We conducted a nested case–control analysis of Mexican mother–infant pairs and found evidence for the presence of higher third-trimester urinary concentrations of phthalate metabolites among pregnant women who delivered pre-term (< 37 weeks gestation) compared with women who delivered at term (≥ 37 weeks). The differences between cases and controls for DBP metabolites (MBP and MCPP; MCPP is an oxidative metabolite of DBP as well as a major metabolite of DOP) and MEHP were not sensitive to correction for urinary dilution using SG or Cr or to the adjustment for potential confounding variables. These results provide the first evidence of an association between phthalate exposure during pregnancy and preterm birth.
Our findings of higher DEHP metabolite concentrations (MEHP and the oxidative metabolites MEOHP, MEHHP, and MECPP) among mothers delivering pre-term are consistent with an Italian study of 84 newborns that reported an inverse association between gestational age and DEHP exposure (Latini et al. 2003
). In that study, MEHP (but not DEHP) in the cord blood of the newborns was associated with decreased gestational age at delivery (OR for absence of detectable MEHP in cord blood associated with a 1-week increase in gestational age = 1.50; 95% CI, 1.01–2.21), and the mean gestational age among infants with MEHP detected in cord blood was 1.2 weeks (8.4 days) less than that of infants with nondetectable MEHP in cord blood (p
= 0.03). However, there are uncertainties over the use of cord blood to measure phthalates in the study, because blood measures of phthalate exposure can be compromised because of contamination by phthalate diesters in sampling tubes and lipases in the blood that can convert the diesters to monoesters (Kato et al. 2003
Our finding that DEHP metabolites were elevated in mothers delivering preterm is also consistent with a recent study of 331 African-American and Dominican mothers and newborns in New York City (Whyatt et al. 2008
). In that study, women with third-trimester spot urine samples with SG-corrected MEHP concentrations in the highest quartile delivered infants with gestational ages 5.1 days (95% CI, 2.1–8.4 days) less than did women in the lowest MEHP quartile (p
< 0.001). In contrast, our results are inconsistent with another recent study of third-trimester urines from 404 women in a multiethnic birth cohort from New York City, which reported increased gestational ages in relation to MEHP and the sum of MBP, MEP, and MiBP concentrations (Wolff et al. 2008
). Differences between the results of the two New York City studies and between our study and the study by Wolff et al. (2008)
may reflect differences in study designs, population characteristics (e.g., age, ethnicity, education, and quality of care), exclusion criteria, and/or exposure levels or sources. Moreover, gestation length, but not preterm birth, was assessed in these previous epidemiologic studies of phthalates and birth outcomes.
There were some differences in phthalate metabolite concentrations among pregnant women who delivered at ≥ 37 weeks in our Mexican cohort compared with those reported in the 2001–2002 National Health and Nutrition Examination Survey for women in the United States (Adibi et al. 2008
; CDC 2005
). The median MBP concentration uncorrected for urine dilution was 2-fold higher in our Mexican cohort than in females from the U.S. population, and the Cr-corrected MBP median was 3-fold higher. Conversely, median MBzP concentrations were about 4-fold lower in the present study compared with U.S. females for uncorrected and Cr-corrected concentrations. Median uncorrected MEHP, MEHHP, MEOHP, MiBP, MCPP, and MEP concentrations were comparable between the two populations, although somewhat lower among women in the present study. The presence of similar to slightly lower MiBP concentrations but higher MBP concentrations in this Mexican population suggests potential differences in patterns or sources of exposure to dibutyl phthalates compared with U.S. women. Based on our preliminary exposure results and evidence for adverse effects of DBP and/or MBP on fetal development (Foster 2006
; Swan 2008
), research designed to assess sources of DBP exposure among pregnant Mexican women is warranted.
Compared with controls, women who had preterm births in the present study had suggestively higher levels of urinary Cr [and SG, because the two were highly correlated (Spearman r
= 0.9)], which served to lessen the differences in phthalate metabolite concentrations after correcting for SG or Cr. Cr clearance increases significantly during pregnancy, in a manner that peaks and then begins to decline several weeks before term (Boeniger et al. 2003; Davison et al. 1980
; Lohsiriwat and Imrittha 2008
; Sims and Krantz 1958
). There is variability in the magnitude and timing of the peaks (as well as the slope of the subsequent decline in Cr clearance) among women (Davison et al. 1980
; Sims and Krantz 1958
), and it may not be appropriate to correct third-trimester urinary phthalate metabolite concentrations by Cr (Adibi et al. 2008
). It is possible that the higher Cr levels among cases may be related to an unmeasured risk factor for preterm birth, but to our knowledge the ability of urinary Cr to predict pre-term birth has not been previously reported. In addition to fluctuations in Cr clearance during pregnancy, there are also cyclic within-woman fluctuations in Cr clearance in relation to normal menstrual cycles that are potentially caused by changes in hormone activity (Davison and Nobles 1981
). Thus, it may also be possible that increased urinary Cr levels could be related to phthalate-induced alterations in endocrine function, although there are currently no data to support this hypothesis.
Preterm birth is likely a syndrome with multiple etiologies (Goldenberg et al. 2008
). Although there is no clear explanation at this time for an association between phthalate exposure and preterm birth, several plausible hypotheses may be put forth. Among the various risk factors identified for preterm birth, maternal inflammation, particularly chorioamnionitis, has been firmly linked to preterm birth in epidemiologic and laboratory studies (Romero et al. 2007
). Because phthalate metabolites stimulate proinflammatory responses in cells in culture, including cytokine release (Jepsen et al. 2004
), activation of the mitogen-activated protein kinase pathway (Pauley et al. 2002
), and activation of the peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ pathways (in a rat placental trophoblast cell line) (Xu et al. 2005
), phthalate exposure may increase risk for preterm birth if it stimulates an inflammatory response in pregnant women, as proposed previously (Latini et al. 2005
). Additionally, prostaglandins are important signaling molecules in parturition, and phthalate metabolites increase expression of prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2) in a rat placental trophoblast cell line (Xu et al. 2005
) and mouse liver cells (Ledwith et al. 1997
). Because PTGS2 is necessary for synthesis of prostaglandins, phthalate-stimulated induction of PTGS2 in gestational tissues such as placenta and the extraplacental membranes could increase risk for preterm birth by increasing premature intrauterine production of prostaglandins. Alternatively, matrix metalloproteinases (MMPs) are activated by inflammatory cytokines, and MEHP activates an MMP in rat testis (Yao et al. 2009
). If phthalate metabolites activate MMPs in extraplacental membranes, it could lead to preterm premature rupture of the membranes (PPROM) and preterm birth.
In addition to inflammation, increased apoptosis in the maternal/fetal membranes is associated with PPROM as well as membrane rupture at term (Kataoka et al. 2002
; Reti et al. 2007
). MEHP stimulates cell responses that can initiate apoptosis, including DNA damage (in human sperm) (Hauser et al. 2007
), oxidative stress (in the testes) (Kasahara et al. 2002
), release of cytochrome c from mitochondria (Kasahara et al. 2002
), and increased expression of Fas ligand, an apoptosis-initiating protein (Richburg and Boekelheide 1996
; Yao et al. 2007
). Oxidative stress is also believed to be involved in the developmental toxicity of DBP (Kim et al. 2002
; Wellejus et al. 2002
). However, most studies of phthalate metabolite–stimulated oxidative stress, DNA damage, and apoptosis have been conducted in males, and studies of effects in females and on pregnancy outcomes are needed.
Alternate mechanisms could involve phthalate impacts on endocrine function. The phthalate diesters DEHP, DBP, and BBzP, and/or their metabolites, are antiandrogenic in males (Foster 2006
; Meeker et al. 2008
; Swan 2008
) and have been shown to reduce estradiol and progesterone production in female rodents and in rat granulosa cells in vitro
(Gray et al. 2006
; Lovekamp and Davis 2003
; Treinen et al. 1990
). Because progesterone plays a key role in maintaining uterine quiescence during pregnancy, it may be plausible that phthalates alter the timing of labor by reducing progesterone production. Furthermore, estradiol is vital in suppressing inflammation and oxidative stress (Straub 2007
; Vina et al. 2006
). However, data in support of the pathways proposed here involving endocrine disturbances, inflammation, and oxidative stress are lacking, and additional research is needed to explore these potential mechanisms.
The present study has several limitations, including its relatively small size, the inability to study high-risk preterm births occurring more remote from term, and the potential for misclassification stemming from maternal recall of last menstrual period to estimate gestational age. Misclassification of preterm cases may be nondifferential with respect to exposure status. However, we cannot rule out differential misclassification. For example, although there is currently no evidence in support of this hypothesized scenario, if phthalate exposure was associated with altered menstrual cycle length, it may have resulted in systematic underestimates of last menstrual period and gestational age among highly exposed women. Another limitation of our study is the potential for uncontrolled biases due to the 1-week difference in median gestational age at the time of urine sample collection between cases and controls. However, although this sample timing was associated with case status, and statistically confounded at least one of the multivariable models and thus was included in the adjusted ORs presented in , it was not associated with uncorrected or dilution-corrected urinary phthalate metabolite concentrations. Our study also made a large number of statistical comparisons, which may have led to chance findings of statistical significance. In addition, the potential for reverse causation cannot be ruled out, whereby other underlying risk factors for delivering preterm could be associated with increased phthalate exposure or altered phthalate metabolism. Finally, our ability to interpret these results to determine which specific phthalates or phthalate metabolites may be the most relevant to preterm birth risk was limited because concentrations of many compounds were higher among preterm cases and the various phthalate metabolites were moderately to strongly correlated with one another.
In conclusion, the present study provides evidence for a potential role for phthalate exposure (DBP, DOP, DEHP, and/or BBzP) in pre-term birth among a group of Mexican women. Additional research, including larger human studies and experimental studies, are warranted to further investigate the relationship between phthalate exposure and preterm birth.