Overall study procedures
The PRIDE Study is a mother-infant cohort based in Cincinnati, Ohio. The pilot wave of the study involved four home study visits; the first visit occurred during the second or third trimester of pregnancy, the second visit occurred at 3–5 week postnatal, the third visit at 24 months and the fourth visit at 36 months (Fig. 1). The purpose of the first study visit was to obtain informed consent, collect data on maternal stress, adversity, and social support during childhood and pregnancy, and collect a hair sample. During the second visit, we assessed infant neurobehavior and collected buccal cells from the infants for DNA methylation analysis. The third and fourth visit continued to collect maternal adversity and protective factors while collecting a buccal sample from children along with child behavior. A small monetary incentive was provided to participants at each study visit.
Population and recruitment
We enrolled 55 mother-infant dyads who were participating in Every Child Succeeds (ECS), a home visiting program which serves the Greater Cincinnati area, including Southwest Ohio and Northern Kentucky, by providing evidence-based services to first-time, at-risk mothers from pregnancy until the child is age 3 years. Enrollment for PRIDE was exclusively from Hamilton County, Ohio. Approximately 25% of eligible mothers in the region participate in the ECS program. Women who enroll prenatally receive weekly, bi-weekly, or monthly home visits depending on the gestational week. Postnatal visits occur with similar frequency and include regular developmental screening using the Ages and Stages Questionnaire (ASQ)-III beginning at age four months. The ASQ-III screens children through age five for developmental delays and disabilities.
ECS home visitors referred all eligible pregnant participants to our PRIDE study team, who contacted each woman to confirm eligibility and schedule the first study visit. In addition to participants in ECS, eligibility criteria for PRIDE included pregnancy prior to 36 weeks,18 years of age or older, and English speaking.
Maternal stress and adversity measures
Several measures of maternal adversity and stress were collected at each study visit. The Adverse Childhood Experiences Scale (ACE) (15), a 10-question self-report measure, captures abuse, neglect, and household dysfunction through age 18. The Edinburgh Postnatal Depression Scale (EPDS) (16) is a 10-item self-report measure of depressive symptoms and is validated for use prenatally.(17) It collects depressive symptoms that occurred over the past week based on a four-point scale indicating frequency and severity. The Brief Symptom Index-18 (BSI-18) is a brief measure used to screen for common psychiatric disorders including depression, anxiety, and somatization.(18) The Pregnancy Experience Scale (PES) Brief version measures pregnancy-specific contributors to psychological state using the top 10 items from the original scale with comparable validity and reliability.(19, 20) The Perceived Stress Scale (PSS), the most widely used instrument to measure perceived stress,(21) was designed for community samples and is easily interpreted. With the exception of the ACE scale and PES, adversity measures were collected again at visits two, three and four.
Neighborhood-level adversity was determined by linking birth address data with socioeconomic variables from the American Community Survey from the U.S. Census Bureau. Variables included the percent of households in the neighborhood with assisted income, percent with a high school education, the mean income, percent with no health insurance, the percent who experience poverty and the percent of vacant housing. In addition, we included a deprivation index which is based on a principal components’ analysis of the six measures.(22, 23) The deprivation index ranges from zero to one.
Biologic measure of stress
For measurement of cortisol accumulation, we collected hair from 30 women. Of the 25 women without a hair sample, a majority were willing but were wearing a wig or weave and therefore unable to provide natural hair. Hair was cut from the occipital vertex using a standard protocol. Our laboratory methods for measuring hair cortisol included duplicate analyses and rigorous quality control standards and are described in detail previously.(24) Briefly, hair is weighed on an analytical balance scale and washed with isopropanol to remove contamination on the external part of the hair. The isopropanol is then dried and the sample is ground to a fine powder. Cortisol is measured using a commercial enzyme immunoassay (Salimetrics) and converted to pg per mg of hair. To determine hair cortisol concentrations the assay readout is converted to pg cortisol per mg sample weight. An LOD taking into account sample weight was calculated for each individual hair sample that, when reconstituted and analyzed, yielded a cortisol value below the overall assay limit of detection (LOD). There were three samples below the weight adjusted LOD. Intra- and inter-assay coefficients of variation for this assay are both < 10%.
Maternal Social Support
The Interpersonal Support Evaluation List (ISEL) (25), a widely used measure of social support, measures 40 items regarding the availability of tangible and emotional support. Scores on four subscales are derived: Appraisal, Belonging, Self-Esteem, and Tangible. The Appraisal scale measures whether individuals have the ability to talk to someone about problems. The Belonging scale measures whether there are people to do things with. The Self-Esteem scale measures whether one has a positive comparison of their self to others and the Tangible scale measures whether there is material aid available. The IESL was collected at each visit.
Infant and child development
Infant neurobehavior was measured using the NICU Network Neurobehavioral Scale (NNNS) at the second study visit (3–5 weeks postnatal).(26) The NNNS measures three components of neurobehavior including: 1) CNS integrity and neurological functions, such as active and passive tone and primitive reflexes; 2) infant behavior to assess neurologic states as well as sensory and interactive responses; 3) signs of stress which can manifest as overt or subtle signals during the course of the examination. The exam was developed based on previous validated infant examinations, in particular the Neonatal Behavioral Assessment Scale (NBAS);(27) however, a major difference is the NNNS incorporates a standardized administrative format developed to minimize the effect of the examiner on the assessment.(26) While the exam was developed for high-risk infants, it is appropriate for all infants regardless of risk for neurobehavioral deficits.(26) There are 114 individual test items. The approximately 30 minute exams were completed by a trained examiner who is also a certified trainer on the NNNS (KY).
Summary scores (domains) were developed using a combined conceptual and statistical approach to aggregate scores from the individual NNNS items to describe 13 dimensions of neurobehavior including: habituation, attention, arousal, self-regulation, special handling required to acquire orientation items, movement quality, excitability, lethargy, non-optimal reflexes, asymmetrical reflexes, hypertonicity, hypotonicity and signs of stress. For all subscales, higher scores reflect a greater tendency toward that dimension regardless of whether it is a positive or negative trait. In addition to evaluating individual dimensions of neurobehavior, we employed previously identified profiles of behavior developed within an independent Cincinnati cohort.(28) Latent profile analyses classified the 13 dimensions to identify infants with profiles described as ‘high-arousal’, ‘hypotonic’ and ‘social’.(29) Our primary outcome variable was having a ‘high-arousal’ infant.
At visits three and four (24 and 36 months), a Child Behavior Checklist (CBCL) was collected. The Child Behavior Checklist (CBCL/1½-5) is a parent-report questionnaire that will be used to measure behavior and emotional functioning including externalizing and internalizing behaviors(30).
Statistical analyses
All variables were examined for errors, inconsistencies, incomplete information and distributional properties. Psychometric assessments were scored based on guidance from the test publishers. Demographics were summarized using means (standard deviations) for continuous variables and number (percent) for categorical variables. A cortisol measure was available for 29 of the 30 hair samples, and one sample outlier was excluded, resulting in 28 samples available for analyses.
To control for potential confounding variables, we employed logistic regression analyses to determine the odds of having a high-arousal infant. Potential covariates included maternal age (years), race (black versus white/Asian/multi-race), and maternal ACEs (< 2, ≥ 2). Linear regression identified associations between maternal adversity and internalizing and externalizing symptoms, as well as the CBCL total score. Interactions between race and maternal ACEs were examined using multiplicative interaction terms and stratification. This research was approved by the Institutional Review Board of Cincinnati Children’s Hospital Medical Center.