The Pregnancy, Infection, and Nutrition Study (PIN) addressed prenatal influences on pregnancy outcomes. Women who sought prenatal care before gestational week 20 at the University of North Carolina Hospitals during 2001–2004 were recruited. Exclusion criteria were not having access to a telephone (<1% ineligible), not speaking English (11% ineligible), being less than 16 years old (<1% ineligible), multiple gestations (4% ineligible), or the woman's healthcare provider did not feel it was in her best interest to participate (<2% ineligible). Before 20 weeks' gestation, women provided saliva and blood at a recruitment visit or, if they agreed to a fasting blood draw, at a research visit to the General Clinical Research Center (visit 1, 14–18 weeks). A second research visit occurred during weeks 24–29 (visit 2). Gestational age was determined via ultrasound when performed before week 22 and otherwise based on reported last menstrual period.
Lazarus' stress-coping model posits that stress is an excess of environmental demand beyond a person's ability to meet it (stressor). Stress is most commonly conceived as a person-environment transaction, the first part of which involves a threat, the second part involves the appraisal of the threat, and the third is a person's response to the threat.
21 A perceived threat is an excess of environmental demand beyond the individual's capacity to meet it, with important perceived consequences.
22 The stressors a person experiences interact with his or her psychological state, personal disposition, and social support, on the background of their physiologic substrate and the social and environmental context.
23 Most psychological measurements involve one of the subparts of this transaction: stressor, appraisal, or response. The scales were chosen to provide information on external stressors (the Sarason's Life Experiences Survey), perceived stress, enhancers of response to external stressors (Trait Anxiety Inventory, and pregnancy-specific anxiety), and buffers of response to external stressors (social support and John Henryism coping).
Psychosocial stress was measured in two telephone interviews and by two self-administered questionnaires (). Details are available online (
www.cpc.unc.edu/projects/pin/docs_3/index.html). Most of the measures were widely used, validated instruments. Except where noted, psychometric data are from validation studies that were conducted on non-pregnant adult populations. A subset of 39 items from Sarason's Life Experiences Survey provided a composite score of life events and the perceived impact of those events.
24 The first interview asked about events since getting pregnant, the second about events since the first interview. For this analysis, two scales were used: the sum of the absolute value of perceived negative impacts and the sum of the total absolute values of the negative and positive perceived impacts. The scale has moderate test-retest reliability for these measures (reliability coefficients of 0.56–0.88) and correlates with depression, personal maladjustment, and academic achievement.
24 The Spielberger State-Trait Anxiety Inventory (STAI)
25 contains two 20-item scales to assess anxiety. The state anxiety scale measures current feelings of anxiety or how the respondent feels “right now,” while trait anxiety is measured by questions that ask how the respondent “generally feels.” Two measures of pregnancy-specific anxiety were used. Rini et al.'s scale focuses on worry about the woman's and her baby's health, labor and delivery, and caring for the baby;
26 six items specific to pregnancy health were taken from the Prenatal Social Environment Inventory of Orr et al.,
27 and four items were added. The “John Henryism” Active Coping Scale includes 12 items that measure coping, overcoming obstacles, and making one's own way in the world.
28 The Medical Outcomes Study (MOS) Social Support Survey assesses perceived social support, including questions about the availability of emotional, informational, tangible, and affectionate support.
29 Item-scale correlations are greater than 0.7, and internal consistency is high for all categories of measures, exceeding 0.50. The scale correlates with measures of loneliness, emotional ties, and family functioning.
29 Women were asked to assess this support since they became pregnant. The Cohen Perceived Stress Scale
30 is designed to measure “the degree to which situations in one's life are appraised as stressful.” The 14-item scale was used at the first interview and the 10-item at the second. Reliability is between 0.84 for short-term and 0.55 for longer-term test-retest.
30 | Table 1.Measurements Taken in the Pregnancy, Infection, and Nutrition Study (PIN) Protocol |
At each clinic visit, a saliva sample was taken to measure cortisol and a blood sample to measure CRH. Samples were taken between 7:30 a.m. and 7:00 p.m. For the purposes of this analysis, we limited the cortisol sample to those taken between 8 a.m. and 10 a.m. For the saliva sample, each study participant was asked to rinse her mouth thoroughly with water 15 minutes before collection. The saliva was collected in a plastic tube and stored at −20°C as soon as possible. Blood was collected in a chilled syringe, transferred to a tube containing EDTA (1

mg/ml of blood) and Aprotinin (500

KIU/ml of blood), and centrifuged at 0°C. The plasma was decanted from the tube, aliquoted into four cryogenic storage tubes, and stored at −70°C until extraction.
Saliva samples were assayed for salivary cortisol using a high-sensitive enzyme immunoassay (Salimetrics, PA). The test uses 25 μl of saliva and has a range of sensitivity of 0.007–1.8

μg/dL; average intra- and inter-assay coefficients of variation were 4.13% and 8.89%, respectively. Eleven percent of the samples were analyzed in duplicate, and the mean of the two values was used.
Fifty μl-plasma samples were assayed for CRH using a competitive enzyme immunoassay. The assay had a minimum detection limit of 0.08

ng/mL and a range of 0–25

ng/mL. Average intra- and inter-assay coefficients of variation were <5% and <14%, respectively. Samples were assayed by Salimetrics, LLC (State College, PA). Seven percent of the samples were analyzed in duplicate, and the mean of the two values was used. Cortisol and CRH results, which were right-skewed, were log-transformed.
All protocols were approved by the UNC School of Medicine Institutional Review Board.
Statistical analysis
As shown in , the amount of missing data on each variable ranged from 3% (Life Experiences Scale 1) to 22% (State Anxiety 2). Complete case analysis was used for each set of calculations. Spearman correlation coefficients were examined among continuous psychosocial measures and stress hormones. The association between psychosocial measures and other factors known to be associated with PTB
31 was then examined using
t-tests for dichotomous variables, analysis of variance (ANOVA) for categorical variables, and correlations for continuous factors. This included demographic variables (age, income as a percent of the poverty line given reported household size, education, race, parity, and marital status), lifestyle variables (smoking, pre-pregnancy body mass index [BMI]), as well as pregnancy complications (pre-eclampsia, pregnancy-induced hypertension, anemia, history of PTB or miscarriage). Finally, we predicted biomarkers using linear regression based on these variables. Models included a quadratic term to examine possible non-linear relationships. Variables were modeled in the forms shown in and ; psychosocial variables were modeled as continuous variables. Because cortisol and CRH increase with gestational age,
32,33 we also examined these models adjusted for gestational age.
| Table 2.Characteristics of Women Participating in the Pregnancy, Infection, and Nutrition Study, 2000–2004 |
| Table 3.Spearman Correlations (r) among Psychosocial Measures and Biomarkers of Stress in North Carolina Pregnant Women, 2000–2004 |
We then examined whether associations between psychosocial measures and hormones changed when these other predictors of PTB were included in the models. Partial correlations were examined, and individual linear models were created, predicting biomarkers by incorporating these variables as well as psychosocial stress variables. We also examined these data using hierarchical linear models to control for the correlation within women. We examined whether the degree of correlation was affected by the time between questionnaire and the blood draw, by examining correlation within groups stratified by time between measurements. We also examined whether John Henryism, social support, and pregnancy-specific anxiety were effect modifiers of the psychosocial stress-stress hormone relationship by including a product term in the models.