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Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure

Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure Research Children’s Health Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure 1 2 3 3 3 4 Shanna H. Swan, Katharina M. Main, Fan Liu, Sara L. Stewart, Robin L. Kruse, Antonia M. Calafat, 5 6 7 8 9 Catherine S. Mao, J. Bruce Redmon, Christine L. Ternand, Shannon Sullivan, J. Lynn Teague, and the Study for Future Families Research Team* 1 2 Department of Obstetrics and Gynecology, University of Rochester, Rochester, Minnesota, USA; Department of Growth and Reproduction, University of Copenhagen, Copenhagen, Denmark; Department of Family and Community Medicine, University of Missouri-Columbia, Columbia, Missouri, USA; National Center for Environmental Health, Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, Georgia, USA; Department of Pediatrics, Division of Endocrinology, Los Angeles Biomedical 6 7 Research Institute at Harbor-UCLA Medical Center, Los Angeles, California, USA; Departments of Pediatrics and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA; Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA; Departments of Surgery (Urology) and Child Health, University of Missouri-Columbia, Columbia, Missouri, USA As with the Colón et al. study, contamination Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in from diesters in laboratory equipment could male rodents. We present data from the first study to examine AGD and other genital measure- not be excluded (Murature et al. 1987). ments in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was More recent studies have examined phtha- obtained in 134 boys 2–36 months of age. AGD was significantly correlated with penile volume late monoester metabolites in urine. Because (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20, urinary metabolites are not likely to be present p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination as the result of contamination, these studies [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We avoid this potential source of measurement examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors error. Duty et al. (2003a) reported dose– of age-adjusted AGI in regression and categorical analyses that included all participants with prena- response relationships between tertiles of tal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phtha- monobutyl phthalate and sperm motility and late (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl sperm concentration, and between tertiles of phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values monobenzyl phthalate (MBzP) and sperm for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP con- concentration. They also reported inverse dose– centration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than response relationships between monoethyl expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for phthalate (MEP) and sperm DNA damage MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age- Address correspondence to S.H. Swan, University of adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009). Rochester, Department of Obstetrics and Gynecology, The associations between male genital development and phthalate exposure seen here are consistent School of Medicine and Dentistry, 601 Elmwood Ave., Box 668, Rochester, NY 14642-8668 USA. with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally Telephone: (585) 273-3521. Fax: (585) 275-7366. exposed rodents. The median concentrations of phthalate metabolites that are associated with E-mail: shanna_swan@urmc.rochester.edu short AGI and incomplete testicular descent are below those found in one-quarter of the female *The Study for Future Families Research Team population of the United States, based on a nationwide sample. These data support the hypothesis included, from the University of Missouri- that prenatal phthalate exposure at environmental levels can adversely affect male reproductive Columbia: E.Z. Drobnis, B.S. Carter, D. Kelly, and development in humans. Key words: anogenital distance, benzylbutyl phthalate, dibutyl phthalate, T.M. Simmons. Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center: diethyl phthalate, monobenzyl phthalate, monoethyl phthalate, monoisobutyl phthalate, mono-n- C. Wang, L. Lumbreras, S. Villanueva, M. Diaz- butyl phthalate, phthalates, prenatal exposure. Environ Health Perspect 113:1056–1061 (2005). Romero, M.B. Lomeli, and E. Otero-Salazar. Cedars- doi:10.1289/ehp.8100 available via http://dx.doi.org/ [Online 27 May 2005] Sinai Medical Center: C. Hobel and B. Brock. University of Minnesota: C. Kwong and A. Muehlen. University of Iowa: A. Sparks, A. Wolf, J. Whitham, Diesters of phthalic acid, commonly referred et al. 2000), and DEHP (Gray et al. 2000; M. Hatterman-Zogg, and M. Maifeld. We thank the health care providers and study par- to as phthalates, are widely used in industry Parks et al. 2000). ticipants at University Physicians Clinic (Columbia, and commerce; they are used in personal care Despite the growing body of literature MO), Fairview Riverside Women’s Clinic (Minne- products (e.g., makeup, shampoo, and soaps), on phthalate reproductive toxicity and data apolis, MN), Los Angeles Biomedical Research plastics, paints, and some pesticide formula- demonstrating extensive human exposure Institute at Harbor-UCLA Medical Center (Los tions. Consistent toxicologic evidence indi- (Silva et al. 2004a), few studies have examined Angeles, CA), Cedars-Sinai Medical Center (Los cates association between several of these the effects of these chemicals on human repro- Angeles, CA), and University of Iowa Hospitals and Clinics (Iowa City, IA). We also thank M. Silva, phthalate esters and reproductive effects. ductive development. Colón et al. (2000) J. Reidy, E. Samandar, and J. Preau for phthalate In particular, dibutyl phthalate (DBP), ben- reported elevated levels of several phthalates analyses and E. Gray, P. Foster, and D. Barr for their zylbutyl phthalate (BzBP), di-2-ethylhexyl [including diethyl phthalate (DEP), DBP, and guidance. phthalate (DEHP), and di-isononyl phthalate DEHP] in serum samples from young girls This work was supported by grants from the U.S. have been shown to disrupt reproductive tract with premature breast development. However, Environmental Protection Agency and the National development in male rodents in an antian- the timing of exposure was unknown and high Institutes of Health (R01-ES09916 to the University of Missouri, MO1-RR00400 to the University of drogenic manner (Parks et al. 2000). Recent exposure levels may have reflected phthalate Minnesota, MO1-RR0425 to Harbor-UCLA studies have reported significant reductions contamination of serum samples (McKee and Medical Center) and by grant 18018278 from the in anogenital distance (AGD) in Sprague- Toxicology Research Task Group 2004). Until State of Iowa to the University of Iowa. Dawley rats after prenatal exposure at high recently, the only study of humans to evaluate The authors declare they have no competing doses to BzBP (Nagao et al. 2000; Tyl et al. phthalate exposure and male reproductive financial interests. 2004), DBP (Barlow and Foster 2003; Foster toxicity measured phthalate diesters in semen. Received 7 March 2005; accepted 25 May 2005. 1056 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance measured using the neutral single-cell gel elec- institutions approved SFFI and SFFII, and all recoveries are near 100%), and precise with trophoresis (comet) assay (Duty et al. 2003b). In participants signed informed consents for each between-day relative standard deviations of this population of men attending an infertility study. < 10%. Quality control (QC) samples and clinic, increased urinary concentration of MBzP Physical examination. After standard laboratory blanks were analyzed along with was also associated with decreased follicle stimu- anthropometric measurements (height, weight, unknown samples to monitor performance of lating hormone, whereas increases in monobutyl head circumference, and skin-fold thickness) the method. The metabolite concentrations phthalate were marginally associated with were obtained, a detailed examination of the reported here are from 85 prenatal maternal increased inhibin-B (Duty et al. 2005). breast and genitals was conducted under the urine samples of a total of 214 that also Newborn male rodents have no scrotum, supervision of pediatric physicians who were included postnatal maternal and baby samples and the external genitalia are undeveloped; trained in its administration. Every attempt from the same mothers and their children. only a genital tubercle is apparent for both was made to standardize the examination, The 214 samples were analyzed for phthalate sexes. The distance from the anus to the which was developed specifically for this study. metabolites in six batches, none of which insertion of this tubercle, the AGD, is andro- These methods included training sessions had to be re-extracted for QC failures. Of the gen dependent and about twice as long in before and during the study and the use of stan- 214 samples, seven were re-extracted using males as in females. The AGD has been dardized equipment. Neither the pediatric < 1 mL of urine because concentrations of shown to be a sensitive measure of prenatal physicians nor the support staff had any knowl- MEP calculated using 1 mL were above the antiandrogen exposure (Rhees et al. 1997). edge of the mother’s phthalate concentrations. linear range of the method. Recently, Salazar-Martinez et al. (2004) stud- Boys’ genital examinations included a Statistical analysis. After examining ied AGD in 45 male and 42 female infants. description of the testes and scrotum, location descriptive and summary statistics for all study They measured the distance from the anus to and size of each testicle, and measurement of variables, we explored models for AGD. We the base of the scrotum in males and from the penis. The placement of each testicle was fit several alternative measures of body size the anus to the base of the genitals (the four- initially coded in six categories; in the present (weight, height, and body mass index) and chette) in females. By these measures, AGD analysis, boys are dichotomized into those with both additive and multiplicative functions of was sexually dimorphic and about twice as normal testicular descent (placement of both these. We defined the anogenital index [AGI = long in males as in females. No other studies testes coded as normal or normal retractile) or AGD/weight (mm/kg)] as a weight-normalized have examined AGD among human males, with incomplete testicular descent (all other index of AGD. although two other studies have evaluated cases). The scrotum was categorized as distinct AGD and AGI were modeled as both lin- AGD in female infants (Callegari et al. 1987; from surrounding tissue or not, and by size ear and quadratic functions of age. For babies Phillip et al. 1996). (small or not). Penile width and (stretched) born at < 38 weeks, age at examination in the length were recorded, and penile volume [pro- first year was calculated from the estimated Materials and Methods 2 × penile length] portional to (penile width/2) date of conception instead of the birth date. Study participants. Women included in our was calculated. We recorded the AGD, meas- Once the best fitting model was identified, we study were originally recruited into the first ured from the center of the anus to the anterior plotted the expected AGI and its 25th and phase of the Study for Future Families (SFFI), a base of the penis. We also recorded the ano- 75th percentiles as a function of age. We cate- multicenter pregnancy cohort study, at prenatal scrotal distance (ASD), measured from the gorized boys in two ways: We dichotomized clinics in Los Angeles, California (Harbor- center of the anus to the posterior base of the boys into those with AGI smaller than or at UCLA and Cedars-Sinai), Minneapolis, scrotum. This latter measurement was used by least as large as expected, and we used the dif- Minnesota (University of Minnesota Health Salazar-Martinez et al. (2004), who refer to it ference between observed and expected AGI to Center), and Columbia, Missouri (University as AGD. define three groups of boys, short (AGI < 25th Physicians), from September 1999 through Phthalate metabolite analysis. Urinary percentile for age), intermediate (25th per- August 2002. Data collection is still ongoing phthalate metabolite analyses were carried centile ≤ AGI < 75th percentile), and long in Iowa, where a center was added late in out by the Division of Laboratory Sciences, (AGI ≥ 75th percentile for age) AGI. We also SFFI, so Iowa participants are not included in National Center for Environmental Health, calculated the proportion of boys in these three this analysis. Methods are described in detail Centers for Disease Control and Prevention groups with normal testicular descent (both elsewhere (Swan et al. 2003). Briefly, couples (CDC), which had no access to participant testes normal or normal retractile) and normal whose pregnancy was not medically assisted data. The analytical approach for the analysis scrotal (scrotum of normal size and distinct were eligible unless the woman or her partner of urinary phthalate metabolites (Silva et al. from surrounding tissue). We calculated the was < 18 years of age, either partner did not 2004b) is a modification of previously pub- correlations between AGD and AGI and penile read and speak Spanish or English, or the lished methods (Silva et al. 2003). The analy- volume, testicular placement and scrotal para- father was unavailable or unknown. All partici- sis involves the enzymatic deconjugation of meters (size and distinctness from surrounding pants completed a questionnaire, most gave the phthalate metabolites from their glu- tissue). Our decision to use AGI as the measure blood samples, and after urine collection was curonidated form, automated on-line solid- of genital development was made, and cut added midway through the study, most also phase extraction, separation with high- points for categorical analyses of outcomes gave a urine sample. performance liquid chromatography, and were selected, before obtaining phthalate Eighty-five percent of SFFI participants detection by isotope-dilution tandem mass metabolite values. agreed to be recontacted, and we invited these spectrometry. This high-throughput method We used general linear models to explore mothers to take part in our follow-up study. allows for the simultaneous quantification in the relationships between phthalate metabolite The family was eligible for the follow-up study human urine of the nine phthalate metabolites concentration (unadjusted for urine concentra- (SFFII) if the pregnancy ended in a live birth, reported in this work. Limits of detection tion) and genital parameters. Most metabolite the baby was 2–36 months of age, and the (LOD) are in the low nanogram per milliliter concentrations were above the LOD; those mother lived within 50 mi of the clinic and range. Isotopically labeled internal standards below the LOD were assigned the value LOD could attend at least one study visit. Here we were used along with conjugated internal stan- divided by the square root of 2, which has been report on results from the first study visit only. dards to increase precision and accuracy of the recommended when the data are not highly Human subject committees at all participating measurements. The method is accurate (spiked skewed, as was the case here (Hornung and Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1057 Swan et al. Reed 1990). Metabolite concentrations were have genital measurements, no frank genital metabolites (other than MEP) were signifi- logarithmically transformed to normalize dis- malformations or disease were detected, and no cantly correlated (p < 0.005). tributions. We examined several potentially parameters appeared grossly abnormal. The Regression analyses. We initially modeled confounding factors including mother’s ethnic- mean age at first examination was 15.9 months, AGD as a linear function of age and weight, = 0.22). ity and smoking status, time of day and season and mean weight was 10.5 kg (Table 2). Mean but this model fit poorly (adjusted R in which the urine sample was collected, gesta- (± SD) AGD was 70.3 ± 11.0 mm, with a dis- We found that using AGI (AGD/weight) as a tional age at sample collection, and baby’s tribution that was well approximated by a nor- function of age provided the best fit, as has weight at examination. mal curve. Overall, 86.6% of boys had both been shown in rodent models (Vandenbergh We also categorized metabolite concentra- testes classified as normal or normal-retractile. and Huggett 1995). The best-fitting model for tions into low (< 25th percentile), intermediate A prenatal urine sample was assayed for AGI includes linear and quadratic terms for (between the 25th and 75th percentiles), and phthalate metabolites for mothers of 85 of these age and is given by AGI = 10.8835 – 0.3798 2 2 high (≥ 75th percentile) categories and exam- boys. These mother–son pairs comprise the (age) + 0.0068 (age ) (adjusted R = 0.61). ined the odds ratio (OR) for smaller than data set for the analysis of AGD and phthalate Using this model, we calculated mean AGI expected AGI for babies with high compared metabolite concentration. Because urine collec- and its 5th, 25th, 75th, and 95th percentiles with low exposure, and medium compared tion began midway through SFFI, mothers with (Figure 1). with low. On the basis of these regression and a stored urine sample were recruited later in We then examined models that included categorical analyses, we identified the phthalate the study, and their sons tended to be younger individual phthalate metabolites. Other than metabolites most strongly associated with AGI. at examination (mean age, 12.6 months; age and age squared, no covariates altered We refer to these as AGI-associated phthalates. interquartile range, 5–16 months). Summary regression coefficients for the phthalate metabo- Because phthalate metabolite concentra- statistics for all boys included in the analysis lites by > 15%, and none were included in final tions are highly correlated, and because our lim- of physical measurements, and the subset of models. All regression coefficients for individ- ited sample size prohibited us from examining boys for whom mothers’ prenatal phthalate ual metabolites (logarithmically transformed to multiway interactions, we constructed a sum- concentrations were also available are shown normalize distributions) were negative (Table mary phthalate score to examine the effect of separately in Table 2. 4). MEP, mono-n-butyl phthalate (MBP), joint exposure to more than one AGI-associated All phthalate metabolites tested were above MBzP, and monoisobutyl phthalate (MiBP) phthalate. For this purpose, we used quartiles of the LOD in > 49% of women, and most tested were (inversely) related to AGI; p-values for metabolite concentration; values in the lowest were above the LOD in > 90% of the samples regression coefficients were between 0.007 quartile did not contribute to the sum, whereas (Table 3). Concentrations spanned four orders and 0.097. We also measured three metabo- higher values increased the sum one unit per of magnitude, from below the LOD (estimated lites of DEHP. Although the hydrolytic quartile. We divided this sum into three cate- value = 0.71 ng/mL) to 13,700 ng/mL for monoester metabolite mono-2-ethylhexyl gories: low (0–1, reflecting little or no exposure MEP. Means ranged from 2.68 for mono- phthalate (MEHP) was unrelated to AGI to AGI-associated phthalates), intermediate 3-carboxypropyl phthalate (MCPP) to 629.8 [regression coefficient = –0.05; 95% confi- (2–10), and high (11–12, reflecting high expo- for MEP. Three of the four AGI-associated dence interval (CI), –0.53 to 0.43], regression sure to all, or almost all, AGI-associated phtha- Table 1. Participants included in present analysis. lates). We examined the magnitude of the Percent Percent residual (observed – expected) AGI as a func- No. potential participants male babies tion of this summary phthalate score. All pregnancy outcomes (CA, MN, and MO) Results Potential participants 654 100 — Eligible for SFFII 477 72.9 — The population for the present analysis was SFFII participant 346 72.5 — identified from families recruited in California, Male babies only (CA, MN, and MO) Minnesota, or Missouri for whom data entry SFFII participant 172 — 100 With AGD, age, and weight 134 — 78 was complete by 17 December 2004, the cutoff Prenatal urine sample 85 — 49 date for the present analysis. At that time, 654 participants from these three centers had A potential participant is an SFFI participant from CA, MO, or MN who gave permission to be recontacted for future studies and for whom all study data were entered by 17 December 2004. Boys in twin births and boys with missing data or AGD completed SFFI and given permission to be measurements considered unreliable by pediatricians excluded. Urine collection began midway through SFFI. recontacted. Of these, 477 (72.9%) were eligi- ble for SFFII and 346 (72.5%) participated Table 2. Characteristics of boys with complete physical examination. (Table 1). SFFII participants were demographi- Percentile cally similar to nonparticipants except that non- Characteristic Mean ± SD 25th 50th 75th participants were more likely to be Hispanic All boys (n = 134) because of a lower eligibility rate (60%) in CA, Age (months) 15.9 ± 8.6 11.0 15.0 23.0 where most participants were Hispanic. Of the Height (cm) 79.1 ± 10.6 72.6 80.0 87.2 172 boys born to these mothers, we excluded Weight (kg) 10.5 ± 2.7 8.7 10.7 12.3 5 boys in twin births, 10 boys with incomplete AGD (mm) 70.3 ± 11.0 63.9 70.3 76.6 AGI (mm/kg) 7.1 ± 1.9 5.8 6.7 7.8 data, and 23 boys for whom AGD was not ASD (mm) 37.4 ± 7.5 31.2 36.8 43.4 recorded [two whose mothers declined the geni- Boys whose mother’s prenatal urine was tal exam, with the remainder older boys (mean assayed for phthalate metabolites (n = 85) age, 19.6 months), for whom the study exam- Age (months) 12.6 ± 6.9 5.0 14.0 16.0 iner felt the measurement was not reliable, Height (cm) 75.6 ± 9.5 66.5 77.6 82.0 usually because of the boys’ activity level]. The Weight (kg) 9.7 ± 2.4 8.4 10.0 11.1 AGD (mm) 68.0 ± 9.7 61.7 66.7 74.4 remaining 134 boys comprise the sample used AGI (mm/kg) 7.4 ± 1.8 6.1 7.0 8.2 for the analysis of AGD and other genital meas- ASD (mm) 35.9 ± 7.1 30.4 35.6 41.4 urements. Among the 134 boys for whom we 1058 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance coefficients for the oxidative monoester MEP, MBzP, and MiBP were 4.7, 3.8, and –0.137). For the other phthalate metabolites, metabolites of DEHP, mono-2-ethyl-5-oxo- 9.1, respectively (all p-values < 0.05). regression coefficients were less significant (all hexyl phthalate (MEOHP), and mono-2-ethyl- Other genital parameters. Degree of testic- p-values between 0.11 and 0.97). 5-hydroxyhexyl phthalate (MEHHP) were of a ular descent was associated with AGD (R = Summary phthalate score. We used magnitude comparable with those for MEP 0.20, p = 0.02). The proportions of boys with the summary phthalate score as defined in and MBzP (p-values = 0.114 and 0.145 for one or both testicles incompletely descended “Materials and Methods” to study the effect of MEOHP and MEHHP, respectively). AGI were 20.0, 9.5, and 5.9% for boys classified as joint exposure to more than one AGI-associated appeared to be independent of the concentra- having short, intermediate, and long AGI phthalate. The summary phthalate score was tions of monomethyl phthalate (MMP) and (p-value for short AGI compared with all other directly related to the proportion of boys with MCPP, metabolites of dimethyl phthalate and boys < 0.001). The proportion of boys with a short AGI (p = 0.001). Of the 10 boys whose di-n-octyl phthalate, respectively. scrotum categorized as small and/or “not dis- phthalate scores were high (score = 11–12), all Categorical analyses. The 25 boys with tinct from surrounding tissue” was also ele- but one had a short AGI. Conversely, of the AGI below the 25th percentile for age were vated for boys with short AGI (p < 0.001). 11 boys whose scores were low (score = 0 or 1), classified as having a short AGI. This group had AGD was significantly associated with penile only one had a short AGI. The ORs for having an AGI that was, on average, 18.3% (range, volume (R = 0.27, p = 0.001), and penile vol- a short AGI for high summary phthalate score 10–32%) shorter than expected based on the ume divided by weight was correlated with compared with low (OR = 90.0; 95% CI, final regression model. Boys with AGI ≥ 75th AGI (R = 0.43, p = 0.001). Testicular volume, 4.88 to 1,659), and high compared with percentile of expected were classified as having a which was measured by orchidometer, is not medium (29.4; 95% CI, 3.4 to 251) were large long AGI, and boys with AGI between the shown here because participating physicians and significant, although the confidence inter- 25th and 75th percentile of expected were con- considered the measurement to be unreliable— vals were very wide. These data are shown sidered intermediate. Boys’ weight and age did a decision made before analyses of phthalate graphically in Figure 1. not differ appreciably among these groups. exposure. Discussion Table 5 shows mean and median values ASD was, on average, 47% as long as for the AGI-associated metabolites for boys in AGD, and these two measurements were cor- In the recent National Health and Nutrition the short, intermediate, and long categories of related (R = 0.47, p < 0.0001). However, the Examination Survey (NHANES 1999–2000), AGI. We calculated the ORs for short AGI for model predicting ASD as a function of baby’s most of the general population in the United = 0.10). each monoester metabolite (Table 6). For high age and weight fit poorly (adjusted R States had measurable exposure to multiple compared with low concentration of MBP, The fit for the model using ASD/weight as a phthalates (CDC 2003; Silva et al. 2004a). the OR for a short AGI was 10.2 (95% CI, function of age and age squared was better The samples in the present study and in 2.5 to 42.2), whereas for medium concentra- (adjusted R = 0.47) but did not fit as well as NHANES were both analyzed using compara- = 0.61). ASD/weight tion compared with low the OR was 3.8 (95% the model using AGI (R ble methods and standards by the same labora- CI, 1.2 to 12.3). The corresponding ORs for was associated with MEP concentration (regres- tory, although the specific metabolites that high compared with low concentration of sion coefficient = –0.429; 95% CI, –0.722 to were measured in the two studies differed somewhat. We compared the medians and 75th percentiles of the AGI-associated phtha- Table 3. Percentiles of phthalate monoester metabolites. late metabolite concentrations among two Percentile (ng/mL) a groups of mothers in our study (those whose Monoester metabolite 25th 50th 75th Percent > LOD boys fell in the short AGI group and all others) Phthalate monoester metabolite with those of females in the NHANES sample MBP 7.2 13.5 30.9 96.5 (Table 7). In the analysis of the NHANES MBzP 3.5 8.3 23.5 94.1 samples, monobutyl phthalate includes both MCPP 0.7 2.1 3.6 69.4 MEP 53.3 128.4 436.9 97.6 MBP and MiBP, which were measured sepa- MiBP 0.7 2.5 5.1 74.1 rately in our study. Metabolite concentrations MMP 0.7 0.7 3.2 49.4 for mothers of boys with short AGI were con- Metabolites of DEHP sistently higher than those of other mothers. MEHHP 6.0 11.4 20.1 97.6 Compared with women in the NHANES sam- MEHP 1.3 3.3 9.0 77.6 ple, metabolite concentrations for our popula- MEOHP 5.1 11.1 19.0 94.1 tion were somewhat lower. However, our LOD for all metabolites was between 0.95 and 1.07 ng/mL. population cannot be directly compared with Phthalate score 0–1 Table 4. Regression analyses of AGI on log monoester metabolite concentration, controlling for age and Phthalate score 2–10 age squared. Phthalate score 11–12 Log monoester metabolite concentration Monoester metabolite Coefficient (SE) p-Value (95% CI) 95% MBP –0.592 (0.269) 0.031 (–1.126 to –0.057) MBzP –0.390 (0.232) 0.097 (–0.851 to 0.072) 75% Mean MCPP –0.264 (0.356) 0.461 (–0.973 to 0.445) 25% MEHHP –0.398 (0.270) 0.145 (–0.935 to 0.140) MEHP –0.051 (0.241) 0.833 (–0.530 to 0.428) 4 5% MEOHP –0.412 (0.258) 0.114 (–0.925 to 0.101) MEP –0.400 (0.164) 0.017 (–0.726 to –0.074) 0 5 10 15 20 25 30 35 40 MiBP –0.765 (0.274) 0.007 (–1.309 to –0.220) Boy’s age (months) MMP –0.283 (0.323) 0.383 (–0.924 to 0.359) Phthalate score –0.0951 (0.035) 0.009 (–0.165 to –0.025) Figure 1. Mean AGI (mm/kg) in relation to boys’ age at examination (months). Phthalate score measures joint exposure to MBP, MBzP, MEP, and MiBP; see “Statistical analysis.” Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1059 AGI (mm/kg) Swan et al. NHANES: the proportion of pregnant women more sensitive to trenbolone by an order of human studies suggesting reproductive toxic- in the NHANES sample is unknown, and age magnitude (Neumann 1976). This greater ity (Colón et al. 2000; Duty et al. 2003b; distributions differ. Nonetheless, these data sensitivity is thought to be a result of rodents’ Main KM, unpublished data). It is therefore demonstrate that the four AGI-associated higher metabolic rate and more rapid inactiva- uncertain whether the absence of data in phthalate metabolites are prevalent in the U.S. tion of toxicants, both of which have been rodents showing reproductive toxicity is the female population, and levels were not unusu- shown to be inversely related to body size result of failure to detect it, unmeasured con- ally high among mothers whose sons had a (White and Seymour 2005). founding in human studies, or interspecies short AGI. In light of the toxicologic literature differences in response to these compounds. Although not identical, AGD in pups is for MBP, MBzP, and MiBP (Ema et al. 2003; DEHP has been shown to shorten AGD most similar to AGD as we defined it in this Foster et al. 1980, 1981; Gray et al. 2000; (Gray et al. 2000) and reduce testosterone study. In rodents, AGD has been shown to be Nakahara et al. 2003), our data suggest that (Parks et al. 2000). Although MEHP was not one of the most sensitive end points for phtha- the end points affected by these phthalates are associated with AGD in our data, the associa- lates such as DBP (Mylchreest et al. 2000) and quite consistent across species. A boy with tions for the oxidative metabolites of DEHP other antiandrogens such as flutamide (Barlow short AGI has, on average, an AGI that is 18% (MEOHP and MEHHP) were of comparable and Foster 2003; McIntyre et al. 2001) and shorter than expected based on his age and magnitude with those for metabolites of DBP finasteride (Bowman et al. 2003). It is difficult weight as well as an increased likelihood of tes- and BzBP, although not statistically signifi- to compare the dose to humans from low- ticular maldescent, small and indistinct scro- cant. Thus, it is unclear whether MEOHP level, ongoing, environmental exposure with tum, and smaller penile size. These changes in and MEHHP are (inversely) associated with that delivered to rodents experimentally in AGD and testicular descent are consistent with AGI, although associations are of borderline a narrow window of gestation. Nonetheless, those reported in rodent studies after high-dose statistical significance because of our sample it is likely that the doses to which our par- phthalate exposure (Ema et al. 2003; Gray size, or whether human and rodent responses ticipants were exposed are lower than those et al. 2000; Mylchreest et al. 2000). The lack to this phthalate and its metabolites differ. used in toxicologic settings, suggesting that of association for MCPP and MMP, which Masculinization of external male genitalia, humans may be more sensitive to prenatal have not been widely studied, is not inconsis- represented by longer AGD, is controlled by phthalate exposure than rodents. This greater tent with the toxicologic literature. dihydrotestosterone (Clark et al. 1990). Ema sensitivity in humans has been observed for With respect to DEP and its metabolite and Miyawaki (2001) demonstrated that other toxicants. For example, humans are MEP, we note that there are three other this metabolite of testosterone is markedly decreased by prenatal administration of MBP, Table 5. Mean (median) phthalate monoester metabolite levels by AGI category. suggesting that MBP acts as an antiandrogen. AGI category [mean (median; ng/mL)] AGD in male rodents is associated with other a b c Monoester metabolite Long (n = 17) Intermediate (n = 43) Short (n = 25) adverse developmental effects (Foster and MBP 13.1 (11.5) 22.2 (13.1) 38.7 (24.5) McIntyre 2002) and some phthalate-induced MBzP 10.6 (6.6) 15.1 (7.7) 25.8 (16.1) changes have been shown to be permanent. MEP 124 (47.1) 592 (112) 1,076 (225) For example, Barlow et al. (2004) report that MiBP 2.3 (1.5) 3.3 (2.1) 7.7 (4.8) prenatal exposure to 500 mg/kg/day DBP a b Long, AGI ≥ 75th percentile of expected AGI. Intermediate, 25th percentile ≤ AGI < 75th percentile of expected AGI. resulted in permanently decreased AGD and Short, AGI < 25th percentile of expected AGI. testicular dysgenesis. They also report that Table 6. OR (95% CI) for AGI less than expected from regression model, by monoester metabolite level. in utero DBP exposure induced proliferative Leydig cell lesions. Follow-up of exposed chil- Monoester metabolite Level (percentile) AGI < expected AGI ≥ expected OR (95% CI) dren until adulthood will be required to MBP Low 5 15 Referent determine whether long-term effects, includ- Medium 24 19 3.8 (1.2 to 12.3) ing testicular dysgenesis, are seen in humans High 17 5 10.2 (2.5 to 42.2) MBzP Low 6 13 Referent after prenatal phthalate exposure. Medium 26 18 3.1 (1.002 to 9.8) Several recent studies of the variability of High 14 8 3.8 (1.03 to 13.9) phthalate monoester concentration in human MEP Low 7 14 Referent samples suggest that phthalate concentration in Medium 25 19 2.6 (0.9 to 7.8) humans is fairly stable, perhaps reflecting High 14 6 4.7 (1.2 to 17.4) habitual use of phthalate-containing household MiBP Low 6 16 Referent Medium 23 18 3.4 (1.1 to 10.5) and consumer products (Colón et al. 2000; High 17 5 9.1 (2.3 to 35.7) Hauser et al. 2004; Hoppin et al. 2002). These studies lend support to the use of a single Low, < 25th percentile; medium, ≥ 25th and < 75th percentile; high, ≥ 75th percentile. sample for exposure assessment. We obtained Table 7. Concentrations of four phthalate metabolites in three groups of women (ng/mL). only a single prenatal urine sample from each This study woman, and most samples were obtained Monoester metabolite Percentile Short AGI Others NHANES quite late in pregnancy (mean = 28.3 weeks). MBP 50th 24.5 12.1 30.0 Therefore, the measured phthalate metabolite 75th 44.8 28.0 59.5 levels may not reflect exposure during the most MBzP 50th 16.1 7.2 16.0 sensitive developmental window, resulting in 75th 27.5 17.8 35.8 some degree of exposure misclassification. MEP 50th 225 90.4 174 However, unless this misclassification varied 75th 551 281 425 MiBP 50th 4.8 2.1 — systematically with outcome, such errors would 75th 12.1 4.3 — bias the effect estimate toward the null. In a b fact, the categorical analysis, which should be Females only (CDC 2003). MBP in the NHANES analysis includes both MBP and MiBP; in this study these metabolites were measured separately. less sensitive to such misclassification, showed 1060 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance stronger associations than did the continuous Parks L. 2000. Perinatal exposure to the phthalates DEHP, genital morphology is altered by antiandro- BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual analysis. gens, including some phthalates. We report differentiation of the male rat. Toxicol Sci 58:350–365. Our analysis is based on a single measure of that AGD, the most sensitive marker of anti- Hauser R, Meeker JD, Park S, Silva MJ, Calafat AM. 2004. Temporal variability of urinary phthalate metabolite lev- AGD, and the reliability of this measurement androgen action in toxicologic studies, is els in men of reproductive age. Environ Health Perspect in humans has not been established. During shortened and testicular descent impaired in 112:1734–1740. two training sessions, three study physicians boys whose mothers had elevated prenatal Hoppin JA, Brock JW, Davis BJ, Baird DD. 2002. Reproducibility of urinary phthalate metabolites in first morning urine each measured AGD in four male infants phthalate exposure. These changes in male samples. Environ Health Perspect 110:515–518. (mean age, 8.1 months). The mean AGD for infants, associated with prenatal exposure to Hornung RW, Reed LD. 1990. Estimation of average concentra- tion in the presence of nondectable values. Appl Occup these measurements was 58.6 mm, SD was some of the same phthalate metabolites that Environ Hyg 5:46–51. (within infant) 4.2 mm, and coefficient of vari- cause similar alterations in male rodents, sug- McIntyre BS, Barlow NJ, Foster PM. 2001. Androgen-mediated ation of 7.2%, suggesting that AGD can be gest that commonly used phthalates may development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal measured reliably. Use of this measurement in undervirilize humans as well as rodents. changes in anogenital distance and nipple retention with larger studies in a range of diverse populations, malformations in androgen-dependent tissues. 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The plasticizer diethylhexyl phtha- Clark RL, Antonello JM, Grossman SJ, Wise LD, Anderson C, late induces malformations by decreasing fetal testos- value in adolescents and adults has yet to be Bagdon WJ, et al. 1990. External genitalia abnormalities in terone synthesis during sexual differentiation in the male male rats exposed in utero to finasteride, a 5 alpha-reduc- determined. rat. Toxicol Sci 58:339–349. tase inhibitor. Teratology 42:91–100. We note that phthalate metabolite levels Phillip M, De Boer C, Pilpel D, Karplus M, Sofer S. 1996. Clitoral Colón I, Caro D, Bourdony CJ, Rosario O. 2000. Identification and penile sizes of full term newborns in two different were highly correlated, and most women were of phthalate esters in the serum of young Puerto Rican ethnic groups. J Pediatr Endocrinol Metab 9:175–179. girls with premature breast development. Environ Health exposed to all metabolites at detectable levels. Rhees RW, Kirk BA, Sephton S, Lephart ED. 1997. 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AGI-associated phthalates, and our results sug- 069X-3-8 [Online 13 September 2004]. 2003b. The relationship between environmental exposures to Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, gest that joint exposure may convey greater phthalates and DNA damage in human sperm using the neu- et al. 2004a. Urinary levels of seven phthalate metabolites in tral comet assay. Environ Health Perspect 111:1164–1169. than additive risk, but larger sample sizes are the U.S. population from the National Health and Nutrition Ema M, Miyawaki E. 2001. Adverse effects on development of Examination Survey (NHANES) 1999–2000. Environ Health needed to confirm this. the reproductive system in male offspring of rats given Perspect 112:331–338. monobutyl phthalate, a metabolite of dibutyl phthalate, Gray and Foster (2003) refer to a “phtha- Silva MJ, Malek NA, Hodge CC, Reidy JA, Kato K, Barr DB, et al. during late pregnancy. Reprod Toxicol 15:189–194. late syndrome” characterized by testicular, epi- 2003. Improved quantitative detection of 11 urinary phtha- Ema M, Miyawaki E, Hirose A, Kamata E. 2003. Decreased late metabolites in humans using liquid chromatography- didymal, and gubernacular cord agenesis as well anogenital distance and increased incidence of unde- atmospheric pressure chemical ionization tandem mass scended testes in fetuses of rats given monobenzyl phtha- as decreased AGD, and stress the importance of spectrometry. J Chromatogr B Analyt Technol Biomed Life late, a major metabolite of butyl benzyl phthalate. Reprod evaluating all components of a syndrome so Sci 789:393–404. Toxicol 17:407–412. Silva MJ, Slakman AR, Reidy JA, Preau JL Jr, Herbert AR, Fisher JS. 2004. Environmental anti-androgens and male repro- that affected animals are not misidentified. It Samandar E, et al. 2004b. 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Studies on the testicular effects and zinc excretion pro- programming and gonadal development during Swan SH, Brazil C, Drobnis EZ, Liu F, Kruse RL, Hatch M, et al. duced by various isomers of monobutyl-o-phthalate in the fetal life. The present findings, though based on 2003. Geographic differences in semen quality of fertile U.S. rat. Chem Biol Interact 34:233–238. males. Environ Health Perspect 111:414–420. Foster PM, McIntyre BS. 2002. Endocrine active agents: implica- small numbers, provide the first data in humans Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Brine DR, et al. tions of adverse and non-adverse changes. Toxicol Pathol linking measured levels of prenatal phthalates to 2004. Reproductive toxicity evaluation of dietary butyl ben- 30:59–65. zyl phthalate (BBP) in rats. Reprod Toxicol 18:241–264. outcomes that are consistent with this proposed Foster PM, Thomas LV, Cook MW, Gangolli SD. 1980. Study of Vandenbergh JG, Huggett CL. 1995. The anogenital distance the testicular effects and changes in zinc excretion pro- syndrome. index, a predictor of the intrauterine position effects duced by some n-alkyl phthalates in the rat. Toxicol Appl This is the first study to look at subtle on reproduction in female house mice. Lab Anim Sci Pharmacol 54:392–398. 45:567–573. patterns of genital morphology in humans in Gray LE Jr, Foster PMD. 2003. Significance of experimental White CR, Seymour RS. 2005. Allometric scaling of mammalian studies for assessing adverse effects of endocrine- relation to any prenatal exposure. It was moti- metabolism. J Exp Biol 208:1611–1619. disrupting chemicals. Pure Appl Chem 75:2125–2141. vated by toxicologic studies showing that Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DNR, Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1061 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Health Perspectives Pubmed Central

Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure

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10.1289/ehp.8100
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Abstract

Research Children’s Health Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure 1 2 3 3 3 4 Shanna H. Swan, Katharina M. Main, Fan Liu, Sara L. Stewart, Robin L. Kruse, Antonia M. Calafat, 5 6 7 8 9 Catherine S. Mao, J. Bruce Redmon, Christine L. Ternand, Shannon Sullivan, J. Lynn Teague, and the Study for Future Families Research Team* 1 2 Department of Obstetrics and Gynecology, University of Rochester, Rochester, Minnesota, USA; Department of Growth and Reproduction, University of Copenhagen, Copenhagen, Denmark; Department of Family and Community Medicine, University of Missouri-Columbia, Columbia, Missouri, USA; National Center for Environmental Health, Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, Georgia, USA; Department of Pediatrics, Division of Endocrinology, Los Angeles Biomedical 6 7 Research Institute at Harbor-UCLA Medical Center, Los Angeles, California, USA; Departments of Pediatrics and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA; Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA; Departments of Surgery (Urology) and Child Health, University of Missouri-Columbia, Columbia, Missouri, USA As with the Colón et al. study, contamination Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in from diesters in laboratory equipment could male rodents. We present data from the first study to examine AGD and other genital measure- not be excluded (Murature et al. 1987). ments in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was More recent studies have examined phtha- obtained in 134 boys 2–36 months of age. AGD was significantly correlated with penile volume late monoester metabolites in urine. Because (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20, urinary metabolites are not likely to be present p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination as the result of contamination, these studies [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We avoid this potential source of measurement examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors error. Duty et al. (2003a) reported dose– of age-adjusted AGI in regression and categorical analyses that included all participants with prena- response relationships between tertiles of tal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phtha- monobutyl phthalate and sperm motility and late (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl sperm concentration, and between tertiles of phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values monobenzyl phthalate (MBzP) and sperm for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP con- concentration. They also reported inverse dose– centration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than response relationships between monoethyl expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for phthalate (MEP) and sperm DNA damage MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age- Address correspondence to S.H. Swan, University of adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009). Rochester, Department of Obstetrics and Gynecology, The associations between male genital development and phthalate exposure seen here are consistent School of Medicine and Dentistry, 601 Elmwood Ave., Box 668, Rochester, NY 14642-8668 USA. with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally Telephone: (585) 273-3521. Fax: (585) 275-7366. exposed rodents. The median concentrations of phthalate metabolites that are associated with E-mail: shanna_swan@urmc.rochester.edu short AGI and incomplete testicular descent are below those found in one-quarter of the female *The Study for Future Families Research Team population of the United States, based on a nationwide sample. These data support the hypothesis included, from the University of Missouri- that prenatal phthalate exposure at environmental levels can adversely affect male reproductive Columbia: E.Z. Drobnis, B.S. Carter, D. Kelly, and development in humans. Key words: anogenital distance, benzylbutyl phthalate, dibutyl phthalate, T.M. Simmons. Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center: diethyl phthalate, monobenzyl phthalate, monoethyl phthalate, monoisobutyl phthalate, mono-n- C. Wang, L. Lumbreras, S. Villanueva, M. Diaz- butyl phthalate, phthalates, prenatal exposure. Environ Health Perspect 113:1056–1061 (2005). Romero, M.B. Lomeli, and E. Otero-Salazar. Cedars- doi:10.1289/ehp.8100 available via http://dx.doi.org/ [Online 27 May 2005] Sinai Medical Center: C. Hobel and B. Brock. University of Minnesota: C. Kwong and A. Muehlen. University of Iowa: A. Sparks, A. Wolf, J. Whitham, Diesters of phthalic acid, commonly referred et al. 2000), and DEHP (Gray et al. 2000; M. Hatterman-Zogg, and M. Maifeld. We thank the health care providers and study par- to as phthalates, are widely used in industry Parks et al. 2000). ticipants at University Physicians Clinic (Columbia, and commerce; they are used in personal care Despite the growing body of literature MO), Fairview Riverside Women’s Clinic (Minne- products (e.g., makeup, shampoo, and soaps), on phthalate reproductive toxicity and data apolis, MN), Los Angeles Biomedical Research plastics, paints, and some pesticide formula- demonstrating extensive human exposure Institute at Harbor-UCLA Medical Center (Los tions. Consistent toxicologic evidence indi- (Silva et al. 2004a), few studies have examined Angeles, CA), Cedars-Sinai Medical Center (Los cates association between several of these the effects of these chemicals on human repro- Angeles, CA), and University of Iowa Hospitals and Clinics (Iowa City, IA). We also thank M. Silva, phthalate esters and reproductive effects. ductive development. Colón et al. (2000) J. Reidy, E. Samandar, and J. Preau for phthalate In particular, dibutyl phthalate (DBP), ben- reported elevated levels of several phthalates analyses and E. Gray, P. Foster, and D. Barr for their zylbutyl phthalate (BzBP), di-2-ethylhexyl [including diethyl phthalate (DEP), DBP, and guidance. phthalate (DEHP), and di-isononyl phthalate DEHP] in serum samples from young girls This work was supported by grants from the U.S. have been shown to disrupt reproductive tract with premature breast development. However, Environmental Protection Agency and the National development in male rodents in an antian- the timing of exposure was unknown and high Institutes of Health (R01-ES09916 to the University of Missouri, MO1-RR00400 to the University of drogenic manner (Parks et al. 2000). Recent exposure levels may have reflected phthalate Minnesota, MO1-RR0425 to Harbor-UCLA studies have reported significant reductions contamination of serum samples (McKee and Medical Center) and by grant 18018278 from the in anogenital distance (AGD) in Sprague- Toxicology Research Task Group 2004). Until State of Iowa to the University of Iowa. Dawley rats after prenatal exposure at high recently, the only study of humans to evaluate The authors declare they have no competing doses to BzBP (Nagao et al. 2000; Tyl et al. phthalate exposure and male reproductive financial interests. 2004), DBP (Barlow and Foster 2003; Foster toxicity measured phthalate diesters in semen. Received 7 March 2005; accepted 25 May 2005. 1056 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance measured using the neutral single-cell gel elec- institutions approved SFFI and SFFII, and all recoveries are near 100%), and precise with trophoresis (comet) assay (Duty et al. 2003b). In participants signed informed consents for each between-day relative standard deviations of this population of men attending an infertility study. < 10%. Quality control (QC) samples and clinic, increased urinary concentration of MBzP Physical examination. After standard laboratory blanks were analyzed along with was also associated with decreased follicle stimu- anthropometric measurements (height, weight, unknown samples to monitor performance of lating hormone, whereas increases in monobutyl head circumference, and skin-fold thickness) the method. The metabolite concentrations phthalate were marginally associated with were obtained, a detailed examination of the reported here are from 85 prenatal maternal increased inhibin-B (Duty et al. 2005). breast and genitals was conducted under the urine samples of a total of 214 that also Newborn male rodents have no scrotum, supervision of pediatric physicians who were included postnatal maternal and baby samples and the external genitalia are undeveloped; trained in its administration. Every attempt from the same mothers and their children. only a genital tubercle is apparent for both was made to standardize the examination, The 214 samples were analyzed for phthalate sexes. The distance from the anus to the which was developed specifically for this study. metabolites in six batches, none of which insertion of this tubercle, the AGD, is andro- These methods included training sessions had to be re-extracted for QC failures. Of the gen dependent and about twice as long in before and during the study and the use of stan- 214 samples, seven were re-extracted using males as in females. The AGD has been dardized equipment. Neither the pediatric < 1 mL of urine because concentrations of shown to be a sensitive measure of prenatal physicians nor the support staff had any knowl- MEP calculated using 1 mL were above the antiandrogen exposure (Rhees et al. 1997). edge of the mother’s phthalate concentrations. linear range of the method. Recently, Salazar-Martinez et al. (2004) stud- Boys’ genital examinations included a Statistical analysis. After examining ied AGD in 45 male and 42 female infants. description of the testes and scrotum, location descriptive and summary statistics for all study They measured the distance from the anus to and size of each testicle, and measurement of variables, we explored models for AGD. We the base of the scrotum in males and from the penis. The placement of each testicle was fit several alternative measures of body size the anus to the base of the genitals (the four- initially coded in six categories; in the present (weight, height, and body mass index) and chette) in females. By these measures, AGD analysis, boys are dichotomized into those with both additive and multiplicative functions of was sexually dimorphic and about twice as normal testicular descent (placement of both these. We defined the anogenital index [AGI = long in males as in females. No other studies testes coded as normal or normal retractile) or AGD/weight (mm/kg)] as a weight-normalized have examined AGD among human males, with incomplete testicular descent (all other index of AGD. although two other studies have evaluated cases). The scrotum was categorized as distinct AGD and AGI were modeled as both lin- AGD in female infants (Callegari et al. 1987; from surrounding tissue or not, and by size ear and quadratic functions of age. For babies Phillip et al. 1996). (small or not). Penile width and (stretched) born at < 38 weeks, age at examination in the length were recorded, and penile volume [pro- first year was calculated from the estimated Materials and Methods 2 × penile length] portional to (penile width/2) date of conception instead of the birth date. Study participants. Women included in our was calculated. We recorded the AGD, meas- Once the best fitting model was identified, we study were originally recruited into the first ured from the center of the anus to the anterior plotted the expected AGI and its 25th and phase of the Study for Future Families (SFFI), a base of the penis. We also recorded the ano- 75th percentiles as a function of age. We cate- multicenter pregnancy cohort study, at prenatal scrotal distance (ASD), measured from the gorized boys in two ways: We dichotomized clinics in Los Angeles, California (Harbor- center of the anus to the posterior base of the boys into those with AGI smaller than or at UCLA and Cedars-Sinai), Minneapolis, scrotum. This latter measurement was used by least as large as expected, and we used the dif- Minnesota (University of Minnesota Health Salazar-Martinez et al. (2004), who refer to it ference between observed and expected AGI to Center), and Columbia, Missouri (University as AGD. define three groups of boys, short (AGI < 25th Physicians), from September 1999 through Phthalate metabolite analysis. Urinary percentile for age), intermediate (25th per- August 2002. Data collection is still ongoing phthalate metabolite analyses were carried centile ≤ AGI < 75th percentile), and long in Iowa, where a center was added late in out by the Division of Laboratory Sciences, (AGI ≥ 75th percentile for age) AGI. We also SFFI, so Iowa participants are not included in National Center for Environmental Health, calculated the proportion of boys in these three this analysis. Methods are described in detail Centers for Disease Control and Prevention groups with normal testicular descent (both elsewhere (Swan et al. 2003). Briefly, couples (CDC), which had no access to participant testes normal or normal retractile) and normal whose pregnancy was not medically assisted data. The analytical approach for the analysis scrotal (scrotum of normal size and distinct were eligible unless the woman or her partner of urinary phthalate metabolites (Silva et al. from surrounding tissue). We calculated the was < 18 years of age, either partner did not 2004b) is a modification of previously pub- correlations between AGD and AGI and penile read and speak Spanish or English, or the lished methods (Silva et al. 2003). The analy- volume, testicular placement and scrotal para- father was unavailable or unknown. All partici- sis involves the enzymatic deconjugation of meters (size and distinctness from surrounding pants completed a questionnaire, most gave the phthalate metabolites from their glu- tissue). Our decision to use AGI as the measure blood samples, and after urine collection was curonidated form, automated on-line solid- of genital development was made, and cut added midway through the study, most also phase extraction, separation with high- points for categorical analyses of outcomes gave a urine sample. performance liquid chromatography, and were selected, before obtaining phthalate Eighty-five percent of SFFI participants detection by isotope-dilution tandem mass metabolite values. agreed to be recontacted, and we invited these spectrometry. This high-throughput method We used general linear models to explore mothers to take part in our follow-up study. allows for the simultaneous quantification in the relationships between phthalate metabolite The family was eligible for the follow-up study human urine of the nine phthalate metabolites concentration (unadjusted for urine concentra- (SFFII) if the pregnancy ended in a live birth, reported in this work. Limits of detection tion) and genital parameters. Most metabolite the baby was 2–36 months of age, and the (LOD) are in the low nanogram per milliliter concentrations were above the LOD; those mother lived within 50 mi of the clinic and range. Isotopically labeled internal standards below the LOD were assigned the value LOD could attend at least one study visit. Here we were used along with conjugated internal stan- divided by the square root of 2, which has been report on results from the first study visit only. dards to increase precision and accuracy of the recommended when the data are not highly Human subject committees at all participating measurements. The method is accurate (spiked skewed, as was the case here (Hornung and Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1057 Swan et al. Reed 1990). Metabolite concentrations were have genital measurements, no frank genital metabolites (other than MEP) were signifi- logarithmically transformed to normalize dis- malformations or disease were detected, and no cantly correlated (p < 0.005). tributions. We examined several potentially parameters appeared grossly abnormal. The Regression analyses. We initially modeled confounding factors including mother’s ethnic- mean age at first examination was 15.9 months, AGD as a linear function of age and weight, = 0.22). ity and smoking status, time of day and season and mean weight was 10.5 kg (Table 2). Mean but this model fit poorly (adjusted R in which the urine sample was collected, gesta- (± SD) AGD was 70.3 ± 11.0 mm, with a dis- We found that using AGI (AGD/weight) as a tional age at sample collection, and baby’s tribution that was well approximated by a nor- function of age provided the best fit, as has weight at examination. mal curve. Overall, 86.6% of boys had both been shown in rodent models (Vandenbergh We also categorized metabolite concentra- testes classified as normal or normal-retractile. and Huggett 1995). The best-fitting model for tions into low (< 25th percentile), intermediate A prenatal urine sample was assayed for AGI includes linear and quadratic terms for (between the 25th and 75th percentiles), and phthalate metabolites for mothers of 85 of these age and is given by AGI = 10.8835 – 0.3798 2 2 high (≥ 75th percentile) categories and exam- boys. These mother–son pairs comprise the (age) + 0.0068 (age ) (adjusted R = 0.61). ined the odds ratio (OR) for smaller than data set for the analysis of AGD and phthalate Using this model, we calculated mean AGI expected AGI for babies with high compared metabolite concentration. Because urine collec- and its 5th, 25th, 75th, and 95th percentiles with low exposure, and medium compared tion began midway through SFFI, mothers with (Figure 1). with low. On the basis of these regression and a stored urine sample were recruited later in We then examined models that included categorical analyses, we identified the phthalate the study, and their sons tended to be younger individual phthalate metabolites. Other than metabolites most strongly associated with AGI. at examination (mean age, 12.6 months; age and age squared, no covariates altered We refer to these as AGI-associated phthalates. interquartile range, 5–16 months). Summary regression coefficients for the phthalate metabo- Because phthalate metabolite concentra- statistics for all boys included in the analysis lites by > 15%, and none were included in final tions are highly correlated, and because our lim- of physical measurements, and the subset of models. All regression coefficients for individ- ited sample size prohibited us from examining boys for whom mothers’ prenatal phthalate ual metabolites (logarithmically transformed to multiway interactions, we constructed a sum- concentrations were also available are shown normalize distributions) were negative (Table mary phthalate score to examine the effect of separately in Table 2. 4). MEP, mono-n-butyl phthalate (MBP), joint exposure to more than one AGI-associated All phthalate metabolites tested were above MBzP, and monoisobutyl phthalate (MiBP) phthalate. For this purpose, we used quartiles of the LOD in > 49% of women, and most tested were (inversely) related to AGI; p-values for metabolite concentration; values in the lowest were above the LOD in > 90% of the samples regression coefficients were between 0.007 quartile did not contribute to the sum, whereas (Table 3). Concentrations spanned four orders and 0.097. We also measured three metabo- higher values increased the sum one unit per of magnitude, from below the LOD (estimated lites of DEHP. Although the hydrolytic quartile. We divided this sum into three cate- value = 0.71 ng/mL) to 13,700 ng/mL for monoester metabolite mono-2-ethylhexyl gories: low (0–1, reflecting little or no exposure MEP. Means ranged from 2.68 for mono- phthalate (MEHP) was unrelated to AGI to AGI-associated phthalates), intermediate 3-carboxypropyl phthalate (MCPP) to 629.8 [regression coefficient = –0.05; 95% confi- (2–10), and high (11–12, reflecting high expo- for MEP. Three of the four AGI-associated dence interval (CI), –0.53 to 0.43], regression sure to all, or almost all, AGI-associated phtha- Table 1. Participants included in present analysis. lates). We examined the magnitude of the Percent Percent residual (observed – expected) AGI as a func- No. potential participants male babies tion of this summary phthalate score. All pregnancy outcomes (CA, MN, and MO) Results Potential participants 654 100 — Eligible for SFFII 477 72.9 — The population for the present analysis was SFFII participant 346 72.5 — identified from families recruited in California, Male babies only (CA, MN, and MO) Minnesota, or Missouri for whom data entry SFFII participant 172 — 100 With AGD, age, and weight 134 — 78 was complete by 17 December 2004, the cutoff Prenatal urine sample 85 — 49 date for the present analysis. At that time, 654 participants from these three centers had A potential participant is an SFFI participant from CA, MO, or MN who gave permission to be recontacted for future studies and for whom all study data were entered by 17 December 2004. Boys in twin births and boys with missing data or AGD completed SFFI and given permission to be measurements considered unreliable by pediatricians excluded. Urine collection began midway through SFFI. recontacted. Of these, 477 (72.9%) were eligi- ble for SFFII and 346 (72.5%) participated Table 2. Characteristics of boys with complete physical examination. (Table 1). SFFII participants were demographi- Percentile cally similar to nonparticipants except that non- Characteristic Mean ± SD 25th 50th 75th participants were more likely to be Hispanic All boys (n = 134) because of a lower eligibility rate (60%) in CA, Age (months) 15.9 ± 8.6 11.0 15.0 23.0 where most participants were Hispanic. Of the Height (cm) 79.1 ± 10.6 72.6 80.0 87.2 172 boys born to these mothers, we excluded Weight (kg) 10.5 ± 2.7 8.7 10.7 12.3 5 boys in twin births, 10 boys with incomplete AGD (mm) 70.3 ± 11.0 63.9 70.3 76.6 AGI (mm/kg) 7.1 ± 1.9 5.8 6.7 7.8 data, and 23 boys for whom AGD was not ASD (mm) 37.4 ± 7.5 31.2 36.8 43.4 recorded [two whose mothers declined the geni- Boys whose mother’s prenatal urine was tal exam, with the remainder older boys (mean assayed for phthalate metabolites (n = 85) age, 19.6 months), for whom the study exam- Age (months) 12.6 ± 6.9 5.0 14.0 16.0 iner felt the measurement was not reliable, Height (cm) 75.6 ± 9.5 66.5 77.6 82.0 usually because of the boys’ activity level]. The Weight (kg) 9.7 ± 2.4 8.4 10.0 11.1 AGD (mm) 68.0 ± 9.7 61.7 66.7 74.4 remaining 134 boys comprise the sample used AGI (mm/kg) 7.4 ± 1.8 6.1 7.0 8.2 for the analysis of AGD and other genital meas- ASD (mm) 35.9 ± 7.1 30.4 35.6 41.4 urements. Among the 134 boys for whom we 1058 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance coefficients for the oxidative monoester MEP, MBzP, and MiBP were 4.7, 3.8, and –0.137). For the other phthalate metabolites, metabolites of DEHP, mono-2-ethyl-5-oxo- 9.1, respectively (all p-values < 0.05). regression coefficients were less significant (all hexyl phthalate (MEOHP), and mono-2-ethyl- Other genital parameters. Degree of testic- p-values between 0.11 and 0.97). 5-hydroxyhexyl phthalate (MEHHP) were of a ular descent was associated with AGD (R = Summary phthalate score. We used magnitude comparable with those for MEP 0.20, p = 0.02). The proportions of boys with the summary phthalate score as defined in and MBzP (p-values = 0.114 and 0.145 for one or both testicles incompletely descended “Materials and Methods” to study the effect of MEOHP and MEHHP, respectively). AGI were 20.0, 9.5, and 5.9% for boys classified as joint exposure to more than one AGI-associated appeared to be independent of the concentra- having short, intermediate, and long AGI phthalate. The summary phthalate score was tions of monomethyl phthalate (MMP) and (p-value for short AGI compared with all other directly related to the proportion of boys with MCPP, metabolites of dimethyl phthalate and boys < 0.001). The proportion of boys with a short AGI (p = 0.001). Of the 10 boys whose di-n-octyl phthalate, respectively. scrotum categorized as small and/or “not dis- phthalate scores were high (score = 11–12), all Categorical analyses. The 25 boys with tinct from surrounding tissue” was also ele- but one had a short AGI. Conversely, of the AGI below the 25th percentile for age were vated for boys with short AGI (p < 0.001). 11 boys whose scores were low (score = 0 or 1), classified as having a short AGI. This group had AGD was significantly associated with penile only one had a short AGI. The ORs for having an AGI that was, on average, 18.3% (range, volume (R = 0.27, p = 0.001), and penile vol- a short AGI for high summary phthalate score 10–32%) shorter than expected based on the ume divided by weight was correlated with compared with low (OR = 90.0; 95% CI, final regression model. Boys with AGI ≥ 75th AGI (R = 0.43, p = 0.001). Testicular volume, 4.88 to 1,659), and high compared with percentile of expected were classified as having a which was measured by orchidometer, is not medium (29.4; 95% CI, 3.4 to 251) were large long AGI, and boys with AGI between the shown here because participating physicians and significant, although the confidence inter- 25th and 75th percentile of expected were con- considered the measurement to be unreliable— vals were very wide. These data are shown sidered intermediate. Boys’ weight and age did a decision made before analyses of phthalate graphically in Figure 1. not differ appreciably among these groups. exposure. Discussion Table 5 shows mean and median values ASD was, on average, 47% as long as for the AGI-associated metabolites for boys in AGD, and these two measurements were cor- In the recent National Health and Nutrition the short, intermediate, and long categories of related (R = 0.47, p < 0.0001). However, the Examination Survey (NHANES 1999–2000), AGI. We calculated the ORs for short AGI for model predicting ASD as a function of baby’s most of the general population in the United = 0.10). each monoester metabolite (Table 6). For high age and weight fit poorly (adjusted R States had measurable exposure to multiple compared with low concentration of MBP, The fit for the model using ASD/weight as a phthalates (CDC 2003; Silva et al. 2004a). the OR for a short AGI was 10.2 (95% CI, function of age and age squared was better The samples in the present study and in 2.5 to 42.2), whereas for medium concentra- (adjusted R = 0.47) but did not fit as well as NHANES were both analyzed using compara- = 0.61). ASD/weight tion compared with low the OR was 3.8 (95% the model using AGI (R ble methods and standards by the same labora- CI, 1.2 to 12.3). The corresponding ORs for was associated with MEP concentration (regres- tory, although the specific metabolites that high compared with low concentration of sion coefficient = –0.429; 95% CI, –0.722 to were measured in the two studies differed somewhat. We compared the medians and 75th percentiles of the AGI-associated phtha- Table 3. Percentiles of phthalate monoester metabolites. late metabolite concentrations among two Percentile (ng/mL) a groups of mothers in our study (those whose Monoester metabolite 25th 50th 75th Percent > LOD boys fell in the short AGI group and all others) Phthalate monoester metabolite with those of females in the NHANES sample MBP 7.2 13.5 30.9 96.5 (Table 7). In the analysis of the NHANES MBzP 3.5 8.3 23.5 94.1 samples, monobutyl phthalate includes both MCPP 0.7 2.1 3.6 69.4 MEP 53.3 128.4 436.9 97.6 MBP and MiBP, which were measured sepa- MiBP 0.7 2.5 5.1 74.1 rately in our study. Metabolite concentrations MMP 0.7 0.7 3.2 49.4 for mothers of boys with short AGI were con- Metabolites of DEHP sistently higher than those of other mothers. MEHHP 6.0 11.4 20.1 97.6 Compared with women in the NHANES sam- MEHP 1.3 3.3 9.0 77.6 ple, metabolite concentrations for our popula- MEOHP 5.1 11.1 19.0 94.1 tion were somewhat lower. However, our LOD for all metabolites was between 0.95 and 1.07 ng/mL. population cannot be directly compared with Phthalate score 0–1 Table 4. Regression analyses of AGI on log monoester metabolite concentration, controlling for age and Phthalate score 2–10 age squared. Phthalate score 11–12 Log monoester metabolite concentration Monoester metabolite Coefficient (SE) p-Value (95% CI) 95% MBP –0.592 (0.269) 0.031 (–1.126 to –0.057) MBzP –0.390 (0.232) 0.097 (–0.851 to 0.072) 75% Mean MCPP –0.264 (0.356) 0.461 (–0.973 to 0.445) 25% MEHHP –0.398 (0.270) 0.145 (–0.935 to 0.140) MEHP –0.051 (0.241) 0.833 (–0.530 to 0.428) 4 5% MEOHP –0.412 (0.258) 0.114 (–0.925 to 0.101) MEP –0.400 (0.164) 0.017 (–0.726 to –0.074) 0 5 10 15 20 25 30 35 40 MiBP –0.765 (0.274) 0.007 (–1.309 to –0.220) Boy’s age (months) MMP –0.283 (0.323) 0.383 (–0.924 to 0.359) Phthalate score –0.0951 (0.035) 0.009 (–0.165 to –0.025) Figure 1. Mean AGI (mm/kg) in relation to boys’ age at examination (months). Phthalate score measures joint exposure to MBP, MBzP, MEP, and MiBP; see “Statistical analysis.” Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1059 AGI (mm/kg) Swan et al. NHANES: the proportion of pregnant women more sensitive to trenbolone by an order of human studies suggesting reproductive toxic- in the NHANES sample is unknown, and age magnitude (Neumann 1976). This greater ity (Colón et al. 2000; Duty et al. 2003b; distributions differ. Nonetheless, these data sensitivity is thought to be a result of rodents’ Main KM, unpublished data). It is therefore demonstrate that the four AGI-associated higher metabolic rate and more rapid inactiva- uncertain whether the absence of data in phthalate metabolites are prevalent in the U.S. tion of toxicants, both of which have been rodents showing reproductive toxicity is the female population, and levels were not unusu- shown to be inversely related to body size result of failure to detect it, unmeasured con- ally high among mothers whose sons had a (White and Seymour 2005). founding in human studies, or interspecies short AGI. In light of the toxicologic literature differences in response to these compounds. Although not identical, AGD in pups is for MBP, MBzP, and MiBP (Ema et al. 2003; DEHP has been shown to shorten AGD most similar to AGD as we defined it in this Foster et al. 1980, 1981; Gray et al. 2000; (Gray et al. 2000) and reduce testosterone study. In rodents, AGD has been shown to be Nakahara et al. 2003), our data suggest that (Parks et al. 2000). Although MEHP was not one of the most sensitive end points for phtha- the end points affected by these phthalates are associated with AGD in our data, the associa- lates such as DBP (Mylchreest et al. 2000) and quite consistent across species. A boy with tions for the oxidative metabolites of DEHP other antiandrogens such as flutamide (Barlow short AGI has, on average, an AGI that is 18% (MEOHP and MEHHP) were of comparable and Foster 2003; McIntyre et al. 2001) and shorter than expected based on his age and magnitude with those for metabolites of DBP finasteride (Bowman et al. 2003). It is difficult weight as well as an increased likelihood of tes- and BzBP, although not statistically signifi- to compare the dose to humans from low- ticular maldescent, small and indistinct scro- cant. Thus, it is unclear whether MEOHP level, ongoing, environmental exposure with tum, and smaller penile size. These changes in and MEHHP are (inversely) associated with that delivered to rodents experimentally in AGD and testicular descent are consistent with AGI, although associations are of borderline a narrow window of gestation. Nonetheless, those reported in rodent studies after high-dose statistical significance because of our sample it is likely that the doses to which our par- phthalate exposure (Ema et al. 2003; Gray size, or whether human and rodent responses ticipants were exposed are lower than those et al. 2000; Mylchreest et al. 2000). The lack to this phthalate and its metabolites differ. used in toxicologic settings, suggesting that of association for MCPP and MMP, which Masculinization of external male genitalia, humans may be more sensitive to prenatal have not been widely studied, is not inconsis- represented by longer AGD, is controlled by phthalate exposure than rodents. This greater tent with the toxicologic literature. dihydrotestosterone (Clark et al. 1990). Ema sensitivity in humans has been observed for With respect to DEP and its metabolite and Miyawaki (2001) demonstrated that other toxicants. For example, humans are MEP, we note that there are three other this metabolite of testosterone is markedly decreased by prenatal administration of MBP, Table 5. Mean (median) phthalate monoester metabolite levels by AGI category. suggesting that MBP acts as an antiandrogen. AGI category [mean (median; ng/mL)] AGD in male rodents is associated with other a b c Monoester metabolite Long (n = 17) Intermediate (n = 43) Short (n = 25) adverse developmental effects (Foster and MBP 13.1 (11.5) 22.2 (13.1) 38.7 (24.5) McIntyre 2002) and some phthalate-induced MBzP 10.6 (6.6) 15.1 (7.7) 25.8 (16.1) changes have been shown to be permanent. MEP 124 (47.1) 592 (112) 1,076 (225) For example, Barlow et al. (2004) report that MiBP 2.3 (1.5) 3.3 (2.1) 7.7 (4.8) prenatal exposure to 500 mg/kg/day DBP a b Long, AGI ≥ 75th percentile of expected AGI. Intermediate, 25th percentile ≤ AGI < 75th percentile of expected AGI. resulted in permanently decreased AGD and Short, AGI < 25th percentile of expected AGI. testicular dysgenesis. They also report that Table 6. OR (95% CI) for AGI less than expected from regression model, by monoester metabolite level. in utero DBP exposure induced proliferative Leydig cell lesions. Follow-up of exposed chil- Monoester metabolite Level (percentile) AGI < expected AGI ≥ expected OR (95% CI) dren until adulthood will be required to MBP Low 5 15 Referent determine whether long-term effects, includ- Medium 24 19 3.8 (1.2 to 12.3) ing testicular dysgenesis, are seen in humans High 17 5 10.2 (2.5 to 42.2) MBzP Low 6 13 Referent after prenatal phthalate exposure. Medium 26 18 3.1 (1.002 to 9.8) Several recent studies of the variability of High 14 8 3.8 (1.03 to 13.9) phthalate monoester concentration in human MEP Low 7 14 Referent samples suggest that phthalate concentration in Medium 25 19 2.6 (0.9 to 7.8) humans is fairly stable, perhaps reflecting High 14 6 4.7 (1.2 to 17.4) habitual use of phthalate-containing household MiBP Low 6 16 Referent Medium 23 18 3.4 (1.1 to 10.5) and consumer products (Colón et al. 2000; High 17 5 9.1 (2.3 to 35.7) Hauser et al. 2004; Hoppin et al. 2002). These studies lend support to the use of a single Low, < 25th percentile; medium, ≥ 25th and < 75th percentile; high, ≥ 75th percentile. sample for exposure assessment. We obtained Table 7. Concentrations of four phthalate metabolites in three groups of women (ng/mL). only a single prenatal urine sample from each This study woman, and most samples were obtained Monoester metabolite Percentile Short AGI Others NHANES quite late in pregnancy (mean = 28.3 weeks). MBP 50th 24.5 12.1 30.0 Therefore, the measured phthalate metabolite 75th 44.8 28.0 59.5 levels may not reflect exposure during the most MBzP 50th 16.1 7.2 16.0 sensitive developmental window, resulting in 75th 27.5 17.8 35.8 some degree of exposure misclassification. MEP 50th 225 90.4 174 However, unless this misclassification varied 75th 551 281 425 MiBP 50th 4.8 2.1 — systematically with outcome, such errors would 75th 12.1 4.3 — bias the effect estimate toward the null. In a b fact, the categorical analysis, which should be Females only (CDC 2003). MBP in the NHANES analysis includes both MBP and MiBP; in this study these metabolites were measured separately. less sensitive to such misclassification, showed 1060 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance stronger associations than did the continuous Parks L. 2000. Perinatal exposure to the phthalates DEHP, genital morphology is altered by antiandro- BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual analysis. gens, including some phthalates. We report differentiation of the male rat. Toxicol Sci 58:350–365. Our analysis is based on a single measure of that AGD, the most sensitive marker of anti- Hauser R, Meeker JD, Park S, Silva MJ, Calafat AM. 2004. Temporal variability of urinary phthalate metabolite lev- AGD, and the reliability of this measurement androgen action in toxicologic studies, is els in men of reproductive age. Environ Health Perspect in humans has not been established. During shortened and testicular descent impaired in 112:1734–1740. two training sessions, three study physicians boys whose mothers had elevated prenatal Hoppin JA, Brock JW, Davis BJ, Baird DD. 2002. Reproducibility of urinary phthalate metabolites in first morning urine each measured AGD in four male infants phthalate exposure. These changes in male samples. Environ Health Perspect 110:515–518. (mean age, 8.1 months). The mean AGD for infants, associated with prenatal exposure to Hornung RW, Reed LD. 1990. Estimation of average concentra- tion in the presence of nondectable values. Appl Occup these measurements was 58.6 mm, SD was some of the same phthalate metabolites that Environ Hyg 5:46–51. (within infant) 4.2 mm, and coefficient of vari- cause similar alterations in male rodents, sug- McIntyre BS, Barlow NJ, Foster PM. 2001. Androgen-mediated ation of 7.2%, suggesting that AGD can be gest that commonly used phthalates may development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal measured reliably. Use of this measurement in undervirilize humans as well as rodents. changes in anogenital distance and nipple retention with larger studies in a range of diverse populations, malformations in androgen-dependent tissues. 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Reprod Toxicol 15:189–194. late syndrome” characterized by testicular, epi- 2003. Improved quantitative detection of 11 urinary phtha- Ema M, Miyawaki E, Hirose A, Kamata E. 2003. Decreased late metabolites in humans using liquid chromatography- didymal, and gubernacular cord agenesis as well anogenital distance and increased incidence of unde- atmospheric pressure chemical ionization tandem mass scended testes in fetuses of rats given monobenzyl phtha- as decreased AGD, and stress the importance of spectrometry. J Chromatogr B Analyt Technol Biomed Life late, a major metabolite of butyl benzyl phthalate. Reprod evaluating all components of a syndrome so Sci 789:393–404. Toxicol 17:407–412. Silva MJ, Slakman AR, Reidy JA, Preau JL Jr, Herbert AR, Fisher JS. 2004. Environmental anti-androgens and male repro- that affected animals are not misidentified. It Samandar E, et al. 2004b. Analysis of human urine for fif- ductive health: focus on phthalates and testicular dysgene- has recently been suggested (Fisher 2004) that teen phthalate metabolites using automated solid-phase sis syndrome. Reproduction 127:305–315. extraction. J Chromatogr B Analyt Technol Biomed Life Sci this “phthalate syndrome” shares many features Foster PM, Cattley RC, Mylchreest E. 2000. Effects of di-n-butyl 805:161–167. phthalate (DBP) on male reproductive development in the with the human testicular dysgenesis syndrome Skakkebaek NE, Rajpert-De Meyts E, Main KM. 2001. Testicular rat: implications for human risk assessment. Food Chem proposed by Skakkebaek et al. (2001) to follow dysgenesis syndrome: an increasingly common develop- Toxicol 38:S97–S99. mental disorder with environmental aspects. Hum Reprod chemically induced disruption of embryonic Foster PM, Lake BG, Thomas LV, Cook MW, Gangolli SD. 1981. 16:972–978. 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The anogenital distance the testicular effects and changes in zinc excretion pro- syndrome. index, a predictor of the intrauterine position effects duced by some n-alkyl phthalates in the rat. Toxicol Appl This is the first study to look at subtle on reproduction in female house mice. Lab Anim Sci Pharmacol 54:392–398. 45:567–573. patterns of genital morphology in humans in Gray LE Jr, Foster PMD. 2003. Significance of experimental White CR, Seymour RS. 2005. Allometric scaling of mammalian studies for assessing adverse effects of endocrine- relation to any prenatal exposure. It was moti- metabolism. J Exp Biol 208:1611–1619. disrupting chemicals. Pure Appl Chem 75:2125–2141. vated by toxicologic studies showing that Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DNR, Environmental Health Perspectives • VOLUME 113 | NUMBER 8 | August 2005 1061

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Environmental Health PerspectivesPubmed Central

Published: May 27, 2005

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