisol gathered from 337 nine-year old children and a cumulative risk index derived from nine risk factors common among families living in poverty (e.g., housing problems, overcrowding, and family turmoil). Thus, the higher the number of sociodemographic risk factors the higher the level of cortisol. In a cross-sectional study of 21 7 children from high, middle, and low socioeconomic environments, Lupien, King, Meaney, and McEwen (2000) reported that basal salivary cortisol was positively associated with SES; at age 10 years, but not at earlier ages, children living in the lowest SES environments had significantly lower resting cortisol levels than children living in middle- or upper-SES environments. These studies highlight the importance of testing whether some factors common to low-income environments are positively associated with functioning of the stress response system whereas other may be negatively associated with the stress response system, including the resting or set point of that system. Third, in many studies of non-human primates and rodents exposure to prenatal stress is associated with atypical patterns of response to stressful stimuli. For example, Weinstock (1997) documented that rat pups whose mothers were exposed to stress during the first trimester of pregnancy demonstrated higher levels of behavioral reactivity to stimuli. Similarly, Schneider and Moore (2000) reported that primate infants exposed to stress prenatally showed less organized behavioral responding to novel stimuli. Coe et al. (2003) demonstrated that prenatal stress in the form of an auditory stimulus was associated with reduced hippocampal volume, which was in turn associated with higher cortisol values after a dexamethasone suppression test in young rhesus monkeys. In addition to demonstrating a prospective relation between exposure to stress prenatally and less optimal response to stress early in life, data from research using animals models suggest that not all stimuli elicit the same type of glucocorticoid response in terms of both the intensity of response (e.g., Fano et al., 2001) and the regions of the brain that are involved in the regulation of the HPA axis system (e.g., Thrivikraman, Nemeroff, & Plotsky, 2000). Thus, comparing and contrasting the association of different types of stimuli on stress reactivity is necessary. Finally, there have been a few prospective studies in which the association between prenatal stress in humans and outcome in the offspring has been examined. O’Connor et al. (2005) reported that individual differences in anxiety during pregnancy were significantly positively correlated with cortisol levels in the offspring upon awakening when the subjects were 10 years of age, even after controlling for maternal post-natal anxiety and depression. In a longitudinal study of Dutch children the association between prenatal stress and broad indices of stress reactivity in the offspring varied across development. For example, perceived stress during pregnancy was positively associated with maternal report of difficultness and observed problems with attention at 3 months of age (Huizink, Robles de Medina, Mulder, Visser, & Buitelaar, 2002), negatively associated with report of restless and disruptive temperament at 27 months of age (Gutteling et al., 2005), and not significantly associated with offspring cortisol levels on the first days of school (Gutteling, de Weerth, & Buitelaar, 2005). Maternal cortisol levels during pregnancy, were unrelated to temperamental problems at 3, 8, or 27 months, but were positively associated with offspring cortisol on the first day of school and in response to a vaccine during the preschool period (Gutteling, De Weerth, & Buitelaar, 2004). The inconsistency in these results may be due to problems resulting from attrition; 230 participants were initially recruited out of 400 who were eligible and less than 30 were available for the preschool assessments. Alternatively, the authors may be encountering unmeasured post-natal effects on offspring behavioral and cortisol response: the cumulative exposure to maternal anxiety or stress over the child’s life may have accounted for the significant associations between maternal prenatal cortisol and cortisol reactivity in the preschooler. This is a critical issue with regard to conceptualizing the mechanism of risk. What needs to be determined is the point in development during which alterations or atypicalities in the stress response system are observed. In summary, the results from these four areas of research suggest that prenatal stress may lead to lasting effects on the stress reactivity system of the offspring. Because stressful experiences are more common in low-income environments, one could expect that pregnant women living in such environments are likely to encounter or experience more stressors. The literature to date in humans, however, leaves unanswered the question, at what point in development is the relation between exposure to stress and effects on the stress response system apparent? It is possible that it is the accumulation of stress experiences over time that leads to a deficient stress response as opposed to starting out life with such deficits. In addition, as evidenced in the animal literature, not all stimuli affect stress reactivity. Poverty and SES are nonspecific variables that require deconstruction in order to generate hypotheses about the mechanism of effect. In the present study we focus on three domains of stressors that are common to families living in low-income environments, may affect stress reactivity in newborns, and have been associated with behavioral and emotional functioning later in

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development. First are socio-demographic factors such as young maternal age at birth and overcrowding in the home, both of which are associated with behavioral and emotional problems in children (Shaw, Vondra, Hommerding, Keenan, & Dunn, 1994; Wakschlag et al., 2000). Overcrowding may affect maternal health during pregnancy in a number of ways including poorer nutrition due to lack of resources and the mothers own stress level. Family composition, which in part reflected family size, was associated with higher than average levels of cortisol in a cross-sectional study of children aged 2 months to 18 years (Flinn & England, 1995). Crowded housing has been associated with altered glucocorticoid functioning and behavior in animals (Brown & Grunberg, 1996; Haller, Baranyi, Bakos, & Halasz, 2004). Maternal age may also affect maternal health during pregnancy via undernutrition of the fetus; young maternal age has been associated with altered reproductive and endocrine functioning in the offspring in mice (Wang and vom Saal, 2000). Second, are maternal psychological health factors including depressive symptoms and life stress, which may affect maternal cortisol production and in turn, fetal and/or infant cortisol secretion (Lundy et al., 1999). Third, are obstetrical complications: women from low-income environments, especially African American women, are more likely to develop medical complications during pregnancy such as gestational diabetes, hypertension and are more likely to deliver by cesarean section (Giscombe & Lobel, 2005). These types of complications can result in fetal stress, which may alter stress reactivity in the newborn (Taylor, Fisk, & Glover, 2000). Atypical patterns of stress response can be operationalized as both lower than average and higher than average in terms of either set points or reactivity. Much of the developmental literature has had a focus on increases in cortisol in response to stimuli, but in several recent studies correlates of lower than average cortisol have been identified. For example, Davis et al. (2004) reported that prematurely delivered children who were treated with betamethasone prenatally had decreases in cortisol in response to the heel stick. Grunau et al. (2005) reported that the number of skin breaking procedures was negatively associated with plasma cortisol response to stimuli. It may be possible, therefore, to observe hypo-responsiveness to a stimulus if that stimulus is experienced following exposure to another acute or repeated stressor. In an earlier set of analyses we explored the utility of the NBAS and heel stick to atypical patterns of stress reactivity (Keenan, Grace, & Gunthorpe, 2003). Those data supported the use of the heel stick to identify hypo-responsiveness. Because the vast majority of neonates demonstrate an increase in cortisol and behavioral distress (e.g., crying) in response to the heel stick, a decrease in cortisol and low levels of behavioral distress reflected atypical responses. The data also supported the use of the NBAS as a means of identifying hyper-responsiveness: neonates typically responded to the NBAS with little to no change in cortisol and minimal behavioral distress. In fact, an association between cortisol and behavior was revealed only when neonates were classified as hypo- or hyper-responsive (Keenan et al., 2003). Because the broader aims of our program of research is to link very early patterns of atypical stress response to later behavioral and emotional dysregulation, we believe that it is important to test whether the hypothesized correlates in the present study are associated with a lower or higher than average stress response. Given the limited literature in this area, however, we do not present hypotheses about the nature of the association between the poverty correlates and stress reactivity. In the present study we examine the association between maternal prenatal and perinatal stressors common to families living in low-income environments and stress reactivity in healthy neonates. Such a test allows us to determine whether an association is observable early in life and to tease apart the construct of poverty by separately examining factors that are common to families living in low-income environments. 1. Methods As part of an ongoing longitudinal study, full term, healthy neonates and their mothers were recruited from the General Care Nursery (GCN) at the University of Chicago. The GCN predominately serves healthy African-American women and their newborns, with the majority of families having incomes below the poverty level. Infants requiring admission to the intensive care nursery, or who were known to have been exposed to illegal substances (e.g., cocaine, marijuana, amphetamine, heroine or methadone) in utero were excluded from the study. Mothers of infants who met the inclusion criteria were approached by one of the project staff. A total of 155 mothers and infants met inclusion criteria for the study and 113 (73%) agreed to participate. Thirteen (8%) of those who agreed to participate were unable to because of technical problems. A total of 100 mothers and infants were enrolled in the study and completed the neonatal assessment.

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All mothers were African-American and received some level of public assistance. At the time of recruitment the average age of the mothers was 22.8 years (S.D. = 4.5 years), 70% had graduated from high school, and 73% were not living with a partner. One third of the births were first births. Mothers in this sample were generally healthy during pregnancy; 92% reported receiving regular prenatal care, 86% did not smoke at all during pregnancy and 96% reported complete abstention from alcohol. The majority of births were unassisted vaginal deliveries (71%). Twelve percent of the deliveries were by planned Cesarean section, 9% were by unplanned Cesarean section, and 8% required assistance. The newborns were full term with a mean gestation of 39 weeks (range = 36–42 weeks, by estimated date of conception) and of average weight (mean = 3220 g; range = 2500–4560 g). The 5-min Apgar scores ranged from 8 to 10 with a mean of 9. Two infants were born between 36 and 37 weeks. Both had Apgar scores of 9 and 9 at 1 and 5 min, respectively, and were over 2500 grams. In addition, the estimated gestational ages by exam were 37 and 38 weeks, respectively. Therefore, they were not excluded from the sample. Almost half of the sample (47%) was male, and all but 4 (8.5%) of the males were circumcised. Circumcisions were typically performed early in the morning without anesthesia. Among the males whose circumcision was performed prior to the heel stick or NBAS and for whom data was available on the time of the circumcision, the average time that elapsed between the circumcision and heel stick was 6 h and 40 min, and the average amount of time between the circumcision and the NBAS was 18 h and 17 min. Two neonates had circumcisions that occurred less than 4 h before the heel stick and two had circumcisions that were performed less than 4 h before the NBAS. These neonates were not included in the analyses of stress reactivity to the heel stick and NBAS, respectively. 1.1. Procedures Data on behavioral and cortisol reactivity to two stimuli, the Neonatal Behavioral Assessment Scale and a heel stick procedure, were gathered within 48 h of birth. Saliva was collected before (pre-) and 20 and 45 min following the offset of each stimulus. The 20-min post-stressor saliva sample was designed to represent the peak stress response and the 45-min sample represented the recovery response. Pre-stressor saliva samples were usually collected within 20 min before the start of the stressor (mean = 4.5 for the NBAS and 18.6 for the heel stick). For a few neonates, an extended elapsed time of more than an hour between saliva collection and the onset of the stimulus occurred. Because the onset of the heel stick was under the control of the phlebotomist, the extended elapsed times occurred more frequently prior to the heel stick (n = 6) than prior to the NBAS (n = 1). We tested whether elapsed times between pre-stressor saliva and onset of stressor were associated with cortisol levels: none of the tests were statistically significant. The Neonatal Behavioral Assessment Scale (NBAS; Brazelton & Nugent, 1995) assesses the infant’s response to his/her environment. A broad range of behaviors is sampled including reflexes, state changes, attention, arousal, and regulatory capacities. In the present study we focus on three ratings: intensity of (i.e., excitability), latency to (i.e. rapidity of build up), and duration of distress (i.e., irritability) each made on nine-point scales. The principal investigator was trained and certified by the Brazelton Institute and then trained the second rater. The NBAS was typically administered during the second day after delivery. Interrater reliability was assessed on 25% of the participants, resulting in Spearman Correlation Coefficients ranging from 0.80 to 0.99. In addition, the neonate’s behavioral response to the stimulus was classified as hyper-reactive or typically reactive using the Kaye criterion (1978). This classification uses three items from the NBAS: rapidity of build-up, irritability, and lability of states, which are combined (alpha = 0.81 in the present sample) and averaged. Neonates who receive a score of 6 or higher are classified as hyper-reactive. Blood drawing from the heel is a state mandated procedure that was typically conducted at 24 h after birth by 1 of approximately 5 technicians. It begins with lancing the heel and is followed by repeatedly squeezing the heel (on average 30 times) in order to generate enough blood for screening for metabolic disorders and elevated bilirubin. The average duration of the heel stick was 5 min. Behavioral response to the heel stick was coded from videotape and included a global rating of intensity of distress (none, mild, moderate, severe) and microanalytic coding of the latency to (in seconds) and duration of distress (proportion of the time exposed to the stressor during which the neonate evidenced moderate to severe distress), using the Video Coding System (James Long Company). Interrater reliability assessed on 20% of the sample was high: 0.86 for intensity, 0.94 for duration, and 0.92 for latency. One participant was not videotaped due to technical difficulties. Videotape coding was conducted by a team of coders not involved in any other data collection and blind to the mother’s

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and infant’s status on other study variables. In order to generate a behavioral index from the heel stick procedure that paralleled the NBAS criteria for behavioral distress in response to the stressor, scores for latency, intensity of, and duration of distress, in response to the heel stick were standardized and combined (alpha = 0.73). The distribution of the summed scores was used to classify neonate’s behavioral response to the stimulus. Neonates were classified as being in any stage of sleep (deep, light, or semi-dozing) or wakefulness (alert, active, or crying) prior to the administration of the NBAS or the heel stick. There were no differences in the proportion of awake or asleep infants across stressor. State of infant prior to the NBAS and the heel stick was not associated with cortisol values pre or post-stressor or behavior (Keenan, Gunthorpe, & Young, 2002). Procedures for collecting saliva were described in detail in an earlier report (Keenan et al., 2003). Briefly, saliva was collected using an unflavored salivette by applying it to the tongue, cheek, and gums of the infant for 2–5 min. Samples were stored at −20 ◦ C until assayed. All samples from each subject were assayed in the same assay batch to minimize variability, and all samples were assayed with reagents from the same lot. Samples were assayed in duplicate using the Salimetrics HS Salivary Cortisol EIA Kit for unbound cortisol. In the present study, the average of the duplicate tests was used in the analyses: approximately 10% of the samples did not have sufficient quantity to run duplicate assays. The intra-assay variance in the present sample was less than 5% and the inter-assay variance was less than 15%. 1.1.1. Sociodemographic factors As described earlier, we focused on maternal age and number of people in the household, which were asked of mothers in a background interview during the immediate post-partum period, because overcrowding and younger maternal age are associated with behavioral emotional and behavioral regulation in children, neuroendocrine functioning in animals, and because younger mothers and overcrowding are more common among families living in poverty. 1.1.2. Obstetrical complications The total number of pregnancy and delivery complications was measured via the Rochester Research Obstetrical Scale (ROS; Sameroff, Seifer, & Zax, 1982). The scale has shown prediction to both neonatal and infant developmental functioning (Molfese & Thomson, 1985). Data for completing the ROS were gathered from the medical record. Examples of pregnancy complications included on the ROS are infections, medication use, and parity. Examples of delivery complications are type of delivery, quality of amniotic fluid, and cord problems (e.g., knotted). 1.1.3. Maternal psychological factors Maternal depressive symptoms during pregnancy were assessed using the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID; First, Spitzer, Gibbon, & Williams, 1995). The SCID was administered in the immediate post-partum period. Mothers were asked to retrospectively report on the symptoms they experienced during their pregnancy. Inter-rater reliability between the research assistant and the principal investigator, assessed on the total number of depression symptoms, was in the moderate range (n = 16, ICC = 0.76). Two mothers did not complete the SCID. Negative life events were measured using the Difficult Life Circumstances Scale (Barnard, Johnson, Booth, & Bee, 1989), which includes 28 yes/no questions about chronic stressful circumstances such as trouble finding an affordable place to live, having a bad credit rating and having problems with a former partner. This scale was developed for use with mothers living in inner-city poverty. The 1-year test re-test correlation for the total score was 0.70 (Barnard et al., 1989). Mothers completed this questionnaire on their own in the immediate post-partum period. 2. Results We first provide descriptive data on cortisol levels and the sociodemographic, obstetrical, and maternal psychological factors, which we refer to collectively as poverty correlates throughout the results section for ease of explication. Next we present the results for the association between the poverty correlates and the average cortisol and behavioral response to the NBAS and the heel stick. Finally, we examine the association between the poverty correlates and the within subject cortisol and behavioral response to the NBAS and heel stick using the conceptualization of the NBAS as stimulus that is best for identifying higher than average responders and the heel stick as a stimulus that is best for identifying lower than average responders.

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Table 1 Descriptive statistics for cortisol and hypothesized correlates Mean (S.D.)

Range

NBAS (n = 6162) Pre-stressor cortisol (␮g/dL) 20 min post-stressor (␮g/dL) 45 min post-stressor (␮/dL)

0.42 (0.4) 0.56 (0.5) 0.49 (0.5)

0.01–3.3 0.01–2.8 0.03–2.2

Heel stick (n = 64) Pre-stressor cortisol (␮g/dL) 20 min post-stressor (␮/dL) 45 min post-stressor (␮g/dL)

0.62 (0.7) 0.82 (0.8) 0.63 (0.6)

0.08–3.6 0.11–4.0 0.08–2.7

Socio-demographic factors Maternal age Number in home

22.8 (4.5) 4.0 (2.1)

17–41 1–12

Obstetrical factors Prenatal Complications (1 or more) Delivery complications

2.3 (1.5)

0–6

Maternal psychological factors Depression symptoms (1 or more) Negative life events

2.8 (1.9)

0–9

N

%

14

14%

27

27%

2.1. Descriptive data on cortisol reactivity and the poverty correlates Extensive descriptive data on cortisol and behavioral reactivity are presented in earlier papers (Keenan et al., 2002, 2003). Raw cortisol values for pre-, 20 min post- and 45 min post-stressor were log transformed to correct skewness. Of the 100 participants recruited for the study, 83 had three sufficient saliva samples (i.e., baseline, 20-, and 45-min post-stressor samples) for at least one of the stimuli (i.e., NBAS or heel stick). A total of 62 participants had complete cortisol data (i.e., three samples) for the NBAS paradigm, and 64 participants had complete data for the heel stick paradigm. Descriptive statistics for the baseline, peak, and recovery cortisol values using untransformed values in ␮g/dL are in Table 1. The average baseline cortisol value was statistically higher in the heel stick paradigm than in the NBAS paradigm (−0.40 versus −0.53, t = 2.39, p < 0.05). The heel stick also occurred somewhat later in the day (mean = 15:06) than the NBAS (mean = 13:52), however this difference in timing of stimulus was not statistically significant. Moreover, the lack of an established circadian rhythm in the neonate reduces the possibility of time of day effects on cortisol levels (Lewis & Ramsay, 1995). Other factors, however, may explain the higher pre-heel stick cortisol values such as the possibility of more frequent handling or other stressful experiences such as diaper changes as the day progressed. In the present study, these daily experiences were not measured. The NBAS differed from the heel stick in terms of the average duration of the stressor (20 min versus 5 min, respectively). Because nearly all the males were circumcised we were unable to test for the effect of circumcision on the infant’s response to either of the stimuli. We were able, however to test for sex differences in cortisol and behavioral response to the NBAS and heel stick; none of the tests were significant. For each point of data collection, we compared cortisol levels for those infants who had generated sufficient saliva for all three samples versus those who had not. No differences in cortisol values were obtained, nor was there a significant effect of the ordering of the stimuli on cortisol response (Keenan et al., 2002). We also compared infants who generated sufficient saliva for three samples and those who did not on the six hypothesized correlates; none of the comparisons were statistically significant. Descriptive statistics for the six poverty correlates also are presented in Table 1. Two correlates were not normally distributed: prenatal complications and depressive symptoms. The vast majority of mothers (86%) had no evidence of pregnancy complications and the remaining 14 were distributed across one, two, and three complications. As a result of this skewed distribution, pregnancy complications were dichotomized as absent or present in all analyses. Similarly, 72 mothers reported no symptoms of depression and 10% met criteria for major depressive disorder, a rate that is similar

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Table 2 Association between poverty correlates and behavioral response to the NBAS

Maternal age Number in home Delivery complications Negative life events

Latency to distress, r

Intensity of distress, r

Duration of distress, r

−0.04 −0.25** 0.33** 0.13

0.01 −0.19* 0.33** 0.11

0.00 −0.16† 0.27** −0.16†

1 or more sxs of depression Yes No

3.6 (2.2)a 3.7 (2.1)a

5.6 (2.0)a 5.6 (1.8)a

5.1 (2.9)a 5.0 (2.5)a

Any prenatal complication Yes No

2.9 (2.0)a 3.7 (2.2)a

5.3 (1.8)a 5.7 (1.9)a

4.5 (2.8)a 5.1 (2.6)a

Pearson correlation coefficient: † p < 0.10; * p < 0.05; ** p < 0.01. a Mean (S.D.).

to other studies of pregnant women living in inner-city, low-income environments (Ritter, Hobfoll, Lavin, Cameron, & Hulsizer, 2000). Because the distribution of depressive symptoms was skewed toward zero, depressive symptoms were dichotomized as absent or present in all analyses. Associations among the poverty correlates were tested using Kendall’s tau-b for non-parametric statistics. Total number of people in the home was positively associated with prenatal complications (correlation coefficient = 0.17, p = 0.048) and negatively associated with delivery complications (correlation coefficient = −0.20, p = 0.011). Delivery complications were negatively associated with depressive symptoms (correlation coefficient −0.20, p = 0.022), and negative life events and symptoms of depression were positively associated (correlation coefficient = 0.26, p = 0.002). Subsequent analyses addressed these patterns of correlations when indicated. 2.2. Relations between poverty correlates and response to the NBAS and heel stick Associations between cortisol and behavior in the context of the NBAS and heel stick and each of the six poverty correlates were tested using Pearson correlations for continuous data and ANOVA for the dichotomous variables prenatal complications and depressive symptoms. In the context of the NBAS, two significant associations were identified: delivery complications were negatively associated with pre-NBAS levels of cortisol (r = −0.22, p = 0.022) and negative life events were positively associated with pre-NBAS levels of cortisol (r = 0.21, p = 0.031). There was a trend for neonates of mothers with at least 1 symptom of depression during pregnancy to have higher pre-NBAS cortisol levels than neonates of mothers who reported no symptoms (−0.37 versus −0.55, p = 0.051). None of the correlates were associated with cortisol levels at 20 or 45 min post-stressor at the univariate level. However, the magnitude of difference between the 45-min post-stressor cortisol levels for infants of mothers with (−0.39) versus without (−0.51) depressive symptoms suggested a meaningfully different pattern. We tested the effect of time on cortisol in each of the two groups: neonates whose mothers reported any depressive symptoms during pregnancy and neonates whose mothers did not report any symptoms. There was a significant quadratic effect of time on cortisol among the infants of mothers without depressed symptoms, indicating that cortisol increased from pre- to 20-min post-NBAS and then decreased by 45 min post-NBAS (F [1,43] = 7.50, p = 0.009). There was no effect of time on cortisol for the infants whose mothers endorsed symptoms of depression. There were a number of significant associations between the poverty correlates and behavioral response to the NBAS. Number of people in the home was significantly negatively associated with latency (r = −0.25, p = 0.006) and intensity (r = −0.19, p = 0.028) and marginally associated with duration of distress (r = −0.16, p = 0.059). Delivery complications were positively associated with all three dimensions of behavioral response (see Table 2), and number of life events was marginally associated with duration of distress (r = 0.16, p = 0.063). Only one significant association was found between the poverty correlates and either cortisol or behavioral response to the heel stick. Prenatal complications were associated with the recovery cortisol levels, with infants whose mothers

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Fig. 1. Interaction of time and prenatal complications on cortisol response to the heel stick (F(1,62) = 4.26, p = 0.043, eta = 0.25).

experienced prenatal complications demonstrating higher cortisol levels at 45 min post-stressor than infants of mothers without prenatal complications (−0.17 versus −0.43). This pattern of response was further examined in a repeated measures linear model shown in Fig. 1 using untransformed cortisol values. The interaction of prenatal complications and time on cortisol level was significant (F (1,62) = 4.26, p = 0.043, eta = 0.25). 2.3. Relations between poverty correlates and within subject response to the NBAS and heel stick Given that the direction of effect varied across the relations described above, with some correlates appearing to be associated with a dampened response (e.g., number of people in the home) and other with a more heightened response (e.g., delivery complications), we tested for associations between the poverty correlates and these atypical patterns of response. In these analyses the dependent measure is a dichotomous variable. For the NBAS, behavioral hyperresponsiveness was operationalized as meeting the Kaye criteria for high distress, and cortisol hyper-responsiveness was operationalized as a level that fell in the upper quartile of the distribution of change scores from pre- to 20 min post-NBAS. For the heel stick, behavioral hypo-responsiveness was operationalized as a behavioral score falling in the lower quartile of the distribution, and cortisol hypo-responsiveness was operationalized as a level that fell in the lower quartile of the distribution of change scores from pre- to 20 min post-heel stick. In response to the NBAS, mothers of behaviorally hyper-responsive neonates experienced a higher number of delivery complications than mothers of other neonates (2.8 versus 2.0; F [1,96] = 6.42, p = 0.013, eta = 0.25). There was a trend for mothers of behaviorally hyper-responsive neonates to have reported a higher number of negative life events during pregnancy than mothers of other neonates (3.2 versus 2.5; F [1,96] = 3.53, p = 0.063, eta = 0.19). None of the poverty correlates were associated with a cortisol hyper-responsiveness to the NBAS. None of the poverty correlates were associated with an atypical cortisol response to the heel stick. One of the six poverty correlates, negative life events, was associated with behavioral hypo-responsiveness to the heel stick. Mothers of behaviorally hypo-responsive neonates reported a higher number of negative life events during pregnancy than mothers of other neonates (3.5 versus 2.5; F [1,96] = 6.00, p = 0.016, eta = 0.25). 3. Discussion One approach to further delineating the role of poverty in increasing the risk for child health and mental health outcomes, is to examine specific factors that are both more likely to occur among families living in poverty and more likely to influence the developmental course of children’s emotional and behavioral functioning and the period during which such relations are evident. In the present study, we tested the association of six factors representing three broad domains of risk: social demographic, obstetrical, and maternal psychological on patterns of stress reactivity within the

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first 2 days of life. Our aim was to provide preliminary data on whether individual differences in very early indices of regulation of the stress response were in fact associated with these three domains. Not surprisingly, some significant associations were found among the poverty correlates, although the magnitude of associations was generally small. Depression was positively associated with negative life events and total number of people in the home was positively associated with delivery complications. A surprising pattern was that of negative associations between depression and delivery complications and total number of people in the home and delivery complications. DiPietro, Novak, Costigan, Atella, and Reusing (2006) recently published a finding that is somewhat consistent with this result: prenatal depressive symptoms, anxiety, and stress, were all positively associated with child development measures at age 2 years. These authors argued that there may be a curvilinear association between prenatal stress and fetal and neonatal growth and development, with mild levels of stress resulting in greater neuromaturation. In the present study, an association between mild levels of stress and neuromaturation may have enabled the fetus to more successfully navigate the birth process. The small sample size and skewed distribution of some of our data prevented us from adequately exploring this hypothesis, however. Few of the poverty correlates were associated with cortisol response to the NBAS using continuous measures of stress reactivity. Delivery complications were associated with lower cortisol levels prior to the administration of the NBAS, whereas maternal report of negative life events was associated with higher pre-NBAS cortisol levels. However, none of these pre-NBAS differences in cortisol led to significant differences in the magnitude of the response or recovery. Although the magnitude of the difference in cortisol level during the recovery period between neonates whose mothers experienced depressive symptoms during pregnancy and those who did not appeared clinically meaningful, the groups did not differ statistically. If pre-stressor cortisol levels are representative of resting levels, we could hypothesize that although both delivery complications and negative life events during pregnancy were associated with cortisol levels, the effect of delivery complications may be more transient than the effect of more chronic stressors. A complicated delivery may have temporarily resulted in the neonate spending more time in a deep sleep state as the neonate recovers from the delivery, during which time cortisol levels would be lower, whereas the exposure to negative life events may have led to a more stable change in the neonate’s set point to a slightly more hyper-aroused state. With regard to behavior, correlates were associated with either a dampening or an increase in the intensity of response. The more people in the home the longer the latency to distress and the less the intensity the distress whereas, the more complicated the delivery the shorter the latency to and the greater intensity and duration of distress. These results suggest that different forms of stress during the perinatal period may result in different patterns of stress response. Thus, based on these very exploratory analyses, combining indices of stress into a cumulative index may not be the most useful approach to understanding the nature of the impact of prenatal stress on stress reactivity in the human neonate. Only one of the six hypothesized correlates, prenatal complications, was associated with cortisol or behavioral response to the heel stick. On average, the nine neonates whose mothers experienced prenatal complication had lower pre-heel stick cortisol levels but higher 20- and 45-min post-heel stick cortisol levels than neonates whose mothers did not experience prenatal complications. If one were only to test differences between groups in resting cortisol, the neonates of mothers who experienced prenatal complications may appear to have a dampened system. Yet the other two data points at 20 and 45 min post-stimulus suggest that these neonates may actually be hyper-reactive, at least in response to a pain stimulus. Since our previous work revealed that the heel stick in particular may be more useful as a paradigm when examining a lower than average response and the NBAS a more useful paradigm for examining a higher than average response, we turned to an examination of whether significant associations would be revealed between the poverty correlates and more extreme stress responses. Interestingly, maternal report of negative life events during pregnancy was associated with atypical behavioral responding to both stimuli, even when an atypical response was operationalized quite differently. By defining atypical responding in the context of the stimulus, significant associations between a specific prenatal stressor and individual differences in patterns of responding to two different types of stimuli were revealed. Although correlates of hypo- and hyper-behavioral responsiveness were revealed in this set of analyses, there were no associations with hypo- and hyper cortisol response. In fact, with the exception of a clear difference in cortisol response to the heel stick for neonates whose mothers did and did not experience prenatal complications, poverty correlates were only associated with pre-stressor cortisol levels but not reactivity. Importantly, much of the literature on prenatal stress and the effect on the stress response in animals indicates that alterations in cortisol response to stress, such as novelty, is not revealed until later in development (Huizink, Mulder, & Buitellar, 2004). Our findings for some association

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between prenatal stress and pre-stressor cortisol levels and much more consistent findings of associations between prenatal stress and behavioral differences suggest that perhaps other systems, such as neurotransmitter systems, may play an important role in the developmental trajectory from prenatal stress to altered stress reactivity in the offspring. Prenatal stress also is associated with the dopaminergic, serotonergic and noradrenergic systems, which are important to behavioral and emotional functioning and the regulation of the HPA axis. It is possible that the observed behavioral differences in the neonates in the present study are more closely associated with alterations in these systems, which negatively impact on the HPA system as the newborn matures. Returning to the overall aims of this study, our results suggest that significant associations between stressors that are more common to low-income environments and offspring response to stress can be observed as early as the first days of life. In addition, the more robust findings were for maternal report of negative life events during pregnancy and obstetrical complications. It makes sense that these two domains of poverty correlates would emerge as those explaining the most variance in stress reactivity in human neonates given the strong empirical support in the animal literature. The fact that in the present study poverty correlates were associated with an increased and a dampened stress response also is consistent with the literature later in development in which problematic behavioral and emotional functioning is associated with lower cortisol levels and reactivity as in the case of conduct disorder (McBurnett, Lahey, Rathouz, & Loeber, 2000) and higher cortisol reactivity as in the case of anxiety (Granger, Weisz, & Kauneckis, 1994). Do the data from the present study support the hypothesis that prenatal stress affects the programming of the stress response system in the human? The fact that prenatal stress was associated with altered behavioral and cortisol response to stimuli in the first days of life, when the impact of the post-natal environment was by definition limited, supports the hypothesis that the observed pattern of responding was a reflection of a adaptation of the stress response system to the uterine environment. Whether the adaptation to a stressful uterine environment sets up the infant for poor developmental outcomes, however, is not at all clear. Much of the literature on prenatal stress leading to programming of the HPA system has been focused on the detrimental effects of exposure of the fetal brain to high levels of gluccorticoids that originate in the maternal compartment (Owen, Andrews, & Matthews, 2005; Welberg & Seckl, 2001). The majority of the animal literature supports such a conceptualization, but the data on humans is limited and, as discussed earlier, less consistent (Mulder et al., 2002). Part of the complexity in the human condition is the extended period of brain development post-natally. In addition, the types of stressors that are most likely relevant for mental health in humans are chronic stressors. In some cases, the organism may adapt to a chronic stressor such as pre-pregnancy anxiety, by being in a heightened state of alert. Adapting to chronic difficult life circumstances may result in dampening of the stress response system. In the context of poverty, one would want to consider the alternative view that some forms of prenatal stress may facilitate the development of a stress response system that will allow for optimal functioning in an environment marked by high levels of chronic stress. The genetic context in which stress is experienced will also be relevant for understanding how the organism adapts. The most useful models, therefore, would include modeling the genetic influence on the fetus, the influence of the gene-environment interaction on the fetus, and whether those influences program the HPA axis of the offspring in a way that enhances or impedes adaptation to the post-natal environment. In the context of such complexity, we recognize that it is a far leap from the results of the present study to the hypothesis that the patterns we observed in the present study are prospectively associated with different types of behavioral and emotional functioning in childhood. Given the small sample size and small effect sizes, it is not clear that what we observed in the human neonate represents reliable individual differences; the possibility of chance findings on the one hand and lack of statistical power to detect true individual differences on the other hand cannot be ruled out. Nor is it clear that the patterns of stress reactivity that we are conceptualizing as hypo- or hyper- are valid operational definitions of atypical stress response. The findings require replication, ideally in a larger and diverse sample, and alternative operational definitions of atypical stress reactivity need to be tested. Even in the event that our observations do reflect reliable individual differences that capture atypical patterns of stress regulation, we recognize that the postnatal environment will have significant effect on the development of the stress response system. In fact, an important long term aim of this program of research is identifying aspects of the post-natal environment that are associated with increasing the capacity of the infant’s ability to regulate stress reactivity. Although factors such as negative life events are not easily modified, the individual’s emotional and behavioral response to such life events is modifiable. Thus, it is possible that assessing negative life events during pregnancy and providing interventions designed to lessen the emotional and physical impact of those events may prove to be one method for reducing the risk for behavioral and emotional problems in children growing up in poverty.

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Acknowledgements This study was supported by grant K01 MH-01484 from the National Institute of Mental Health to Dr. Keenan, the Walden and Jean Young Shaw Foundation, and grant M01 RR-00055 from the National Institutes of Health to the Clinical Research Center at the University of Chicago. The authors would also like to acknowledge the intellectual contribution of the members of a Translational Science grant (R21 MH-068449) from the National Institute of Mental Health to Dr. Delia Vazquez The authors thank the mothers and babies who participated in the Chicago Baby Project. We would like to dedicate this paper to our colleague and friend Dr. Marguerite Herschel, without whom the Chicago Baby Project could not have been successful.

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