Study population

Data come from a population-based birth cohort established in the city of Sabadell (Catalonia, Spain) as part of the Infancia y Medio Ambiente (INMA) Environment and Childhood Project. Between July 2004 and July 2006, 657 pregnant women who visited the public primary care center of Sabadell for an ultrasound in the first trimester of pregnancy were recruited. A total of 99.5% of Spaniards have public health insurance, and an estimated 70-90% of women use public health services during pregnancy.

The participation rate was 60% (657/1097 eligible women). The educational achievement of women who refused to participate was lower than that of participants (58% had secondary education or less, compared with 28% in participants), although no differences in age were found. Among the participants, 504 mothers who had CRP levels measured in serum during pregnancy and provided complete outcome data on their children up to their 14th month visit (76.7%) were included in the present analysis.

Information on education, social class (using the International Standard Classification of Occupations [ISCO-88] code), demographic factors, marital status, maternal disease and obstetric history, parity, alcohol consumption and smoking habits during pregnancy, and physical activity was obtained by questionnaires administered by trained interviewers at recruitment, during the third trimester and at delivery. Gestational age and anthropometric measures were collected from clinical records. Informed consent was obtained from all participants, and the study was approved by the Clinical Research Ethical Committee of the Municipal Institute of Health Care, Barcelona.

Offspring wheezing and LRTIs

Information about the presence of respiratory symptoms and physician-confirmed diagnosis of LRTIs was obtained from parents through questionnaires completed when the child was 6 and 14 months old. At 6 months parents were asked whether the infants had ever experienced “whistling or wheezing from the chest but not noisy breathing from the nose” since birth. Subsequently when children were 14 months old, parents were asked whether the infants had ever experienced “whistling or wheezing from the chest but not noisy breathing from the nose” since the last interview.

Children were classified as “never wheezing” (no episodes of wheezing during first 14 months of life), “infrequent wheezing” (any episode of wheeze either during the first 6 months or from 6 to 14 months) and “recurrent wheezing” (episodes of wheeze both at 6 and at 14 months of age). Occurrence of an LRTI was defined as a positive answer to the question, “Has a doctor ever told you that your son/daughter has had a chest infection?” A second question referred specifically to a doctor diagnosis of either bronchitis, bronchiolitis, or pneumonia. Agreement between the 2 questions was almost perfect.

Similarly, children were classified as “never LTRI” (never diagnosed with having a chest infection), “infrequent LRTI” (diagnosed by a doctor with chest infections either during the first 6 months or from 6 to 14 months) and “recurrent LRTI” (diagnosed by a doctor with a chest infection both at 6 and at 14 months of age).

CRP level measurement and CRP genotyping

Maternal blood was collected at recruitment (mean ± SD, 13.4 ± 1.7 weeks of gestation). Serum CRP values were determined by turbidimetric assay using a Hitachi modular analyzer system (Roche Modular DPP, Hitachi Ltd, Tokyo, Japan) at the Laboratori de Referencia de Catalunya. The minimum detectable concentration of CRP was 0.2 mg/dL. For participants who had a CRP value below the detection limit (15.9%), we imputed a value of half of the detection limit.

SNP rs1205 (2042C>T) was selected based on its known association with CRP plasma levels. CRP genotyping deoxyribonuclease (DNA) extraction from maternal total blood was performed at the Spanish National Genotyping Centre using the Chemagic Magnetic Separator technology (Chemagen). DNA samples were quantified using PicoGreen double-stranded DNA fluorescent detection (Molecular Probes, Eugene, OR) and normalized to the same concentration. The rs1205 variant was genotyped by TaqMan allelic discrimination (C_7479334-10; Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. Replicate quality and positive controls were included and genotyped with 100% concordance. The rs1205 genotypes were in Hardy-Weinberg equilibrium both in the total analyzed cohort and among 412 mothers of European white descendent.

Statistical analysis

Accepting an alpha risk of 0.05 in a 2-sided test with 307 subjects in the control group (never diagnosed of LRTI) and 61 children reporting recurrent diagnosis of LRTI, the statistical power was 0.82 to recognize as statistically significant the difference in proportions from 0.27 in the control group (proportion of children never diagnosed of LRTI in the highest tertile of CRP) to 0.47 (proportion of children diagnosed of recurrent LRTI in the highest tertile of CRP).

Statistical analyses were performed with the Stata 8.0 statistical software (StataCorp, College Station, TX). Because maternal CRP serum concentrations were not normally distributed, we used a natural logarithmic transformed variable and geometric mean with 95% confidence intervals are reported. In addition, maternal serum CRP concentrations were categorized into tertiles to assess dose-response relation with offspring outcomes studied. Multinomial logistic regression analysis was conducted to model the adjusted association between maternal CRP concentrations during pregnancy and the development of wheezing and LRTIs in offspring at early age.

All variables significantly related with wheezing or LRTIs in the bivariate models ( P < .2) were included in the multivariate model and retained only if they had an at least marginally significant association ( P < .1) or modified the coefficient of maternal CRP levels by at least 5%.

Final multivariate models were adjusted for sex, birthweight, prematurity, small for gestational age, maternal smoking during pregnancy (number of cigarettes per day), parity as an indicator of having elder siblings at home (0, 1, ≥1), maternal body mass index before pregnancy (kilograms per square meter) (<20, 20-24.99, 25-29.99, ≥30), maternal total immunoglobulin (Ig) E (log transformed), maternal alcohol consumption in pregnancy (no, yes), day care attendance (no, yes), breast-feeding duration (never, 0-16 weeks, 14-24 weeks, and more than 24 weeks), and maternal dichlorodiphenyldichloroethylene serum levels in pregnancy (log transformed).

Allowance for other possible confounding variables (eg, maternal age, maternal social class, maternal education level, and maternal physical activity) did not change effect estimates and were therefore not included in the regression model. Significance was accepted at P < .05.