In this large nationally representative cross-sectional study, in which we used urinary phthalate metabolite concentrations as biomarkers of exposure to four parent phthalates, we observed weak positive associations for urinary MBP, and weak inverse associations for urinary MEHP, in relation to self-reported history of endometriosis and leiomyomata. Our analyses of the two oxidative metabolites of DEHP—MEHHP and MEOHP—yielded findings that were consistent with our findings for MEHP with respect to endometriosis, but not with respect to uterine leiomyomata. We found little evidence for associations of urinary MEP and MBzP with each of these outcomes.
Our results for MEHP and endometriosis do not agree with findings from the three case–control studies on this subject, all which identified positive associations between markers of DEHP exposure and risk of endometriosis. Cobellis et al. (2003)
reported higher plasma DEHP concentrations [median, 0.57 μg/mL; interquartile range (IQR), 0.06–1.23] among women with endometriosis than among controls (0.18 μg/mL; IQR, 0–0.44; p
= 0.005). However, plasma MEHP concentrations were generally low and did not differ between the outcome groups: for cases the median MEHP was 0.38 μg/mL (IQR, 0.1–0.97), and for controls the median was 0.58 μg/mL (IQR, 0.34–0.71). Our MEHP results also diverge from a subsequent case–control study by Reddy et al. (2006)
, in which women with laparoscopy-confirmed endometriosis had significantly higher plasma concentrations of DEHP compared with controls, and concentrations appeared more elevated with increasingly severe stage of endometriosis. In a third case–control study by Itoh et al. (2009)
, laparoscopy-confirmed cases were more likely than controls to have “elevated” creatinine-corrected urinary MEHP concentrations [above the controls’ median (4.2 ng/mg)], although this association was not significant. Findings were weaker for urinary MEHHP, MEOHP, and the molar sum of the three DEHP metabolites. Itoh et al. also found significantly higher uncorrected concentrations of all three DEHP metabolites with increasingly severe stage of endometriosis (p
< 0.02), but these trends diminished upon creatinine correction. The discrepant findings may stem from differences in exposure assessment and/or case definition. For measures of exposure to DEHP, the first two case–control studies used blood-based concentrations of DEHP, which is no longer considered a valid approach for assessing exposure because of concerns about contamination from laboratory equipment and other external sources. The correlation between serum DEHP and MEHP concentrations was weak in the Cobellis et al. (2003)
= 0.16, p
≥ 0.3), raising questions about the utility of the serum DEHP measurements, including the possibility that DEHP measurements were affected by external contamination (Hauser and Calafat 2005
). Blood-based concentrations of MEHP are also prone to contamination from DEHP that is also present in the specimen, because serum enzymes can hydrolyze DEHP to MEHP during storage (Kato et al. 2003
). For their case definitions, all three case–control studies enrolled women with laparoscopically confirmed endometriosis. Cases in the Cobellis et al. (2003)
study underwent further histologic confirmation. However, because all participants were enrolled in clinical settings, it is possible that DEHP and MEHP concentrations were affected by supplies used in very recent medical procedures associated with the clinical conditions (Hauser and Calafat 2005
). For example, at least some of the specimens in the Cobellis et al. (2003)
study were collected immediately before anesthesia for laparoscopy, when the women were likely on an intravenous therapy line, a potential source of DEHP exposure (U.S. Food and Drug Administration 2002
). This may explain why plasma MEHP concentrations in the Cobellis et al. (2003)
study were about 100 times the urinary MEHP concentrations among the women in our study. It is also possible that controls recruited from a clinical setting—such as those seeking treatment for infertility in the Itoh et al. (2009)
study—had conditions that were themselves influenced by exposure to phthalates, leading to biased findings (Hernán et al. 2004
In contrast, our results for MBP agree with results from the Reddy et al. (2006)
study in which women with endometriosis had significantly higher plasma concentrations of DBP (the parent compound of MBP) compared with controls (Reddy et al. 2006
). Nonetheless, blood measures of DBP entail limitations as described for DEHP, and even in the absence of external contamination, there remains the possibility that some of the DBP could be missed because of its hydrolysis to MBP (Kato et al. 2003
). In the study by Itoh et al. (2009)
, endometriosis was not significantly associated with having an “elevated” creatinine-corrected urinary MnBP concentration (above the median among the controls, 43.4 ng/mg), and there was no apparent trend in urinary MnBP across severity classes of the condition.
Our null findings for MBzP and endometriosis were consistent with those of Itoh et al. (2009)
but diverged from those of Reddy et al. (2006)
in which plasma BzBP concentrations among cases were significantly higher than those among controls. Similar to our findings for MEP, Itoh et al. (2009)
did not find a significant association between creatinine-corrected urinary MEP and endometriosis.
Our weakly inverse association between MEHP and leiomyomata agrees with the case–control study by Luisi et al. (2006)
, in which significantly lower serum DEHP and MEHP concentrations were found among hysterectomy-confirmed cases of leiomyomata relative to controls (confirmed by ultrasound to be free of disease). The measurement concerns we described above also pertain to this case–control study, but they would likely serve to overestimate DEHP exposures among the cases, rendering the estimates from this case–control study conservative. However, this study may have reduced external contamination through its use of glass collection and storage materials.
Our results are also consistent with animal data showing positive associations of exposure to DEHP and its metabolite MEHP with anovulation or delayed ovulation, longer estrous cycles, decreased synthesis of estradiol, and decreased serum progesterone levels (Davis et al. 1994a
; Lovekamp and Davis 2001
), all of which would predict a protective effect on uterine leiomyomata and endometriosis. The pathophysiology of leiomyomata is believed to depend on the biological activity of the endogenous sex steroid hormones estrogen (estradiol, estrone, estriol) and progesterone (Rein 2000
; Rein and Nowak 1992
; Rein et al. 1995
), as well as locally derived growth factors (Andersen 1998
). The hormone-dependent nature of leiomyomata is supported by the following observations: They do not occur before menarche, they have an increased concentration of estrogen and progesterone receptors compared with normal myometrium (Strauss and Coutifaris 1999
), and they shrink in volume after menopause or with suppression of ovarian function via gonadotropin-releasing hormone (GnRH) agonist therapy or oophorectomy (Friedman et al. 1990
). The literature on the relation of endogenous sex hormones to leiomyomata is limited and mixed. Although an earlier study reported than serum estrogen levels did not differ significantly between leiomyomata cases and controls (Potgieter et al. 1995
), a subsequent study reported that urinary concentrations of many sex hormones, including 17β-estradiol, were significantly higher among leiomyomata cases than controls (Jung et al. 2004
). Several epidemiologic studies show an increased risk of leiomyomata associated with reproductive and hormonal factors, including early age at menarche and nulliparity, which suggests that increased menstrual cycling is a risk factor (Schwartz 2001
; Wise et al. 2004
Similarly, endometriosis is associated with abnormal steroid production and activity (Dizerega et al. 1980
; Eskenazi and Warner 1997
), with diagnoses occurring postmenarchally (Houston 1984
) and symptoms abating after menopause. Endometriotic implants are dependent on estrogen for their maintenance and growth (Dizerega et al. 1980
; Gurates and Bulun 2003
), and reduction of estradiol production, via either surgical (oophorectomy) or hormonal (GnRH agonist analogues) intervention, causes atrophy of endometriosis lesions and is effective in treating pain symptoms. High-dose synthetic progestins are effective in the treatment of endometriosis through suppression of luteinizing hormone and FSH secretion that inhibits estradiol production, direct antiestrogenic effects on the lesions, and induction of pseudodecidualization (Taylor and Lebovic 2009
). Estradiol production is up-regulated in endometriotic implants, whereas estradiol metabolism is impaired because of a deficiency of the enzyme 17β-hydroxysteroid dehydrogenase type 2 (Gurates and Bulun 2003
). Also, the progesterone receptor subtype B is deficient in endometriosis tissue (Attia et al. 2000
). In epidemiologic studies, endometriosis is positively associated with early age at menarche, shorter cycle length, decreased parity, and not breast-feeding (Missmer et al. 2004a
), all of which can affect menstrual cycling and lifetime cumulative exposure to estrogens.
Nonetheless, although our findings for DEHP’s oxidative metabolites provided support for an inverse association between DEHP exposure and endometriosis, they raised doubts about an association with leiomyomata. In the subset of women evaluated in 2001–2004, we compared findings for urinary concentrations of MEHHP and MEOHP with findings for MEHP, and both MEHHP and MEOHP were detectable in nearly all specimens in our study, including those in which MEHP was undetectable. The associations of the two oxidative metabolite concentrations with endometriosis were inverse and remarkably similar in magnitude to the corresponding association for MEHP. However, the associations between the oxidative metabolites and uterine leiomyomata were not consistent with the inverse (albeit nonsignificant) corresponding association for MEHP. The association for MEHHP was essentially null [OR = 0.97 (95% CI, 0.55–0.71) for women in the highest quartile vs. the lowest three quartiles], and although the associations for MEOHP and MECPP suggested a positive (nonsignificant) association, further analyses at alternative threshold concentrations were more consistent with null associations [e.g., OR = 0.92 (95% CI, 0.39–2.15) comparing highest sextile of urinary MEOHP vs. the lowest five]. Although these findings emanated from a subset of participants, they suggest that the inverse association between MEHP and leiomyomata may be a chance finding.
Our study has three principal limitations that warrant consideration. First, with its cross-sectional design, the assessment of exposure to phthalates may have occurred several years after diagnosis of the outcomes. It is uncertain whether diagnosis would directly influence urinary phthalate concentrations by altering the metabolism of phthalates, although recent data from 60 women in the Early Pregnancy Study indicate little variation in urinary phthalate metabolite concentrations across phases of the menstrual cycle (Baird et al. 2009
), suggesting that changes in a woman’s endogenous hormonal milieu (which might occur with age, oral contraceptive use, or hysterectomy) have minimal effects on phthalate metabolism. However, it remains possible that diagnoses could indirectly affect urinary phthalate concentrations via exposures resulting from diagnosis, as described above. However, given the short half-lives of the metabolites, it seems unlikely that diagnosis-related procedures occurring more than a few days before the NHANES examination would be an important source of exposure. Moreover, although medications have recently been identified as an important source of DBP and DEP exposures (Hauser et al. 2004
; Hernández-Díaz et al. 2009
), medications typically indicated for endometriosis and leiomyomata are not likely to contain these compounds because they are not coated for timed release. Even in the absence of reverse causation, the increasing interval between the relevant and measured exposures is a form of measurement error—nondifferential with respect to exposure or disease status—that could attenuate the observed associations. The associations between MBP and uterine leiomyomata and between MEHP and endometriosis became stronger when we confined analyses to women whose diagnoses or hysterectomies occurred within 7 years of their NHANES evaluation, suggesting that this type of exposure measurement error may have affected our results.
Our exposure measure is limited in that phthalates do not bioaccumulate and have short half-lives in the body. Therefore, measurements taken at a single point in time are less reliable indicators of typical exposures than, for example, a series of measurements taken over several weeks. Yet emerging data suggest some consistency of phthalate exposures over time among reproductive-age women. In their study of 46 women 35–49 years of age who provided first-morning urinary voids on 2 consecutive days, Hoppin et al. (2002)
found intraclass correlations (ICCs) ranging from 0.53 to 0.80 for creatinine-corrected urinary concentrations of MBP, MEP, MEHP, and MBzP. Over longer time intervals, these correlations may be weaker, as suggested by data from two studies: the Early Pregnancy Study, which reported ICCs for these urinary metabolites ranging from 0.36 to 0.53 for measurements taken over 2-week intervals (Baird et al. 2009
); and a study of 28 pregnant women (Adibi et al. 2008
), which reported ICCs for the creatinine-corrected urinary phthalate concentrations ranging from 0.21 to 0.65 for measurements taken repeatedly over 6 weeks. Therefore, it is possible that we have underestimated the true associations, where they exist, between phthalate exposures and endometriosis and uterine leiomyomata. Nonetheless, our exposure measures do represent an advance over measures used in previous work in that we have used urinary measures of phthalate metabolites, which are far less vulnerable to contamination than phthalate diester measurements in blood. Moreover, we have evaluated exposures to four phthalates, conferring a degree of specificity to our findings.
Finally, an important limitation of our study is that measures of both outcomes were based solely on self-report of physician diagnosis. Prevalence estimates of these conditions in our study population are similar to studies based on hospital-discharge and prospective cohort data (Eskenazi and Warner 1997
; Missmer and Cramer 2003
; Schwartz and Marshall 2000
; Wise et al. 2005
). Two large prospective studies of self-reported leiomyomata that validated the reports against medical records indicated a low proportion of false positives (Marshall et al. 1997
; Wise et al. 2004
); therefore, specificity of diagnosis is likely to be high. False reports of endometriosis diagnosis may occur more frequently (Missmer et al. 2004b
), but the proportion of undiagnosed endometriosis cases is likely to be smaller (Zondervan et al. 2002
). For both outcomes, it seems unlikely that misclassification of disease status was influenced by degree of phthalate exposure, and such nondifferential misclassification would have attenuated our findings. Our findings for endometriosis may have been further influenced by phthalate effects that are specific to different types of underlying pathology. Estrogen-related risk factors appear to vary by endometriosis that is concurrent with and independent of infertility (the latter which is often accompanied by pain) (Missmer et al. 2004a
). However, in the absence of data on infertility and symptomatic pain, the findings reflect an averaged association of the phthalate measures with these two subgroups.
In addition to our study’s strengths in its exposure measurements, our study also benefited from its large sample size, its consideration of several important potential confounders, and its representativeness of the general population of reproductive-age women living in the United States. In conclusion, we have identified preliminary evidence suggesting that exposure to DBP may be associated with increased risk of endometriosis and uterine leiomyomata, and that exposure to DEHP may be associated with reduced risk of these conditions. These findings warrant investigation in future prospective studies.