Rossner et al. immune–coagulation interactions. Activated immune cells express various molecules that are important in the formation of a dysfunc- tional placenta, resulting in excessive tropho- blast apoptosis, shallow trophoblast invasion, and impaired spiral artery remodeling (Li and Huang 2009). These processes cause adverse pregnancy outcomes. The aim of our study was to investigate factors associated with LBW and IUGR risks in a group of 891 newborns born in two dis- tricts of the Czech Republic with different levels of air pollution. The analyzed factors included the levels of air pollutants [benzo[a] pyrene (B[a]P), PM2.5] during pregnancy, 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-ox- odG) levels in placental DNA, and a panel of 576 single nucleotide polymorphisms (SNPs) in 97 genes related to DNA repair, oxidative stress, xenobiotic metabolism, and immune functions. We hypothesized that air pollution induces oxidative DNA damage, which, in turn, increases the risk of adverse pregnancy outcomes. These effects may be modulated by SNPs in selected genes. Our principal aim was to determine whether oxidative stress was a major factor affecting LBW and IUGR in our cohort. Materials and Methods Subjects and samples. For our study, 1,200 subjects were randomly selected from a large case–control study conducted between 1994 and 1999 in two districts in the Czech Republic (Teplice and Prachatice) that have different levels of air pollution (Dejmek et al. 1999, 2000). The Teplice district is located in Northern Bohemia, and at the time of the study it was one of the most polluted regions of the Czech Republic. Air pollution arose mostly from chemical industry and brown coal power plants. The District of Prachatice is a rural region in Southern Bohemia with little industry and low levels of air pollu- tion [Supplemental Material, Tables 2 and 3 (doi:10.1289/ehp.1002470)]. The aim of the original study was to evaluate the effect of ambient PAHs and fine particles on preg- nancy outcome. The criteria for the selection of samples for this study were that the pro- portion of both IUGR- and LBW-affected newborns would be 10–15%. After removing the samples for which all relevant information was not available, the final cohort consisted of 891 subjects, among them 15.8% newborns with IUGR and 14.5% newborns with LBW. All mothers included in the study signed informed consent forms and could cancel their participation at any time, according to the Helsinki II declaration. The ethical commit- tee of the Institute of Experimental Medicine approved the study. The subjects analyzed in our study were single births that occurred in each district between April 1994 and March 266 1999. Preterm births (< 37 weeks gestation) were not included. The subjects were of two ethnic origins: European (749 newborns) or Romani (142 newborns). Detailed health, per- sonal, and lifestyle information was obtained from self-administered maternal question- naires and from medical records. The Romani ethnicity was determined according to the answer in the maternal questionnaires. An IUGR birth was defined as one in which the newborn had a birth weight below the 10th percentile, by sex and gestational age, for live births in the Czech Republic (Dejmek et al. 1999). The gestational age was estimated by gynecologists using each woman’s information on her last menstrual period, prenatal visits, and ultrasound examinations (Dejmek et al. 1999). LBW newborns had a birth weight < 2,500 g. Placentas were collected at the time of birth. The villus parenchyma sections were obtained by dissecting a 1.5-cm cubic-shaped segment (approximately 5 cm away from the site of cord insertion) and then splitting it into three equal parts: maternal (including thin basal plate), middle, and fetal (including the chorionic plate). The middle sections (one sample per placenta) were frozen and stored at –80°C until further processing. Air pollution monitoring. Concentrations of ambient air pollutants in both districts were measured using a Versatile Air Pollution Sampler (VAPS) (Pinto et al. 1998). The VAPS continuously monitored the levels of c-PAHs and PM2.5. PAHs were extracted from filters, and a quantitative chemical analysis of c-PAHs, including benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, B[a]P, dibenzo[a,h]anthracene, benzo[g,h,i] perylene, and indeno[1,2,3-cd]pyrene was per- formed by high performance liquid chroma- tography with fluorescence detection according to the U.S. Environmental Protection Agency method (U.S. Environmental Protection Agency 1999). DNA isolation and analysis of 8-oxodG adducts. DNA was isolated from the placenta essentially as described previously (Rossner et al. 2009). Vacuum-dried DNA pellets were stored at –80°C until further processing. Oxidative DNA damage, measured as levels of 8-oxodG in placental DNA, was analyzed using competitive ELISA with the primary antibody N45.1 (concentration 0.2 µg/mL; JaICA, Haruoka, Japan) as described (Rossner et al. 2009). Each sample was analyzed in triplicate. 8-OxodG levels were expressed as the number of 8-oxodG molecules per 105 guanosine molecules (8-oxodG/105 dG). Analysis of genetic polymorphisms. DNA samples for the analysis of genetic polymorphisms were dissolved in TE buf- fer (10 mM Tris base, 0.1 mM EDTA, pH 8.0) and diluted to a final concentration of 50 ng/µL. The samples were stored at –80°C until analysis. Genetic polymorphisms were assessed in 97 genes. The presence/absence of the glutathi- one S-transferase M1 (GSTM1) and GSTT1 genes was assessed by the TaqMan Real-Time PCR Assay [TaqMan Gene Copy Number Assays (PN4331182); Applied Biosystems, Carlsbad, CA, USA]. Although the method allows for the analysis of the exact number of the respective genes in the sample, for the purposes of our study we split our samples into two groups: GSTM1/GSTT1–null and GSTM1/GSTT1–positive samples (this group contained all samples having at least one copy of the gene). The assay was performed on a 7900HT system (Applied Biosystems) accord- ing to the manufacturer’s recommendation. SNPs in the remaining 95 genes were ana- lyzed by the GoldenGate genotyping assay developed by Illumina (San Diego, CA, USA) on custom-made 96-sample panels. SNPs that could be potentially associated with adverse pregnancy outcomes, oxidative stress, or the response of the organism to air pollutants were selected to be analyzed. They included genes related to the protection of the organ- ism against oxidative damage, genes respon- sible for the metabolism of xenobiotics, DNA repair genes, and genes affecting immune and inflammatory responses. SNPs in these genes were selected from the SNP500Cancer Database (http://snp500cancer.nci.nih.gov/). The criterion for the selection of an SNP was that the frequency of the minor allele should not be < 5%. We selected a total of 768 SNPs for a custom-made panel. We per- formed the GoldenGate genotyping assay on a BeadStation 500GX system (Illumina) according to the manufacturer’s instructions and analyzed the results using the BeadStudio software. We first excluded samples and SNPs that were not successfully analyzed based on the following parameters: p10 GC, all sam- ples with a score < 0.40 were removed; Call Frequency, all SNPs with a score < 0.60 were excluded from further analysis. The SNPs and samples that remained were checked manu- ally for proper clustering, and the final results were exported to an Excel file. After analyzing the results, we removed 138 DNA samples for which we were not able to determine the genetic polymorphism and 40 SNPs that did not cluster properly. Statistical analysis. Although we started our study with 1,200 samples, statistical analysis was performed on 891 samples for which all relevant data were available. We first compared 8-oxodG levels between districts for both ethnicities and for pregnancy outcomes using the Mann–Whitney U test. We checked the distribution of alleles for individual SNPs for Hardy–Weinberg equilibrium using a chi-square test and removed an additional 153 SNPs that were not in equilibrium. The ovlume 119 | number 2 | February 2011 • Environmental Health Perspectives