In the recent National Health and Nutrition Examination Survey (NHANES 1999–2000), most of the general population in the United States had measurable exposure to multiple phthalates (CDC 2003
; Silva et al. 2004a
). The samples in the present study and in NHANES were both analyzed using comparable methods and standards by the same laboratory, although the specific metabolites that were measured in the two studies differed somewhat. We compared the medians and 75th percentiles of the AGI-associated phthalate metabolite concentrations among two groups of mothers in our study (those whose boys fell in the short AGI group and all others) with those of females in the NHANES sample (). In the analysis of the NHANES samples, monobutyl phthalate includes both MBP and MiBP, which were measured separately in our study. Metabolite concentrations for mothers of boys with short AGI were consistently higher than those of other mothers. Compared with women in the NHANES sample, metabolite concentrations for our population were somewhat lower. However, our population cannot be directly compared with NHANES: the proportion of pregnant women in the NHANES sample is unknown, and age distributions differ. Nonetheless, these data demonstrate that the four AGI-associated phthalate metabolites are prevalent in the U.S. female population, and levels were not unusually high among mothers whose sons had a short AGI.
Concentrations of four phthalate metabolites in three groups of women (ng/mL).
Although not identical, AGD in pups is most similar to AGD as we defined it in this study. In rodents, AGD has been shown to be one of the most sensitive end points for phthalates such as DBP (Mylchreest et al. 2000
) and other antiandrogens such as flutamide (Barlow and Foster 2003
; McIntyre et al. 2001
) and finasteride (Bowman et al. 2003
). It is difficult to compare the dose to humans from low-level, ongoing, environmental exposure with that delivered to rodents experimentally in a narrow window of gestation. Nonetheless, it is likely that the doses to which our participants were exposed are lower than those used in toxicologic settings, suggesting that humans may be more sensitive to prenatal phthalate exposure than rodents. This greater sensitivity in humans has been observed for other toxicants. For example, humans are more sensitive to trenbolone by an order of magnitude (Neumann 1976
). This greater sensitivity is thought to be a result of rodents’ higher metabolic rate and more rapid inactivation of toxicants, both of which have been shown to be inversely related to body size (White and Seymour 2005
In light of the toxicologic literature for MBP, MBzP, and MiBP (Ema et al. 2003
; Foster et al. 1980
; Gray et al. 2000
; Nakahara et al. 2003
), our data suggest that the end points affected by these phthalates are quite consistent across species. A boy with short AGI has, on average, an AGI that is 18% shorter than expected based on his age and weight as well as an increased likelihood of testicular maldescent, small and indistinct scrotum, and smaller penile size. These changes in AGD and testicular descent are consistent with those reported in rodent studies after high-dose phthalate exposure (Ema et al. 2003
; Gray et al. 2000
; Mylchreest et al. 2000
). The lack of association for MCPP and MMP, which have not been widely studied, is not inconsistent with the toxicologic literature.
With respect to DEP and its metabolite MEP, we note that there are three other human studies suggesting reproductive toxicity (Colón et al. 2000
; Duty et al. 2003b
; Main KM, unpublished data). It is therefore uncertain whether the absence of data in rodents showing reproductive toxicity is the result of failure to detect it, unmeasured confounding in human studies, or interspecies differences in response to these compounds.
DEHP has been shown to shorten AGD (Gray et al. 2000
) and reduce testosterone (Parks et al. 2000
). Although MEHP was not associated with AGD in our data, the associations for the oxidative metabolites of DEHP (MEOHP and MEHHP) were of comparable magnitude with those for metabolites of DBP and BzBP, although not statistically significant. Thus, it is unclear whether MEOHP and MEHHP are (inversely) associated with AGI, although associations are of borderline statistical significance because of our sample size, or whether human and rodent responses to this phthalate and its metabolites differ.
Masculinization of external male genitalia, represented by longer AGD, is controlled by dihydrotestosterone (Clark et al. 1990
). Ema and Miyawaki (2001)
demonstrated that this metabolite of testosterone is markedly decreased by prenatal administration of MBP, suggesting that MBP acts as an antiandrogen. AGD in male rodents is associated with other adverse developmental effects (Foster and McIntyre 2002
) and some phthalate-induced changes have been shown to be permanent. For example, Barlow et al. (2004)
report that prenatal exposure to 500 mg/kg/day DBP resulted in permanently decreased AGD and testicular dysgenesis. They also report that in utero DBP exposure induced proliferative Leydig cell lesions. Follow-up of exposed children until adulthood will be required to determine whether long-term effects, including testicular dysgenesis, are seen in humans after prenatal phthalate exposure.
Several recent studies of the variability of phthalate monoester concentration in human samples suggest that phthalate concentration in humans is fairly stable, perhaps reflecting habitual use of phthalate-containing household and consumer products (Colón et al. 2000
; Hauser et al. 2004
; Hoppin et al. 2002
). These studies lend support to the use of a single sample for exposure assessment. We obtained only a single prenatal urine sample from each woman, and most samples were obtained quite late in pregnancy (mean = 28.3 weeks). Therefore, the measured phthalate metabolite levels may not reflect exposure during the most sensitive developmental window, resulting in some degree of exposure misclassification. However, unless this misclassification varied systematically with outcome, such errors would bias the effect estimate toward the null. In fact, the categorical analysis, which should be less sensitive to such misclassification, showed stronger associations than did the continuous analysis.
Our analysis is based on a single measure of AGD, and the reliability of this measurement in humans has not been established. During two training sessions, three study physicians each measured AGD in four male infants (mean age, 8.1 months). The mean AGD for these measurements was 58.6 mm, SD was (within infant) 4.2 mm, and coefficient of variation of 7.2%, suggesting that AGD can be measured reliably. Use of this measurement in larger studies in a range of diverse populations, with many more such training sessions, will be needed to obtain normative data.
Although it might have been ideal to examine babies shortly after birth, the timing of grant funding did not allow this. Babies were born to SFFI mothers as early as January 2000, and the first baby visits did not occur until April 2002. To maximize the number of children participating, we allowed recruitment over a range of ages. On the other hand, because the use of AGD in humans is new, the optimal timing for this measurement is not known. Our data suggest that measurements are reliable and informative in young children at least until 18 months, when AGD becomes more difficult to obtain reliably. Its value in adolescents and adults has yet to be determined.
We note that phthalate metabolite levels were highly correlated, and most women were exposed to all metabolites at detectable levels. Gray et al. (2000)
suggested that risk assessments for phthalate-induced reproductive toxicity should consider phthalates as a group and include exposures from multiple sources. The score we use reflects joint exposure to the four AGI-associated phthalates, and our results suggest that joint exposure may convey greater than additive risk, but larger sample sizes are needed to confirm this.
Gray and Foster (2003)
refer to a “phthalate syndrome” characterized by testicular, epididymal, and gubernacular cord agenesis as well as decreased AGD, and stress the importance of evaluating all components of a syndrome so that affected animals are not misidentified. It has recently been suggested (Fisher 2004
) that this “phthalate syndrome” shares many features with the human testicular dysgenesis syndrome proposed by Skakkebaek et al. (2001)
to follow chemically induced disruption of embryonic programming and gonadal development during fetal life. The present findings, though based on small numbers, provide the first data in humans linking measured levels of prenatal phthalates to outcomes that are consistent with this proposed syndrome.
This is the first study to look at subtle patterns of genital morphology in humans in relation to any prenatal exposure. It was motivated by toxicologic studies showing that genital morphology is altered by antiandrogens, including some phthalates. We report that AGD, the most sensitive marker of antiandrogen action in toxicologic studies, is shortened and testicular descent impaired in boys whose mothers had elevated prenatal phthalate exposure. These changes in male infants, associated with prenatal exposure to some of the same phthalate metabolites that cause similar alterations in male rodents, suggest that commonly used phthalates may undervirilize humans as well as rodents.