Approximately 4 million neonatal deaths occur each year globally, o\ue004 which more than one-quarter are caused by preterm birth (Lawn et al. 2005). Although 99% o\ue004 neo- natal deaths arise in low- or medium-income countries, preterm birth has also emerged as a public health priority in the United States and other industrialized nations. Te U.S. rates o\ue004 preterm birth have increased by > 30% since 1981 and by 18% since 1990, and as o\ue004 2004 accounted \ue004or 12.8% o\ue004 live births (Martin et al. 2008). Preterm birth is associated with more than one-third o\ue004 in\ue004ant deaths in the United States, making it the leading cause o\ue004 neona tal mortality (Mathews et al. 2004). In addition, many complications \ue004rom preterm birth develop into chronic health conditions such as blindness, dea\ue004ness, cerebral palsy, and lower IQ (Slattery and Morrison 2002).

Tere is growing concern \ue004or the potential role o\ue004 environmental pollutants in preterm birth, yet this area remains poorly understood (Institute o\ue004 Medicine 2006). Only a \ue004ew envi- ronmental agents have been investigated \ue004or associations with preterm birth in well-con- trolled epidemiologic studies, including the insecticide DD\ue003 (dichlorodiphenyltrichloro- ethane) (Longnecker et al. 2001), secondhand tobacco smoke (Jaakkola et al. 2001; Kharrazi et al. 2004), and lead (Andrews et al. 1994). \ue003here is \ue004urther evidence \ue004or the contribu- tion o\ue004 environmental exposures on preterm

birth through observations o\ue004 increased risk among individuals with polymorphisms in genes involved in xenobiotic metabolism (Nesin 2007).

Phthalates are a group o\ue004 chemicals used in a wide range o\ue004 industrial applications and consumer products, and this widespread use results in exposure among most o\ue004 the gen- eral population (Hauser and Cala\ue004at 2005; Silva et al. 2004). Sources o\ue004 exposure to low- molecular-weight phthalates [e.g., diethyl phthalate and di-n-butyl phthalate (DBP)] may include personal care products such as per\ue004umes, lotions, and cosmetics; through their use as solvents; or as coatings on timed- release pharmaceuticals (Hauser and Cala\ue004at 2005). Exposure to higher-molecular-weight phthalates such as di(2-ethylhexyl) phthalate (DEHP) and butylbenzyl phthalate (BBzP) may be related to their use as plasticizers in \ue004lexible vinyl plastic consumer products (e.g., construction materials, foor covering, and vinyl wall paper), \ue004ood packaging, and medical devices (Hauser and Cala\ue004at 2005). Exposure to DEHP was associated with decreased gestational age in studies conducted in Italy (Latini et al. 2003) and in New York City (Whyatt et al. 2008). Conversely, a di\ue004- \ue004erent study conducted in New York City reported a positive association between ges- tational age and urinary concentrations o\ue004 metabolites o\ue004 DEHP as well as the phthalate

metabolites mono-n-butyl phthalate (MBP) and monoethyl phthalate (MEP) (Wol\ue004\ue004 et al. 2008).

Although previous studies investigated the association between phthalate exposure and gestational age among primarily term births, the relationship with preterm births has not yet been addressed. In the present study we conducted a nested case\u2013control analysis o\ue004 the association between urinary phthalate metabolites and preterm birth in a Mexico City birth cohort.

within a Mexican birth cohort study in which women were recruited during prenatal visits at one o\ue004 \ue004our clinics o\ue004 the Mexican Institute o\ue004 Social Security (IMSS) in Mexico City between 2001 and 2003 (Ettinger et al. 2009). Te clin- ics serve a low- to moderate-income population. Women were eligible i\ue004 they had a con\ue002rmed pregnancy o\ue004 no more than 14 weeks\u2019 gesta- tion, did not present with a high-risk pregnancy (including daily consumption o\ue004 alcoholic bev- erages; addiction to illegal drugs; continuous use o\ue004 prescription drugs; or diagnosis o\ue004 mul- tiple pregnancy, preeclampsia, renal or heart disease, gestational diabetes, or seizures that require medical treatment), planned to reside in Mexico City \ue004or approximately 5 years, and were willing to participate in a \ue004ollow-up study protocol. Gestational length was estimated by maternal recall o\ue004 the date o\ue004 last menstrual period, because early ultrasound is not routinely per\ue004ormed at the IMSS clinics. In\ue004ormation on demographic, socioeconomic, and other \ue004actors

Address correspondence to J. Meeker, Department o\ue002 Environmental Health Sciences, University o\ue002 Michigan School o\ue002 Public Health, 6635 SPH \ue001ower, 109 S. Observatory St., Ann Arbor, MI 48109 USA. \ue001elephone: (734) 764-7184. Fax: (734) 936-7283. E-mail: meekerj@umich.edu

We thank M. Silva, E. Samandar, J. Preau, and \ue001. Jia [Centers \ue002or Disease Control and Prevention (CDC)] \ue002or technical assistance in measuring the concentrations o\ue002 phthalate metabolites.

\ue001his work was supported by R01 ES 007821, P42 ES 05947 Project 1, and a pilot project grant \ue002rom the Department o\ue002 Environmental Health Sciences, University o\ue002 Michigan School o\ue002 Public Health.

\ue001he \ue002indings and conclusions in this report are those o\ue002 the authors and do not necessarily represent the views o\ue002 the CDC.

of Statistics, Center for Evaluation Research and Surveys, National Institute of Public Health, Cuernavaca, Mexico;3Centers for Disease
and Control and Prevention, Atlanta, Georgia, USA;4Department of Environmental Health, Harvard School of Public Health, Boston,
Massachusetts, USA;5Ministry of Health, Mexico City, Mexico

U.S. \ue001emales, although in the present study mono-n-butyl phthalate (MBP) concentrations were higher and monobenzyl phthalate (MBzP) concentrations lower. In a crude comparison be\ue001ore cor- recting \ue001or urinary dilution, geometric mean urinary concentrations were higher \ue001or the phthalate metabolites MBP, MBzP, mono(3-carboxylpropyl) phthalate, and \ue001our metabolites o\ue001 di(2-ethyl- hexyl) phthalate among women who subsequently delivered preterm. Tese di\ue000erences remained, but were somewhat lessened, a\ue001ter correction by specifc gravity or creatinine. In multivariate logis- tic regression analysis adjusted \ue001or potential con\ue001ounders, elevated odds o\ue001 having phthalate metabo- lite concentrations above the median level were \ue001ound.

that could con\ue004ound the relationship between phthalate exposure and gestational length was collected through questionnaire. Te study was described in detail to all participating moth- ers, and all study participants gave in\ue004ormed consent. \ue003he research protocol was approved by the Ethics and Research Committees o\ue004 all participating institutions. \ue003he involve- ment o\ue004 the Centers \ue004or Disease Control and Prevention (CDC) laboratory was limited and was determined not to constitute engage- ment in human subjects research. O\ue004 the 1,853 women approached, 670 (36%) agreed to par- ticipate in the cohort study. O\ue004 these, archived third-trimester urine samples were available \ue004or 518 nonsmoking women because urine col- lection did not commence until a\ue004ter the ini- tiation o\ue004 study recruitment had begun. In the present nested case\u2013control study conducted within these 518 women, 30 cases were selected randomly among 44 women who delivered be\ue004ore the completion o\ue004 37 weeks o\ue004 gesta- tion. Controls (n = 30) were selected randomly among women who had completed \u2265 37 weeks o\ue004 gestation at the time o\ue004 delivery.

ond morning void) urine sample was collected \ue004rom each woman during a third- trimester visit to the project\u2019s research center. Phthalate metab- olites were measured in urine to avoid potential sample contamination \ue004rom the parent diester and because the metabolites, as opposed to the parent diesters, seem to be the active toxicants (Li et al. 1998; Peck and Albro 1982). Te \ue004ol- lowing 11 phthalate metabolites were measured at the CDC (Atlanta, GA, USA): mono(2- ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), MBP, MEP, mono-isobutyl phthalate (MiBP), three oxidized metabolites o\ue004 DEHP [mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-ox- ohexyl) phthalate (MEOHP), and mono(2- ethyl-5-carboxypentyl) phthalate (MECPP)],

mono(3-carboxypropyl) phthalate [MCPP; an oxidized metabolite o\ue004 both DBP and di-n-octyl phthalate (DOP)], and monocarboxyisooctyl phthalate (MCOP) and monocarboxyisononyl phthalate (MCNP) (oxidized metabolites o\ue004 diisononyl phthalate and diisodecyl phthalate, respectively).

\ue003he analytical approach involved enzy- matic deconjugation o\ue004 the metabolites \ue004rom their glucuronidated \ue004orm, solid-phase extrac- tion, separation with high-per\ue004ormance liquid chromatography, and detection by isotope-di- lution tandem mass spectrometry (Silva et al. 2007). Te limits o\ue004 detection (LODs) were in the low nanogram per milliliter range \ue004or each phthalate metabolite. Isotopically labeled internal standards and conjugated internal standards were used to increase precision o\ue004 measurements. Along with the unknown sam- ples, each analytical run included calibration standards, reagent blanks, and quality control materials o\ue004 high and low concentration to monitor \ue004or accuracy and precision. Analysts at the CDC were blind to all in\ue004ormation concerning subjects.

Urinary phthalate metabolite concentra- tions were corrected \ue004or urine dilution by speci\ue004ic gravity (SG) using the \ue004ormulaPc =P[(1.014 \u2013 1)/SG \u2013 1)], wherePc is the SG-corrected phthalate metabolite concentra- tion (micrograms per liter),P is the observed phthalate metabolite concentration, 1.014 is the median SG value among the present study population, andSG is the speci\ue002c grav- ity o\ue004 the individual urine sample. SG was measured using a handheld digital re\ue004rac- tometer (A\ue003AGO Company Ltd., \ue003okyo, Japan). Phthalate metabolite concentrations were also corrected by creatinine (Cr; phtha- late metabolite concentrations expressed as micrograms per gram Cr), which was mea- sured using a MicroLab A\ue003 Plus robotic liquid handler (Hamilton Co., Reno, NV,

USA) and Microplate Spectrophotometer (SpectraMax 340; Molecular Devices, Sunnyvale, CA, USA). Finally, we calculated (in nanomoles per milliliter) the sum o\ue004 con- centrations o\ue004 DEHP metabolites that were measured (i.e., MEHP, MEHHP, MEOHP, and MECPP).

analysis using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Maternal and preg- nancy characteristics were tabulated and compared between cases and controls using Student\u2019st-tests and chi-square tests when appropriate. For phthalate metabolite concen- trations below the LOD, we used an imputed value equal to one-hal\ue004 the LOD. In prelimi- nary crude analyses to assess whether women who experienced a preterm birth had higher phthalate metabolite concentrations than controls, we compared geometric mean values between the two groups usingt-tests. \ue003o take into account potential con\ue004ounding variables, we used multivariable logistic regression to model the odds o\ue004 having high (above the median) phthalate metabolite concentrations among the preterm women compared with controls. Variables considered in this analysis included maternal age, prepregnancy body mass index (BMI), parity, education, marital status, the in\ue004ant\u2019s sex, and gestational age at the time o\ue004 urine sample collection. Variables that did not di\ue001er between cases and controls and did not statistically con\ue004ound (change in e\ue004\ue004ect estimate o\ue004 > 10%) in any o\ue004 the models were not retained. Te variables that did appreciably change (> 10%) the e\ue004\ue004ect estimate in at least one o\ue004 the models were included in all models \ue004or consistency.

\ue003able 1 presents maternal and pregnancy characteristics o\ue004 the 30 selected preterm cases and the 30 term controls. Cases and con- trols did not di\ue004\ue004er by age, BMI, parity, or history o\ue004 previous preterm deliveries. Cases and controls di\ue004\ue004ered slightly with regard to marital status, education, and gestational age at the time the urine sample was collected. \ue003able 2 presents distributions o\ue004 uncorrected phthalate concentrations measured in urine o\ue004 controls. Similar to recent U.S. stud- ies, phthalate metabolites were detected in all samples. Among monoesters, the lower- molecular-weight phthalate metabolites MBP and MEP had median values o\ue004 33.4 and 108 \u00b5g/L, respectively. In comparison, the higher-molecular-weight monoester MBzP had a median concentration o\ue004 2.85 \u00b5g/L. Among DEHP metabolites, the oxidative metabolites MEOHP, MEHHP, and MECPP had median concentrations o\ue004 13.6, 17.1, and 38.2 \u00b5g/L, respectively, which, consistent with previous reports (CDC 2005), were higher than that o\ue004 the monoester MEHP (3.0 \u00b5g/L).

Correlations between phthalate metabo- lites were moderate to strong. Spearman cor- relation coe\ue000cients between metabolites \ue004rom di\ue004\ue004erent parent diesters ranged \ue004rom 0.21 (between MEHP and MEP) to 0.68 (between MECPP and MCPP). \ue003he Spearman cor- relation between MEHP and MBP was 0.44. Among the DEHP metabolites, Spearman correlations ranged \ue004rom 0.80 to 0.84 between MEHP and the oxidized metabo- lites (MEHHP, MEOHP, and MECPP) and were \u2265 0.91 between the oxidized DEHP metabolites. Among metabolites o\ue004 DBP, the Spearman correlation between MBP and MCPP was 0.85.

\ue003able 3 presents crude results comparing phthalate metabolite concentrations between cases and controls \ue004or phthalate concentra- tions that were uncorrected \ue004or urine dilu- tion, SG corrected, and Cr corrected. When phthalates were uncorrected \ue004or dilution, concentrations o\ue004 all metabolites detected in at least 80% o\ue004 samples were higher among cases compared with controls, and concen- trations \ue004or many metabolites among cases were nearly double those o\ue004 controls. Te larg- est di\ue004\ue004erence in uncorrected phthalate uri- nary metabolites was observed with MBP, \ue004or which median concentrations o\ue004 cases were 3-\ue004old higher than controls (p < 0.005). Cases had higher SG (p = 0.07) and Cr (p = 0.06) values than did controls, indicating that the urine samples o\ue004 women who delivered at term were more diluted than samples \ue004rom women who delivered preterm. Tese di\ue001er- ences in urine dilution somewhat reduced the observed di\ue001erences in phthalate metabolite concentrations a\ue004ter correcting by SG and Cr, but concentrations o\ue004 all metabolites remained higher in cases than in controls.

\ue003able 4 presents results \ue004rom multivari- able logistic regression analysis adjusted \ue004or potential con\ue004ounding variables. Preterm birth cases had elevated odds \ue004or having phthalate metabolite concentrations above the median \ue004or all metabolites. For metabolite concentra- tions uncorrected \ue004or dilution, the greatest odds ratios (ORs) and 95% con\ue004idence intervals (CIs) were observed \ue004or the DBP and diisobu- tyl phthalate metabolites MBP (OR = 10.7; 95% CI, 2.4\u201347.4), MiBP (3.6; 1.1\u201312.2), and MCPP (6.3; 1.8\u201321.9); \ue004or the DEHP metabolites MEHP (3.5; 1.0\u201312.9), MEHHP (4.6; 1.3\u201316.7), and MEOHP (7.1; 1.9\u201326.5); and \ue004or the sum o\ue004 DEHP metabolites (5.0; 1.4\u201318.0). Correction o\ue004 metabolite concen- trations by SG or Cr attenuated these ORs, although they remained elevated \ue004or most metabolites, particularly MBP and MEHP.

We conducted a nested case\u2013control analysis o\ue004 Mexican mother\u2013in\ue004ant pairs and \ue004ound evi- dence \ue004or the presence o\ue004 higher third-trimester

urinary concentrations o\ue004 phthalate metabolites among pregnant women who delivered pre- term (< 37 weeks gestation) compared with women who delivered at term (\u2265 37 weeks). Te di\ue001erences between cases and controls \ue004or

DBP metabolites (MBP and MCPP; MCPP is an oxidative metabolite o\ue004 DBP as well as a major metabolite o\ue004 DOP) and MEHP were not sensitive to correction \ue004or urinary dilu- tion using SG or Cr or to the adjustment \ue004or