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ORIGINAL RESEARCH |
From the Departments of Epidemiology, and Pediatrics and Human Development, Michigan State University, East Lansing, Michigan; and the Department of Obstetrics and Gynecology, University of Texas, San Antonio, Texas.
Address reprint requests to: Claudia Holzman, DVM, PhD Department of Epidemiology Michigan State University 4660 South Hagadorn, Suite 600 East Lansing, MI 48823 E-mail: holzman{at}msu.edu
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Abstract |
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Methods: This nested case-control study included two case groups (97 women who delivered before 35 weeks’ gestation, 144 who delivered at 35–36 weeks’ gestation), and a control group (244 women who delivered at or after 37 weeks’ gestation) frequency matched by ethnicity (black, white) and by alpha-fetoprotein levels (normal, unexplained high). Corticotropin-releasing hormone was evaluated in stored maternal sera collected at 15–19 weeks’ gestation from cases and controls.
Results: Delivery before 35 weeks’ gestation was associated positively with a second trimester, ethnic-specific CRH above 1.5 multiples of the median in white women [odds ratio (OR) 2.3, 95% confidence interval (CI) 1.1, 5.1] and black women (OR 5.0, 95% CI 1.8, 13.3). Sensitivity was 29% in whites and 41% in blacks; specificity was 84% in whites and 80% in blacks. We estimated the positive and negative predictive values to be 6% and 97%, respectively, in white women, and 16% and 93%, respectively, in black women. It was also noted that, within case and control groups, black women had consistently lower CRH levels than white women.
Conclusion: Factors that lead to a premature increase in placental CRH production and are associated with an increased risk of preterm birth are evident as early as 15–19 weeks’ pregnancy. When considering potential links between stressors, placental changes, CRH levels, and preterm birth, it might be important to stratify or adjust for ethnicity.
The excessively high rate of preterm birth among disadvantaged women suggests that important etiologic clues might be found in psychosocial and biologic factors. One biologic factor, corticotropin-releasing hormone (CRH), has stimulated interest among investigators from diverse backgrounds who have been studying the hormone as a mediator of parturition and human stress response. Levels of CRH in maternal blood increase exponentially from the second through the third trimesters of a normal pregnancy1,2 because of CRH production in the decidua, fetal membranes, and placenta.3–6 Corticotropin-releasing hormone’s precipitous rise in maternal blood during the month before delivery1,2 has led investigators to consider the effect of CRH as a proximate predictor, and possible mediator, of term and preterm parturition.7–9 Evidence also suggests that maternal CRH levels might serve as a more distal predictor (mediator or marker) that can identify women in the second trimester who are at increased risk of preterm delivery.1,10,11 The value of such a distal predictor would be its application to clinical care, and as an aid for uncovering antecedent factors related to preterm birth.
This study was designed to test the hypothesis that women who deliver preterm are more likely to have elevated maternal serum CRH levels in the 15th–19th weeks of pregnancy. We selected the 15th–19th weeks because they predate parturition by more than 4 weeks in most preterm births, and therefore are less likely to include labor-associated rises in CRH (a proximate predictor). The 15–19-week serum samples are collected routinely as part of prenatal screening, making this sample convenient for CRH assessment.
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Materials and Methods |
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Pregnancy outcomes were assessed by linking cohort women to their infants’ birth certificates. Women were excluded before or after the linkage for ethnicity other than black or white; diabetes before pregnancy; multiple fetuses; fetal structural defects or chromosomal abnormalities; or infants born before the 37th week of pregnancy whose birth weights were above the 99th percentile for gestational age.12 Gestational age was determined antenatally, using the first day of last menstrual period (LMP) recorded on the prenatal screening form, or in the absence of an LMP, the gestational age estimate from an early ultrasound. When both LMP and early ultrasound age were recorded, and they differed by more than 2 weeks, the ultrasound gestational age was given precedence. Overall, 49% of white women and 56% of black women were listed as having ultrasounds at or before 19 weeks’ gestation.
Cohort women who delivered preterm were stratified by gestational week at delivery (before week 35, 35–36), and selected as cases if adequate stored serum remained from their prenatal screens. Cases that met this criterion included 61% (N = 97) of cohort women who delivered under 35 weeks’ gestation, and 55% (N = 144) of cohort women who delivered at 35–36 weeks’ gestation, a total of 241 cases. Controls (N = 244) were a random sample of cohort women with stored serum who delivered at term (at or after 37 weeks’ gestation). They were frequency matched to cases by ethnicity, MSAFP level (normal, unexplained high), and date of MSAFP screen. Stored serum samples collected at 15–19 weeks’ gestation from cases and controls were evaluated for CRH levels by laboratory technicians who were masked to case-control status. Study sera had been stored at -20C for 6–10 years, and many samples were thawed and refrozen (74% once, 26% twice) when used to assess other biomarkers.
Before measuring CRH levels in stored (-20C) sera of cases and controls, studies were done to determine the stability of CRH in human whole blood processed as serum and ethylenediaminetetra-acetic acid (EDTA)-plasma under varied conditions: held at room temperature or 4C for 1, 2, 4, 6, and 24 hours; collected in siliconized versus nonsiliconized tubes; stored at -20C for 1 day, 1 week, or 1 month; repeat freeze-thaw cycles; and long-term storage for at least 2 years. The methanol extraction method also was compared with the classical Sep-Pak C18 chromatography extraction method13 by testing the same samples using both methods.
Preliminary tests indicated that CRH was stable in blood subsequently processed as EDTA plasma or serum over 24 hours and held at room temperature or 4C. The recovery of CRH when collected and processed in nonsiliconized glass was similar to using siliconized glass. Samples stored frozen (-20C) for 1 day, 1 week, 1 month, or 6 months had no loss of CRH. Samples assayed for CRH within 6 months of collection using the classical extraction procedure had similar CRH concentrations after repeated freeze-thaw cycles and when reassayed more than 2 years later using methanol extraction.
In the assays of case and control sera, methanol (3.5 mL) was used to extract CRH from serum (0.5 mL). The precipitate was separated by centrifugation and the methanol-extracted CRH was dried. The residue was suspended in assay diluent (250 µL) before the assay. Extraction efficiency was 87.5% ± 2.0%. Corticotropin-releasing hormone was measured using a specific and sensitive radioimmunoassay procedure similar to that described by Siler-Khodr et al14 for GnRH, except CRH in these samples was first extracted free of the CRH binding protein as described. Aliquots of resuspended extract were assayed in duplicate. Antiserum to CRH (TS-6) was a gift from Dr. Chrousos (NIH, Bethesda, MD) and used at a final dilution of 1/60,000. Standard CRH was purchased from Sigma Chemical Co. (St. Louis, MO) and corrected for water content. Antiserum (100 µL) and sample or standard (100 µL) were preincubated for 2 days at 4C, then 125I-CRH (10 pg/mL) was added. Tyr-CRH (Sigma Chemical Co.) was radioiodinated by the method of Hunter and Greenwood.15 After addition of label, incubation was continued for 3 days at 4C. Separation of bound and free CRH was done with antirabbit gamma globulin conjugated to magnetic beads (0.5 mL, Perceptive Diagnostics, Cambridge MA). Assay sensitivity was 5 pg/tube or approximately 30 pg/mL when corrected for extraction loss. Intra-assay and interassay coefficients of variation were 3% and 10%, respectively.
Distribution of CRH levels was skewed, so we present the median as the measure of central tendency, and the first and third quartiles as summaries of the degree of variability. Statistical analyses were conducted using Stata Release 6 (Stata Corp., College Station, TX). Median CRH levels in cases and controls, and in whites and blacks, were compared by median regression (least absolute value regression) using procedure bsqreg.16 Confidence intervals (CIs) and sample sizes were calculated using bootstrap standard errors with 1000 replications.17 Median comparisons were adjusted for gestational week at prenatal screen. To compute CRH multiples of the median (MoM), a median regression line was fitted to the plot of median CRH levels by gestational week at prenatal screen using data from controls. The coefficient of the regression slope was 0.3 pg/mL per week for blacks, and 2.3 pg/mL per week for whites. Gestational week-specific CRH medians were generated from the regression equation, and used to calculate CRH MoMs for cases and controls. Odds ratios (OR) and their 95% CIs were calculated to assess the association between CRH MoMs and preterm delivery.
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Results |
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. Comparisons within the ethnic-MSAFP sampling strata showed that age, education level, Medicaid insurance status, parity, and gravidity were not significantly different between cases and controls, with two exceptions. White women with normal MSAFP who delivered preterm were more likely to be primiparous, and black women with high MSAFP who delivered preterm were less likely to be covered by Medicaid insurance.
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). By contrast, median CRH levels in cases delivered before 35 weeks’ gestation were higher than controls within all ethnic and MSAFP strata, but the difference was not statistically significant in white women. Cases and controls were frequency matched for MSAFP status, and the relationship between maternal CRH levels and risk of preterm birth was similar among high and normal MSAFP strata, so MSAFP strata were combined to improve statistical power. In the combined strata, the median CRH level in white women who delivered before 35 weeks’ gestation was 13 pg/mL higher than white controls (89.7 versus 77.1 pg/mL, P = .10). In black women who delivered before 35 weeks’ gestation, the CRH median was 23 pg/mL higher than that of black controls (73.6 versus 50.7 pg/mL, P < .001), but there remained considerable overlap in the CRH distribution of cases and controls (Figure 1).
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), although a statistically significant difference in medians was noted only between black women who delivered at 29–34 weeks’ gestation (79.3 pg/mL) and their term controls (50.7 pg/mL). The lack of statistical significance in the other preterm groups might be explained in part by the small samples that resulted from partitioning the earliest deliveries. The study power to detect the observed 24-pg/mL difference in median CRH between the very preterm and term white women was 69%. The study power to detect the observed 16-pg/mL difference between very preterm and term black women was 28%.
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). The ORs for delivering at 35–36 weeks’ gestation were more modest (1.5–1.6) in white and black women with CRH levels above 1.5 MoM, and 95% CIs included 1.0. For these analyses, our sample size calculations showed that we had 80% power to detect ORs from 2.4 to 4.6. The narrow range of CRH levels at the 15th–19th weeks of pregnancy meant that few women had CRH levels that exceeded 2.0 MoM (Figure 1
), and a larger sample was needed to adequately assess cut-points above 1.5 MoM.
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Within control groups (high and normal MSAFP), the CRH medians of black women were significantly lower (P < .05) than those of white women (Figure 2). The same pattern was noted in case groups, although statistical power was limited, and ethnic differences were not always statistically significant. The ethnic differences in CRH medians within case and control groups persisted when the analysis, adjusted for gestational week at prenatal screen, included only women who had an early ultrasound, or separately considered women with Medicaid and non-Medicaid insurance.
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Discussion |
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Several theories have been proposed for the role of CRH in human parturition. Placental CRH might stimulate the fetal pituitary-adrenal axis directly,20,21 and fetal adrenal production of cortisol and dehydroepi-androsterone,22 or CRH might have an autocrine-paracrine effect by stimulating prostaglandin production in the decidua, fetal membranes, and placenta,23 and by binding to the myometrium24 and potentiating oxytocin.25 Little is known about the causes of elevated CRH in the second trimester and its association with preterm birth, although higher CRH levels have been noted in pregnancies complicated by preeclampsia26,27 and pregnancy-induced hypertension.28,29 In studies of women with preterm labor, Warren et al9 and Korebrits et al8 did not find an association between CRH levels and infection-related preterm delivery, whereas Petraglia et al30 detected higher CRH levels in preterm births complicated by microbial contamination of amniotic fluid. Based on the current belief that very preterm births are more likely to have an underlying infection etiology, we further subdivided preterm cases and analyzed CRH levels in women who delivered before 29 weeks’ gestation. In this crude test of association between second trimester CRH levels and infection-related preterm delivery, CRH levels of study women who delivered before 29 weeks’ gestation were higher than those who delivered at term, although the difference was not statistically significant, perhaps because of the limited number of very preterm deliveries.
Our finding of lower CRH levels in black women compared with white women, within case and control groups, was unexpected and needs to be explored in future studies. If ethnic differences in maternal CRH prevail, analyses that disregard this difference might result in reverse confounding, attenuating the true relationship between CRH levels and risk of preterm delivery. Few in vivo studies have been published on factors that affect maternal CRH levels, making it difficult to explain observed ethnic differences. Cortisol suppresses CRH in the hypothalamus, and some in vitro studies report that cortisol31,32 and norepinephrine33 stimulate CRH production in the placenta and fetal membranes. One current hypothesis that maternal stress increases placental CRH is counterintuitive to our observed ethnic differences in CRH. That hypothesis assumes maternal stressors raise cortisol levels, although that assumption might not be true in the case of chronic stress leading to periods of negative affect. A recent study of disadvantaged, pregnant adolescents (98% white women) reported lower CRH levels at 9–21 weeks’ gestation in relation to symptoms of depression.34
Our study had several potential limitations, one being the use of birth certificate data to determine pregnancy outcomes. Without more detailed information on women and their pregnancies, we could not elaborate on the circumstances that preceded premature deliveries. We also lacked data on factors such as placental features (eg, size, vascular supply, pathology) and hormone levels (eg, cortisol, catecholamines, progesterone) that might have explained variations in CRH levels between cases and controls, and between ethnic groups.
We were initially concerned that the serum samples would be another limitation in this study. Blood collected routinely for prenatal screening, subjected to variations in processing and temperature during handling and shipping, was not the ideal specimen for CRH assessment. However, our laboratory validation studies correctly predicted that CRH was recoverable in our stored serum samples, contrary to our concerns. Our results might accurately reflect the "real world" scenario of using prenatal screening samples as a source for CRH testing, although our samples were stored for long periods. It would be of interest to evaluate CRH levels in a large number of prenatal screening samples within 6 months of being stored at -70C, and compare week-specific medians at 15–19 weeks’ gestation with CRH medians generated from long-term stored samples.
According to our data, CRH above 1.5 MoM at 15–19 weeks’ gestation has a low positive predictive value (6% white women, 16% black women) for predicting delivery before 35 weeks’ gestation in an unselected population (eg, no prerequisite preterm labor). Despite high negative predictive values (93–97%), the probabilities of delivering at less than 35 weeks’ gestation given a negative test (3% for white women, 7% for black women) were similar to our population estimates of risk without the added CRH screen information, ie, 3.6% in white and 8.7% in black women. The more optimistic view of CRH screening in a recent study10 might be caused in part by their inclusion of CRH evaluated later in pregnancy, ie, 24–30 weeks’ gestation. That period does not coincide with most routine prenatal screening. Prediction of preterm birth later in pregnancy can blur the distinction between elevated CRH as an indicator of preterm-related pathology and elevated CRH as an epiphenomenon of the preparation for preterm parturition (consider the rise in CRH as early as 4 weeks before term deliveries). That distinction might be particularly important in unraveling the causes of preterm birth, and in determining the fruitfulness of interventions at the time of prediction.
In view of our results that showed a strong association between CRH evaluated at 15–19 weeks’ gestation and preterm delivery, we cannot rule out the possibility that early CRH screening might be clinically useful when applied to a unique group of high-risk women, or when coupled with other biomarkers. Our results indicate that second trimester CRH might be most helpful in etiologic research on preterm birth.
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Footnotes |
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Received October 24, 2000. Received in revised form December 7, 2000. Accepted December 12, 2000.
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