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The insecticide DDT is still used for disease control in some areas, resulting in high levels of human exposure. The main degradation product of DDT is 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), an antiandrogen. In animal experiments, in utero exposure to DDE decreases anogenital distance in male offspring. In these models, anogenital distance serves as a measure of fetal androgen action. The authors designed the present study to examine the hypothesis that in utero exposure to DDE decreases anogenital distance in newborn human males. A cross-sectional study of 781 newly-delivered male infants was conducted in 2002–2003 in Chiapas, México, where DDT had recently been used for malaria control. Measurements were taken of anogenital distance and penile dimensions, and a sample of the mother’s blood was drawn. In this population the range of serum DDE levels was large (0.8–398 μg/L). The authors, using two-sided tests, found no evidence that exposure in utero to DDE was related to reduced androgen action as reflected by anogenital distance or penile dimensions at birth. If DDE has important antiandrogenic action in humans, it may be manifest only at higher levels of exposure or via effects on other outcomes.
The insecticide DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) is still used in some countries for disease-vector control, resulting in high levels of human exposure (1). The toxic effects of DDT and its degradation products, however, have not been adequately characterized in humans (2), and additional data are needed to inform policy regarding use. DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), the main degradation product of DDT, has been reported to be a potent antiandrogen, and one of the effects seen in animal models is that in utero exposure decreases anogenital distance at birth (3). Among the potential clinical effects of human exposure to an antiandrogen early in life are cryptorchidism, hypospadias, and reduced fertility.
In animal experiments anogenital distance is used as a measure of fetal androgen action (4–6); anogenital distance usually tracks through life, varies by dose of antiandrogen, and can be predictive of other androgen-responsive outcomes (5, 7). In human males, testicular volume and penile dimensions have traditionally been used as indicators of androgenicity (8, 9), and use of anogenital distance as an outcome has been rare (4, 10). Recent human data suggest, however, that anogenital distance may be responsive to antiandrogenic exposures in utero (10), and it is measured more reliably than penile dimensions (11).
The present study was designed to examine the effect of in utero exposure to DDE on anogenital and penile dimensions in relatively highly exposed newborn males. DDT was used in the study area for agriculture until 1991 (12), and for malaria control until 2000 (13).
A cross-sectional study of newly-delivered male infants and their mothers was conducted in 2002–2003 in Tapachula, a city in the state of Chiapas, México. Women were recruited during the postpartum period at both of the city’s hospitals, which also serve the surrounding area. About fifty percent of births in Tapachula occur in these hospitals (14). Women at the Social Security Hospital were more frequently urban, and had, on average, higher socioeconomic status and more education than women at the General Hospital (data not shown). If the eligibility criteria were met, the mothers were invited to participate and sign an informed consent form. The study protocol was approved by Institutional Review Boards at the Instituto Nacional de Salud Pública (National Institute of Public Health) in México and the National Institute of Environmental Health Sciences in the United States.
The eligibility criteria were chosen to exclude subjects for whom complicating medical conditions might have affected anogenital distance in male offspring or our ability to measure it. These criteria were determined a priori, before any data or determinants of anogenital distance were available. Exclusion criteria for the mother were age greater than 35 years; pre-eclampsia or pregnancy-related diabetes or hypertension; seizure disorders requiring daily medication; history of repeated urinary tract infections; psychiatric, kidney, or cardiac disease; and non-speaker of Spanish. Infants were excluded if female, gestational age at delivery as estimated by the Capurro scale (15) or the medical record (based on last menstrual period) was < 36 weeks, birthweight was < 2,500 g, pregnancy was not singleton, Apgar score at 5 minutes was ≤ 6, or child was admitted to the neonatal intensive care unit. Of the subjects who were invited to participate, 95 percent did so, resulting in 872 mother-infant pairs. Of these, the first 91 were enrolled when a preliminary anthropometric measurement protocol was in place. We excluded these from the present analysis because their measurements were not comparable, leaving 781 observations.
A questionnaire was administered to the mothers about socio-demographic characteristics, reproductive history, maternal health status, and various exposures. Maternal serum DDE and DDT was quantitated after solid phase extraction (C18 column purification), using gas chromatography and mass spectrometry (16, 17). For DDE the limit of detection was 0.2 μg/L; the recovery was 97 percent; and the between-assay coefficient of variation at 10 μg/L was seven percent. For DDT the limit of detection was 0.2 μg/L; the recovery was 97 percent; and the between-assay coefficient of variation at 2.5 μg/L was six percent. Total serum lipid was estimated based on levels of serum cholesterol and triglycerides (18), which were measured using standard enzymatic methods. Measurements of weight and height were performed on the mothers and newborns. In addition, we measured infant anogenital distance and penis size.
The technique for measurement of anogenital distance and penis size has been described in detail elsewhere (11) and is summarized briefly here. Three measures of anogenital distance were taken: anterior base of penis to anus (AGD1), posterior base of penis to anus (AGD2), and posterior of scrotum to anus (ASD) (see figure 1). In addition, we measured penile width (PW) and stretched penile length (PL). The anogenital and PW measurements were performed using Swiss Precision Cali Max Vernier calipers, from Bel-Art Products, Pequannock, NJ, USA. The calipers were read in increments of 1 mm. The penile measurements were done when the newborn’s penis was flaccid. Each measurement was taken on two occasions; the first set of readings was recorded in the questionnaire, and after these were completed, the second set was taken and noted on a sheet that was attached to each subject’s file. On each of these two occasions, PL was usually measured in duplicate, yielding up to four recorded values. Over 80 percent of children were examined before they were 6 hours old. With one exception, all examinations were conducted before 34 hours of age; the remaining examination was conducted 7 days after birth. The anthropometrists received special training before measuring anogenital and penile dimensions, length, and weight, and they received periodic retraining during the study (equipment for length and weight measures have been reported previously (11)). Twenty-two anthropometrists participated in the study. The reliabilities of the measures (the fraction of the variability that is true variability rather than measurement variability) were: AGD1, 0.91; AGD2, 0.88; ASD, 0.85; PW, 0.77; and PL, 0.76 (11). The small variation in measures among replicates, and due to observers, is described in detail elsewhere (11).
All 781 infants had measurements of each of the three anogenital distances; in all but two (AGD1 and AGD2) or three (ASD) cases, duplicate measurements were taken. All infants also had PW available (in duplicate in all but three cases). For 541 infants, all four PL measurements were available. Of the remainder, 43 infants had two measurements on the first and one on the second occasion, 188 had two measurements on the first and none on the second, three had one on each occasion, five had only one on the first occasion, and one had no measurements.
The goal of the analysis was to test the hypothesis that maternal DDE concentration is associated with anogenital distances and penile measurements in newborn male infants. Significance tests were based on F-tests in linear regression models, testing the hypotheses that the coefficient(s) were zero. All tests were two-sided.
Means of the replicates were used for all analyses. However, subjects were excluded from analyses of a particular anthropometric measurement if replicates differed by 30 percent or more; there were two such cases for ASD, one for PW, and three for PL. In the primary analysis, DDE was expressed on a per gram serum lipid basis, and subjects were placed in categories defined by < 3 μg/g, 3-<6 μg/g, 6-<9 μg/g, and ≥ 9 μg/g. These categories were chosen without examination of outcomes; the top category was chosen to contain a reasonable fraction of the children, with the remaining categories being of equal width. Finer categories (width of 1 ug/g rather than 3 ug/g, with the top categories collapsed to contain at least 20 children), were examined subsequently (see below). Analyses were also conducted with DDE concentration or logarithm of DDE concentration included as a linear term in the models.
When examining the relationship of anogenital and penile dimensions to DDE, we looked at both crude and adjusted relationships. Adjustment factors were those items that had previously been shown to be related to the anthropometric measurements in these data (11); potential predictors examined included infant birth weight and length, gestational age, maternal height and prepregnancy body mass index, maternal age, parity, maternal education, maternal marital status, household income per capita, urban or rural residence, and hospital. Those that were significant at p<0.20 in models including all non-DDE predictors were included in the present models (19). For anogenital distances, these factors were birth weight (included as a linear term), gestational age (categorized as 36–37, 38, 39, 40, 41+ weeks), urban vs rural residence, and hospital. For penile measurements, they were birth weight (linear), maternal age (linear), maternal height (linear), and parity (categorized as 1, 2–3, 4+). In both cases, we also adjusted for anthropometrist as a random effect, to account for observer-to-observer variability. Linear regression models were fitted using the MIXED procedure in SAS, version 9.00 (SAS Institute, Inc., Cary, North Carolina). All categorical variables were modeled with indicator variables.
Levels of DDT and DDE were highly correlated (Spearman r = 0.84), so we avoided fitting models that included both terms. However, we also conducted analyses for DDT similar to those described above, and in addition examined models that included DDE and the ratio of DDT to DDE.
The mothers were relatively young (table 1) and the median height (152.4 cm) was close to the national median (153.0 cm) for women of reproductive age (20). Median body mass index (23.1 kg/m2) was below the national median (25.0 kg/m2) (20). Most of the mothers had had children previously. They were about evenly split between the two hospitals. More than half lived in urban areas, primarily Tapachula, with the remainder in surrounding villages. Only 32 percent had gone beyond ninth grade (not shown). Twenty-nine percent reported living in houses that had been sprayed with DDT. Given the eligibility criteria, which excluded infants under 2500 grams or born before 36 weeks gestation, the range of birth weight among the babies was as expected. The Spearman correlation of DDE (lipid basis) with gestational age was 0.00, with birth weight was 0.03, and with birth length was 0.05.
The range of serum DDE levels among the mothers was large (table 2) and the median was about 10-fold greater than recent measures among women in the U.S. (0.26 μg/g lipid) (21). The relatively low ratio of DDT to DDE reflects that the study was done several years after DDT was last used. The median level of DDT was more than 20 times greater than U.S. median levels (<0.02 μg/g lipid (21)). The major determinant of DDE concentration was rural residence (median rural 4.0 μg/g, urban 2.1 μg/g). Conditional on residence, reported spraying of the individual home had much less influence (among rural residents, median if sprayed 4.4 μg/g, if not 3.6 μg/g; among urban residents, median if sprayed 2.4, if not 2.1 μg/g). The differences between hospitals essentially disappeared conditional on residence (among rural residents, median for both hospitals 4.0 μg/g; among urban residents, median Social Security 2.2 μg/g and General 2.1 μg/g). DDE concentrations rose with age and declined with parity (details not shown). As virtually all children in this population are breastfed, parity and lactation were confounded.
The variability of anogenital and penile measures is also shown in table 2. These outcome measures were all approximately normally distributed.
Mean anogenital distance was similar across categories of serum DDE, and adjustment for potentially confounding factors had essentially no effect on results (table 3). Modeling DDE as a continuous variable (table 3) and use of finer categories of DDE also indicated no association (figure 2). Similarly, for penis length and width, important variation in mean values across categories of DDE was not seen (table 3).
When similar analyses were performed for DDT instead of DDE, the findings were essentially the same as those shown. In analyses with DDE and the ratio of DDT to DDE included in the same model, neither showed any relationship to the outcomes.
In this population with relatively high in utero exposure to DDE, we found no evidence that exposure was related to reduced androgen action as reflected by anogenital distance or penile dimensions at birth. If DDE has important antiandrogenic action in humans, it may be manifest only at higher levels of exposure or via effects on other outcomes.
Among African women who lived in homes regularly sprayed with DDT the median serum DDE level was about 100 μg/L (22). In our study population 29 percent reported living in DDT-sprayed homes, and accordingly the median serum DDE level in our whole population was about 20 μg/L (n = 781, see table 2). The half-life of DDE is roughly 5–10 years in adults, although lactation shortens this by several years (23, 24). Thus, even though use of DDT in the area we studied was stopped shortly before enrollment began, we had many subjects with relatively high exposure. Furthermore, in our population the distribution of exposure was unusually wide, increasing the power to detect associations. Nonetheless, the possibility exists that in a population of women living in sprayed homes, with higher exposures, the power to detect an effect, if any, would be greater. It is also possible that in humans anogenital distance is not affected by minor changes in the androgen environment in utero. As noted above, however, recent data on in utero phthalate exposure in humans, suggest otherwise (10).
On the other hand, imprecision in assessment of exposure or outcome was a less likely explanation for failure to detect an association. History of spraying homes with DDT, conditional on residence (urban vs. rural) may not have predicted levels well because the subjects may not have detailed knowledge of what their homes were sprayed with, but serum levels of DDE were measured with reasonable precision, a single measure of DDE at birth corresponds well with levels during pregnancy (25), and maternal serum levels of DDE are closely related to those in umbilical cord blood (26–28). Thus, a cross-sectional study design, with exposure and outcome ascertained at birth should provide unbiased estimates of effect unless the subgroup of subjects susceptible to DDE effects was lost before birth (29, 30). Although the subjects in our study were not a random sample of those in the Tapachula area, we have no reason to suspect that the associations examined among our subjects would be biased. Furthermore, our earlier study showed that the outcomes were measured with high reliability (11), which was slightly greater for the anogenital distance measures than for the penile dimensions.
In rodents as well as humans, the critical period for male sexual development is androgen-dependent and occurs before birth. Thus, even though rodents are less mature at birth than humans, the animal model is useful for understanding the potential effects of antiandrogenic exposures during the embryonic and fetal stages. Fetal rats are more sensitive than adults to the antiandrogenic effects of DDE (3, 31). In animal models exposure to DDE in utero affects anogenital distance (3, 32), but does not cause cryptorchidism or hypospadias (7). Human data also indicate that exposure to DDE in utero is not associated with cryptorchidism or hypospadias (33–35). In adult humans, some data suggest that extremely high exposure to DDE has adverse effects on male reproductive hormone levels and semen quality, but the data are from small studies and are inconsistent (36–39).
In our data, antiandrogenic effects of DDE were not evident. Other outcomes, however, such as fertility following in utero exposure, may be more sensitive to such effects. Furthermore, it is possible that outcomes mediated via other mechanisms could be a more important health consideration among those living in homes sprayed with DDT.
This study was supported in part by a contract from the National Institute of Environmental Health Sciences, National Institutes of Health (N01-ES-15468) and in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
We wish to thank the nurses and other personnel from the two participating hospitals in Tapachula (Hospital General de Zona # 1 del IMSS, and Hospital General de Tapachula, de la Secretaría de Salud de Chiapas) for their support during data collection. We also wish to thank Catherine S. Mao, Harbor-UCLA Medical Center, and David K. Walmer, Duke University Medical Center, for their help in developing the anthropometric measures.
Conflict of interest: none declared.