1 Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
2 Division of Epidemiology, Statistics, and Prevention Research, National Institute of Child Health and Human Development, Rockville, MD.
3 National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA.
4 Department of Biostatistics, University of North Carolina, Chapel Hill, NC.
5 Epidemiologic Research and Information Center, Veterans Affairs Medical Center, Durham, NC.
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ABSTRACT |
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abnormalities; androgen antagonists; androgens; cryptorchidism; DDE; hypospadias; nipples
Abbreviations: CI, confidence interval; DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene; DDT, 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane
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INTRODUCTION |
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Normal development of male external genitalia depends on androgen action (3, 4
). For example, genetic defects that interfere with androgen action in humans can result in crypt-orchidism (failure of one or both testicle(s) to descend into the scrotum) or hypospadias (a urethral opening on the underside of the penis or on the perineum) (5
). In addition, in male rodents, the destruction of mammary rudiments requires androgen (6
, 7
), and treatment with DDE in utero leads to retained nipples (1
, 8
). In humans, extra nipples are called supernumerary nipples or polythelia. We hypothesized that in-utero exposure to the androgen antagonist DDE could be related to the frequency of cryptorchidism, hypospadias, and polythelia among boys (9
). To test this hypothesis, we studied a population with relatively high serum DDE levels: persons born in the United States in the early 1960s.
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MATERIALS AND METHODS |
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The characteristics of women in the sample at registration were essentially the same as those in the sampling frame (10). Four percent of subjects who enrolled were lost to follow-up before delivery. Once the women were enrolled, nonfasting blood samples were collected from them approximately every 8 weeks, as well as at delivery and 6 weeks postpartum. Serum samples were stored in glass at -20°C, with no recorded thaws. Approximately 42,000 women were enrolled in the study, and 55,000 children were born into the study. The children were systematically assessed for the presence of birth defects and other outcomes through the age of 7 years. Follow-up to age 7 years was completed for approximately 75 percent of subjects born into the study.
We used a nested case-control design to examine the association between maternal serum DDE level and risk of cryptorchidism, hypospadias, and polythelia among sons. Eligibility criteria were that the infant had to be 1) male, 2) liveborn, and 3) a singleton and that 4) a 3-ml aliquot of third-trimester maternal serum had to be available. We considered subjects to have cryptorchidism if they were classified as having an undescended testicle(s) at any time during the first year of life. Subjects first observed to have an undescended testicle(s) after the first year of life were not considered cryptorchid, because they might have had retractile testes. For hypospadias and polythelia, diagnosis at any time up to age 7 years was accepted. Among the 28,444 boys in the Collaborative Perinatal Project, 267 were not liveborn and 441 were not singletons; for 5,389, no maternal blood sample was available.
Among the eligible 22,347 boys, there were 241 cases of cryptorchidism, 214 cases of hypospadias, and 185 cases of polythelia. The eight subjects with more than one of these defects were included in each defect group that applied. From the eligible subjects, we selected 599 controls at random, so that the control:case ratio would be more than 2:1 for each defect under study. Other than restriction of the study to males, there were no other matching criteria. A laboratory result for DDE was not obtained for 100 of the 1,239 potentially eligible subjects (8 percent), mainly because the measured value did not meet quality control criteria for acceptance (11). Two additional subjects were excluded because no laboratory results for serum lipid were available. Thus, in the final analysis, there were 219 boys with crypt-orchidism, 199 with hypospadias, and 167 with polythelia and 552 controls. The proportion of subjects without a DDE measure was similar across these groups (cryptorchidism, 8 percent; hypospadias, 9 percent; polythelia, 7 percent; controls, 9 percent).
Among boys diagnosed as cryptorchid during the first year of life, the study records indicated that the testicles were descended at birth in 103. Because the cremasteric reflex (which causes retraction of the testicles) is not well developed in the first year of life (3), we assumed that these 103 subjects had been misclassified as normal on their birth examination. To evaluate this assumption, we considered these subjects separately in a sensitivity analysis (described below). Among boys with at least one undescended testicle at birth (n = 138), all but one also had a subsequent observation of the abnormality in at least one of the three subsequent physical examinations (ages 4 months, 1 year, and 7 years) or orchidopexy. Regarding hypospadias, the Collaborative Perinatal Project records did not note the degree. For polythelia, the presence of mammary tissue or areola or the number of supernumerary nipples was not recorded. Of the eight subjects who had more than one of the defects, five subjects had cryptorchidism and hypospadias, two had cryptorchidism and polythelia, and one had hypospadias and polythelia.
The socioeconomic index calculated for subjects in the Collaborative Perinatal Project was the mean of three percentile scores (for education, occupation, and family income), where education was that of the head of the household, occupation was that of the head of the household or the chief wage-earner, and the score used to calculate the percentile for an occupation was based on the percentiles of education and income among persons with the same occupation (12).
Laboratory assays
Serum levels of p,p'-DDT and p,p'-DDE were measured in 19971999 after solid-phase extraction, cleanup, and dual-column gas chromatography using electron capture detection (11). The proportion of DDE in the samples recovered by extraction averaged 70 percent (range, 5785 percent), and the between-batch coefficient of variation in recovery was 8.2 percent (n = 312). The within-batch variation in recovery was not estimated; without this, it was not possible to know whether adjustment of DDE levels for proportion of material recovered gave estimates that were more accurate and precise than the unadjusted values. Thus, results are presented with and without recovery adjustment. All subjects had serum DDE values above the method detection limit (0.61 µg/liter). Among the analytical batches that included a subject with a given type of birth defect, 91 percent or more included at least one male control from the same center, and 87 percent of batches contained an aliquot from a single large pool, used to calculate the between-assay coefficient of variation. The between-assay coefficient of variation was 19 percent at 29 µg DDE/liter (n = 291). The order of specimens within-batch was determined by a random process. The laboratory personnel were masked with respect to the type of sample. Serum levels of cholesterol and triglycerides were measured using standard enzymatic methods (13
). Serum sodium was measured by inductively coupled plasma-atomic emission spectroscopy (14
), to assess desiccation in the samples.
We determined DDE levels in serum from the third trimester, because these samples were the most complete. In 67 women selected at random from the Collaborative Perinatal Project, the Pearson's correlation coefficient between lipid-adjusted DDE levels measured in the first and third trimesters was 0.86 (15). DDE crosses the placenta, and in a US study conducted around 1980, levels in the mother's serum at delivery and the child's umbilical cord serum were correlated at r = 0.79 (16
).
Statistical methods
To divide subjects into categories based on DDE exposure, we identified a set of four equally spaced cutpoints that yielded at least 20 controls per group and a large contrast in exposure between those in the lowest and highest categories. We estimated the odds of having a birth defect in relation to DDE level using conditional logistic regression, conditional on study center (12 strata). Serum levels of DDE are proportional to those of lipid; thus, serum triglycerides and cholesterol were included as continuous variables in models wherein DDE was expressed per unit volume of serum. In one set of models, we expressed DDE on a per-unit-serum-lipid basis and did not include the lipid levels as independent variables. To express DDE on a per-unit-serum-lipid basis, we estimated the lipid content of serum using the formula given by Phillips et al. (17) and the level of total cholesterol and triglycerides in each sample. Furthermore, race was related to the occurrence of polythelia and was associated with DDE levels (Blacks had more polythelia and higher DDE levels than Whites), so all models were adjusted for race (White, Black, other).
Confounding was evaluated by comparing the coefficient for DDE from models including lipids and race with the coefficients in a model with an additional factor. DDE was modeled as a continuous variable and as a categorical variable, and results from both types of models were used to evaluate the change in estimate. If the odds ratio per 15 µg/liter of DDE changed by 15 percent or more or the odds ratio for the contrast between the highest exposure categories and the lowest changed by 15 percent or more, the factor was considered a confounder. The factors considered as potentially confounding variables were season of birth, mother's age, parity, socioeconomic index, body mass index (weight (kg)/height (m)2) before pregnancy, weight gain during pregnancy, smoking during pregnancy, hyperemesis gravidarum, hypertension, age at menarche, history of infertility, menstrual cycle irregularity, estrogen use during pregnancy, progesterone use during pregnancy, method of delivery, and serum sodium level. We similarly considered the effect of adjustment for preterm birth, birth weight, smallness for gestational age, and gestational age, even though these were potentially intermediate variables. Using a similar approach but with cross-product terms, we evaluated effect modification by race, birth weight, gestational age, hormone use, and study center. Evaluation of effect modification by categorical variables with more than two categories was supplemented by comparing the model-fit statistics for models with and without the cross-product terms. For effect modification, the p values associated with the interaction terms or likelihood ratio tests all had values greater than 0.10, and thus the degree of potential effect modification was not considered further. All statistical analyses were conducted using the SAS statistical software package, version 8.0 (SAS Institute, Inc., Cary, North Carolina).
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RESULTS |
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For cryptorchidism and hypospadias, the adjusted odds ratio among boys in the highest category of serum DDE compared with boys in the lowest was modestly elevated, but the confidence intervals were wide (table 2). For polythelia, the adjusted odds ratio for the same exposure contrast was moderately increased, though again the confidence interval was wide. For all three birth defects, when DDE was modeled as a continuous variable (scaled to odds ratio per 15-µg/liter increase), all odds ratios were greater than 1 but were small and imprecise. The addition of a quadratic term for DDE to models that included DDE as a continuous variable did not improve the fit. The crude and adjusted results were essentially the same, although for polythelia the crude odds ratio was increased in the highest exposure category because of confounding by race. Further adjustment for the other factors considered as potentially confounding variables had no substantial effect on results (the largest change in the odds ratio due to the inclusion of any factor was 11 percent); as mentioned above, notable effect modification was not observed.
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DISCUSSION |
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Among mothers of controls in the present study, representing 12 US cities in 19591966, the median serum recovery-adjusted DDE level was 34.3 µg/liter. More recently reported serum DDE levels in the United States have been lower (e.g., 1.3 µg/liter (18) and 7.1 µg/liter (19
)), which is consistent with the known decrease that has taken place since about 1970 (20
). In approximate terms, then, the median exposure level in the Collaborative Perinatal Project was more than five times greater than that which exists at present. By comparison, in a South African population whose homes are sprayed annually with DDT for mosquito control, the mean serum DDE level is 103 µg/liter (21
).
Steady increases in rates of cryptorchidism and hypospadias have been reported in the United States since 1970, when data first became available (22). In the United States, at the same time that rates of cryptorchidism and hypospadias increased, background-level exposure to DDE was decreasing (20
). These trends, being in opposite directions, suggest that background-level DDE exposure had little effect on risks of cryptorchidism and hypospadias. Nonetheless, if a maternal serum recovery-adjusted DDE level of
85.6 µg/liter (the highest category shown in table 3) did slightly increase risk, the contribution to the number of US cases would have been small, given the relatively small proportion of subjects (about 5 percent) exposed at that level. Thus, an effect of DDE on trends in crypt-orchidism or hypospadias, if any, could easily have been lost among the effects of other determinants of trend that either affected a greater proportion of the population or had larger effects.
In animals, antiandrogenic agents clearly can cause crypt-orchidism, hypospadias, and retained nipples (23). The first report on the antiandrogenic effect of DDE (1
) showed that a large dose of DDE given to pregnant rats caused male offspring to have reduced anogenital distance and retained nipples. In a subsequent study (8
), DDE effects were found to vary across rat species, and, in sensitive species, dose-response analyses revealed that DDE was a relatively weak antiandrogen. In rodents, the outcome most sensitive to DDE is retained nipples, and these were seen in the offspring of rat dams whose mean serum DDE level was 110 µg/liter while pregnant, 2 days after their last dose (8
). Because our results were more suggestive of an association for polythelia than for the other defects, and because our results for polythelia were most suggestive in the group with recovery-corrected DDE levels of
85.6 µg/liter (the highest exposure category in table 3), the animal and human data show interesting parallels.
In recent studies of risk factors for cryptorchidism and hypospadias (2426
), both defects were directly linked to preterm birth and smallness for gestational age and were inversely associated with parity. This suggests that the etiology of these two male defects is partly shared (24
). In multivariate analyses of our data, preterm birth and smallness for gestational age were also associated with an increased risk for these conditions, but the study was relatively small and most effect estimates were imprecise (data not shown). Risk factors for polythelia have been less well studied, although race and urogenital malformations have been consistently related to risk (27
, 28
).
The greater risk of polythelia among Blacks could be due in part to DDE exposure (Blacks have higher DDE levels (2)). All studies of polythelia among Blacks were US studies, beginning in the 1960s. In our data, Black race remained an important predictor of polythelia when DDE was included in the model. The odds ratio for polythelia among Blacks as compared with Whites (in a model adjusted for triglycerides, cholesterol, and "other" race) was 3.5 (95 percent CI: 1.9, 6.5) prior to adjustment for DDE (five categories) and 3.3 (95 percent CI: 1.7, 6.2) after adjustment. The Pearson correlation coefficient between race (Black vs. other) and loge DDE was 0.33that is, not high enough that keeping both in the model would obscure an effect of DDE.
In the Collaborative Perinatal Project, birth defects were ascertained through the age of 7 years. A greater proportion of cases than of controls were followed to age 7 (82 percent vs. 75 percent). The greater loss to follow-up among controls could have led to a relative underdiagnosis of birth defects. However, such defects were sufficiently rare that if they had been undiagnosed, the resulting misclassification would not have affected our results much. Furthermore, at least 85 percent of the known defects were diagnosed during the first year of life. Median DDE levels among all cases and controls followed to age 7 years were 25.0 mg/liter and 23.8 µg/liter, respectively; and among boys not followed to age 7 years, median DDE levels among all cases and controls were 27.4 mg/liter and 26.9 µg/liter, respectively. These figures, and the combination of the relatively complete follow-up and the predominance of early diagnosis of birth defects, suggest that bias from cases' being misclassified as controls because of loss to follow-up would be positive but negligible in magnitude. The rates of cryptorchidism and hypospadias reported in the Collaborative Perinatal Project (29) were higher than those in other US reports (22
). In the Collaborative Perinatal Project, cases were identified in a series of systematic examinations, whereas data in birth defect registries are typically based on routine reports and records only (29
). We excluded as cases boys for whom cryptorchidism was initially diagnosed after the first year of life, to reduce the possibility of mixing boys with retractile testes into our case group. For polythelia, the systematic examination used in the Collaborative Perinatal Project was not especially effective in detecting cases. When newborns have been examined specifically for polythelia (30
, 31
), the rate has been higher than that seen in the Collaborative Perinatal Project. In any event, the diagnoses of the three defects studied were made by personnel who had no information about the subject's DDE exposure, so any bias due to misclassification of cases probably caused the odds ratios to be closer to the null than they would have been if such error had been absent (32
). Similarly, if an association was present, nondifferential error in exposure measurement probably caused the odds ratio per unit of change in serum DDE to appear smaller than it actually was (33
).
Expression of serum organochlorine levels on a per-unit-serum-lipid basis has been recommended (34), and we did so here to provide results in a format familiar to some readers. The usual method underlying the lipid-basis metric is estimation of total serum lipids from total cholesterol and triglyceride levels using a formula (17
). The origins of the formula, however, have never been clearly described. If one applies the formula in the population used to derive it (35
), using the mean total cholesterol and triglyceride levels in that population, total serum lipid levels are not especially well predicted. Furthermore, whether the formula works well enough to estimate total lipids in pregnant women (or any other population, for that matter) is unclear. The goal here was to examine associations with organochlorine levels after accounting for variation in organochlorine levels due to the concentration of lipids, and that goal was better achieved by letting our own data determine the best coefficients for lipid adjustment. Moreover, because a substantial portion of organochlorine in serum is not associated with lipid, expressing concentration on a per-unit-serum-lipid basis is misleading (36
). The attenuation of the odds ratios for the high:low exposure category comparison in table 4 as compared with table 3 may be entirely due to misclassification resulting from expression of exposure levels on a per-unit-serum-lipid basis. Similarly, for the reasons stated above (see "Laboratory assays"), we have little faith that recovery adjustment of our exposure values improved DDE measures in these data. Our adjusted measures were useful, however, when comparing our DDE levels with those reported by other investigators.
In summary, these data alone provide no clear evidence of an effect of DDE on male development, but given the experimental and emerging human evidence (37) of DDE as an androgen antagonist, there remains the suspicion that high maternal levels of DDE may affect the development of male offspring.
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NOTES |
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REFERENCES |
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