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American Journal of Epidemiology, doi:10.1093/aje/kwm136
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American Journal of Epidemiology Copyright © 2007 by the Johns Hopkins Bloomberg School of Public Health All rights reserved; printed in U.S.A.

Original Contribution

Time to Pregnancy and Secondary Sex Ratio in Men Exposed Prenatally to Diethylstilbestrol

Lauren A. Wise1, Linda Titus-Ernstoff2,3, Julie R. Palmer1, Robert N. Hoover4, Elizabeth E. Hatch5, Kimberly M. Perez6, William C. Strohsnitter7, Raymond Kaufman8, Diane Anderson9 and Rebecca Troisi2,4

1 Slone Epidemiology Center, Boston University, Boston, MA
2 Departments of Community and Family Medicine and of Pediatrics, Dartmouth Medical School, Lebanon, NH
3 The Hood Center for Children and Families, Dartmouth-Hitchcock Medical Center, Lebanon, NH
4 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD
5 Department of Epidemiology, Boston University School of Public Health, Boston, MA
6 Department of International Health, Boston University School of Public Health, Boston, MA
7 Department of Obstetrics and Gynecology, New England Medical Center, Boston, MA
8 Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX
9 Department of Obstetrics and Gynecology, University of Chicago, Chicago, IL

Correspondence to Dr. Lauren A. Wise, Slone Epidemiology Center, Boston University School of Public Health, 1010 Commonwealth Avenue, Fourth Floor, Boston, MA 02215 (e-mail: lwise{at}bu.edu).

Received for publication November 8, 2006. Accepted for publication March 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Little is known about the influence of prenatal diethylstilbestrol (DES) exposure on time to pregnancy or secondary sex ratio in men. The authors evaluated these associations among men participating in the DES Combined Cohort Follow-up Study for whom exposure status was confirmed by medical record. In 2001, men provided data on their reproductive histories. Demographic, behavioral, and medical data were collected in 1994, 1997, and 2001. Cox's proportional hazards models with frailty were used to estimate fecundability ratios for time to pregnancy in relation to DES. Generalized estimating equations were used to estimate odds ratios for fathering a male birth in relation to DES. Models included potential confounders and accounted for multiple pregnancies contributed by each man. Overall, DES was not associated with a delay in time to pregnancy (fecundability ratio = 0.95, 95% confidence interval: 0.86, 1.06). The odds ratio for fathering a male birth was 0.92 (95% confidence interval: 0.80, 1.04) comparing the exposed with the unexposed. In conclusion, prenatal DES exposure was not associated with a significant decrease in either fecundability or secondary sex ratio.

diethylstilbestrol; estrogens; fertility; males; reproduction; sex ratio

Abbreviations: CI, confidence interval; DES, diethylstilbestrol; FR, fecundability ratio


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Diethylstilbestrol (DES) is a synthetic estrogen that was prescribed for over 2 million pregnant women during the 1940s–1960s to prevent pregnancy loss. Prenatal DES exposure in the daughters of these women has been associated with several adverse reproductive events, including vaginal and cervical clear cell adenocarcinoma (1), reproductive tract anomalies (2, 3), infertility (4, 5), and poor pregnancy outcomes (4, 6).

Studies in men provide mixed results regarding the association between prenatal DES exposure and reproduction. Prenatal DES exposure is associated with an excess rate of structural and functional malformations of the male genitalia (7–14), including cryptorchidism (8, 12, 13), varicocele (11), and epididymal cysts (11, 12, 15). Some studies report lower sperm counts (11, 12), decreased sperm motility (12), and abnormal sperm morphology (12) in exposed than unexposed men, but other studies report no differences in sperm motility or morphology (11) or in any semen parameters examined (16, 17). Finally, while some studies report a positive association between prenatal DES exposure and male infertility (8, 11, 18, 19), most studies show null (7, 13, 16, 17) or weak (20) associations.

Little is known about the influence of prenatal DES exposure on time to pregnancy (7) or secondary sex ratio in men. Time to pregnancy—defined as the number of menstrual cycles (or months) from the time a couple stops contraception until pregnancy occurs—is considered a sensitive indicator of a couple's fertility (21). Cigarette smoking (22), increasing age (23), and environmental contaminants such as lead (24) and polychlorinated biphenyls (25) have been associated with delayed time to pregnancy in men. Only one study investigated the relation of prenatal DES exposure with time to pregnancy in men and found no association, but data were limited to the couples' most recent pregnancy (7).

The secondary sex ratio (proportion of male births) is a prevalence measure reflecting sex programming at the time of conception and survival until birth (26). There is accumulating evidence that endocrine disruptors with properties similar to those of DES (e.g., polychlorinated biphenyls and dioxin) may influence the sex ratio in men (27–41), with most studies showing declining sex ratios attributed to these chemicals (27–29, 35, 38, 40, 42, 43). One hypothesis is that endocrine disruptors cause differential preclinical loss of males. Male rats exposed to polychlorinated biphenyls and dioxin-like chemicals in utero have permanently altered sperm-transit time (44, 45), which can hinder the process of fertilization and implantation (44, 46, 47) and disproportionately affect male conceptuses (48). Alternatively, endocrine disruptors may influence paternal hormones around the time of conception (49), with high levels of testosterone and estrogen increasing the probability of a son and high levels of gonadotropins and progesterone increasing the probability of a daughter. In support of the latter hypothesis, prenatal DES exposure is associated with decreased testosterone levels in male rodents (50–52).

The present report examines the relation of prenatal DES exposure with time to pregnancy and secondary sex ratio in men, by use of data from the National Cancer Institute's DES Combined Cohort Follow-up Study (20). We hypothesize that men exposed prenatally to DES will have delayed time to pregnancy and a reduced proportion of male births.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Study population and follow-up
The present analysis includes male participants from the National Cancer Institute's DES Combined Cohort Follow-up Study, a multicenter cohort study initiated in 1992 (13, 15). Methods of the original studies from which individual cohorts were assembled are described elsewhere (13, 15, 16, 53–55). Briefly, the combined cohort consists of exposed and unexposed 1) men whose mothers participated in a clinical trial of DES use during pregnancy in the early 1950s ("Dieckmann cohort" (53)); 2) men whose mothers were treated in an infertility clinic near Boston, Massachusetts, from 1933 to 1975 ("Horne cohort" (55)); 3) men whose mothers were identified by obstetrics record review at the Mayo Clinic from 1939 to 1962 ("Mayo cohort" (15)); and 4) men whose mothers were identified by obstetrics record review at two New England hospitals and a private obstetrics office from 1940 to 1960 ("Women's Health Study cohort" (54)). The study protocol was approved by the institutional review boards of all participating centers and the National Cancer Institute.

Of the 4,101 men (2,001 exposed, 2,100 unexposed) who were potentially eligible for study in 1992, 242 (110 exposed, 132 unexposed) were untraceable, 136 (102 exposed, 34 unexposed) had died, and 898 (384 exposed, 514 unexposed) did not respond to the baseline questionnaire or had declined participation prior to study initiation. Surviving and traceable men who previously had not declined participation were contacted by mailed questionnaire in 1994. Follow-up questionnaires were mailed in 1997 and 2001. Completed 1994 questionnaires were obtained from 1,405 (70.2 percent) exposed and 1,420 (67.6 percent) unexposed members of the initial cohort. Of these, 1,253 (89.2 percent) exposed and 1,277 (89.9 percent) unexposed men completed the 1997 questionnaire, and 1,239 (88.2 percent) exposed and 1,233 (86.8 percent) unexposed men completed the 2001 questionnaire.

Exclusionary criteria
We excluded 1,178 men who did not complete a questionnaire in 1994 or 1997, as well as 182 men who provided incomplete reproductive histories or reported having sex with men only. We further excluded 245 men who did not complete the 2001 questionnaire on which we asked about offspring sex and time to pregnancy, leaving 2,496 men eligible for analysis (1,246 exposed, 1,250 unexposed).

Assessment of exposure, outcomes, and covariates
DES exposure status was verified in all cohorts by medical record. The Dieckmann cohort was exposed to high cumulative doses of DES (median = 12 g), following the regimen recommended by Smith and Smith (56). Dose in the Mayo cohort was difficult to estimate because of incomplete data but ranged from approximately 1.5 to 2.5 g (15). Doses were generally higher in the Horne cohort and were unavailable in the Women's Health Study cohort. Information on DES dose was available for 42.1 percent of the exposed men. We used 5 g as a cutpoint because the distribution was bimodal, with peaks at about 2 and 12 g (20).

Data on gestational age at first DES exposure (in weeks), available for 74.2 percent of the exposed men, were used to assess whether biologic susceptibility was related to the timing of DES exposure. A cutpoint of 11 weeks' gestation was chosen because the male genitalia are considered most susceptible to teratogens before the end of the ninth week of gestation, corresponding to about 11 weeks after the last menstrual period (7, 57, 58).

The 1994 questionnaire elicited information on demographic and lifestyle factors, screening behaviors, and medical history. The 1997 and 2001 questionnaires updated data on these factors. In 2001, men provided a detailed account of their reproductive histories, including whether a woman had ever become pregnant by them, the date and outcome of each pregnancy fathered (live- or stillbirth, spontaneous abortion, therapeutic abortion, other), the sex of each livebirth, whether the pregnancy was planned, and, if so, the number of months it took to conceive. Participants reported whether they had ever tried to father a pregnancy for 12 or more months without success (1994 and 2001), their age at the start of this period (2001 only), whether they had ever sought medical assistance for infertility (1994 and 2001), and the suspected cause of infertility (2001 only): "problem with male" (male factor), "problem with female" (female factor), "problem with both" (male and female factors), or "unknown reason" (unexplained infertility).

Data analysis
Analyses of time to pregnancy
The fecundability ratio represents the cycle-specific probability of conception among the exposed divided by that among the unexposed. A fecundability ratio below one indicates reduced fecundability, or increased time to pregnancy, among the exposed relative to the unexposed. Analyses included all planned pregnancies that resulted in a live- or stillbirth. Using STATA, version 9.2, software (59), we fit a Cox proportional hazards model with frailty to estimate fecundability ratios and 95 percent confidence intervals for DES in relation to time to pregnancy. The frailty model explicitly models the correlation between time-to-pregnancy values for a given participant by a random-effect term (gamma distributed, with mean = 1 and variance = unknown), accounting for multiple pregnancies contributed by each man (60–62).

Men who reported unsuccessful pregnancy attempts (e.g., conception-free cycles leading up to miscarriage or therapeutic abortion) were included in the regression model but were censored at their reported time to pregnancy (21). Men who never fathered a pregnancy but reported trying for 12 or more months to conceive without success were censored at 12 months. To avoid bias due to digit preference, we censored time-to-pregnancy models at 14 months instead of 12 months (21), the usual amount of time after which couples seek medical assistance for infertility. Fecundability ratio estimates were insensitive to different censoring cutoffs (e.g., 12, 10, or 7 months; 14 vs. 12 months for men who never fathered a pregnancy). We conducted secondary analyses in which we used time-to-pregnancy data from 1) the first pregnancy, which is not susceptible to bias from previous pregnancies (21); and 2) the most recent pregnancy for comparability with previous studies (7). In both cases, we used a discrete-time analogue of the Cox model (SAS, version 9.1, software) (63, 64). We used the likelihood ratio test to assess departure from the proportional hazards assumption, comparing models with and without a cross-product term between cycle number (<6, ≥6 months) and exposure.

Of the 4,649 pregnancies contributed by 2,496 men, we excluded 1,488 unplanned pregnancies, 447 pregnancies with missing data on planning, 521 planned pregnancies with missing time-to-pregnancy estimates, and 20 planned pregnancies with missing dates. The proportion of unplanned pregnancies did not vary appreciably by exposure status (34.6 percent exposed vs. 36.2 percent unexposed). Of the time-to-pregnancy estimates included, 1,990 (89 percent) resulted in livebirth, seven (0.3 percent) in stillbirth, 166 (7.4 percent) in miscarriage, and 10 (0.5 percent) in therapeutic abortion. An additional 63 men (2.8 percent) who tried to conceive for 12 or more months without success were included (total n = 2,236).

For secondary analyses in which the first planned pregnancy was used (n = 1,132), we retained all pregnancies for men with one pregnancy (n = 353), the first pregnancy for men with more than one pregnancy (n = 716), and one observation for men trying 12 or more months without success (n = 63). For analyses in which the most recent planned pregnancy was used (n = 1,154), we retained all pregnancies for men with one pregnancy (n = 353), the most recent pregnancy for men with more than one pregnancy (n = 738), and one observation for men trying 12 or more months without success (n = 63).

Analyses of secondary sex ratio
Of the 4,200 livebirths contributed by 2,496 men, we excluded multiple-gestation births (n = 52) and births with missing data on child's sex (n = 122) or birth date (n = 375), leaving 3,651 singleton livebirths for analysis. To account for nonindependence, we used generalized estimating equations with an unstructured correlation matrix to estimate odds ratios and 95 percent confidence intervals for the association of DES with sex ratio (SAS, version 9.1, software) (64, 65).

Assessment of confounding and interaction
Both sets of analyses were adjusted for cohort (Dieckmann, Horne, Mayo, Women's Health Study) and putative risk factors for the outcome that were associated with DES. Thus, time-to-pregnancy models further adjusted for paternal age at pregnancy completion or age at first pregnancy attempt if unsuccessful (<25, 25–29, 30–34, ≥35 years) and education (less than college, college, graduate school, missing). Sex ratio models further adjusted for paternal age at birth (<30, 30–34, ≥35 years), birth order (1, 2, ≥3), and year of child's birth (before 1985, 1985–1989, 1990 or later). Time-to-pregnancy models were conducted with and without control for semen analysis (abnormal, normal, not assessed) and genitourinary anomaly (yes, no) to observe whether these factors explained differences in time to pregnancy. We also treated time to pregnancy as an explanatory variable in analyses of sex ratio as some studies (26, 66), but not all (67), have shown an association. We used likelihood ratio tests to evaluate statistical interaction, comparing models with and without cross-product terms between DES and selected covariates. All p values were two sided.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
DES-exposed men were younger and more likely to have graduated from college than were unexposed men (table 1). Exposed and unexposed men were similar with regard to body mass index, smoking, alcohol consumption, marital status, and sexual history. Exposed men were more likely to report a genitourinary anomaly or abnormal semen analysis than were unexposed men. Although distributions of pregnancies and livebirths were similar for both exposure groups, DES-exposed men were more likely to report trying to conceive for 12 or more months without success, seeking medical assistance for infertility, male factor infertility, and later age at first birth (table 1). Most men, regardless of exposure status, reported having at least one physical examination in the 5 years prior to baseline. Urologic examinations, while less frequent, also were similar for the exposed and unexposed.


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  		TABLE 1. Characteristics of men, by prenatal DES* exposure, DES Combined Cohort Follow-up Study, 1994–2001{dagger}

 
Figure 1 shows the conditional probability of pregnancy per month, by exposure status. Time to pregnancy ranged from 1 to 96 months. There was evidence of digit preference at 6, 9 (unexposed only), and 12 months. The same pattern emerged within each of the four cohorts and among first and most recent pregnancies. The cumulative probabilities of pregnancy after 6 and 12 months were 75.5 percent and 81.7 percent for exposed men and 75.1 percent and 84.1 percent for unexposed men.


Figure 1
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  		FIGURE 1. Conditional probability of pregnancy per month among men attempting to conceive, by exposure status (censored at 14 months), DES Combined Cohort Follow-up Study, 2001. DES, diethylstilbestrol.

 
The adjusted fecundability ratio comparing DES-exposed with unexposed men was 0.95 (95 percent confidence interval (CI): 0.86, 1.06) (table 2). Control for abnormal semen analysis attenuated the associations (fecundability ratio (FR) = 0.99, 95 percent CI: 0.90, 1.08), but genitourinary anomaly had little effect (data not shown). There was no interaction by cohort (pinteraction = 0.58) or paternal age (pinteraction = 0.56), and the fecundability ratios were similar according to DES dose and timing (table 3). Although the fecundability ratio differed significantly by cycle number (<6-month FR = 1.00, 95 percent CI: 0.91, 1.11; ≥6-month FR = 0.75, 95 percent CI: 0.59, 0.94; pinteraction = 0.01), there was no evidence of interaction when time-to-pregnancy values of 9 for unexposed men were omitted from the analysis (overall FR = 0.93, 95 percent CI: 0.84, 1.04; <6-month FR = 0.95, 95 percent CI: 0.86, 1.05; ≥6-month FR = 0.86, 95 percent CI: 0.67, 1.09; pinteraction = 0.48). Likewise, no interaction was found when we omitted time-to-pregnancy values of 9 for exposed and unexposed men (overall FR = 0.94, 95 percent CI: 0.84, 1.04; <6-month FR = 0.96, 95 percent CI: 0.87, 1.06; ≥6-month FR = 0.82, 95 percent CI: 0.64, 1.05; pinteraction = 0.26).


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  		TABLE 2. Prenatal DES* exposure and time to pregnancy, overall and by selected factors, DES Combined Cohort Follow-up Study, 2001

 

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  		TABLE 3. Prenatal DES* exposure and time to pregnancy, by dose and gestational timing of first exposure, DES Combined Cohort Follow-up Study, 2001{dagger}

 
Multivariable results for time to pregnancy were similar whether we included pregnancy losses as events (FR = 0.95, 95 percent CI: 0.87, 1.04), conditioned analyses on ever having conceived (FR = 0.94, 95 percent CI: 0.80, 1.10), or ignored the correlation of time-to-pregnancy values (FR = 0.96, 95 percent CI: 0.88, 1.05). Fecundability ratios were similar for the first pregnancy (FR = 0.93, 95 percent CI: 0.80, 1.09) and most recent pregnancy (FR = 0.96, 95 percent CI: 0.83, 1.12). Finally, results were similar whether time-to-pregnancy values of 0 and 1 were grouped together (main results), separated out (FR = 0.96, 95 percent CI: 0.88, 1.07), or omitted from analysis (FR = 0.96, 95 percent CI: 0.85, 1.09).

DES-exposed men fathered fewer boys than girls (sex ratio = 0.497), while unexposed men fathered fewer girls than boys (sex ratio = 0.517), yielding an adjusted odds ratio for male birth of 0.92 (95 percent CI: 0.80, 1.04) (table 4). Neither time to pregnancy nor the report of an abnormal semen analysis was associated with sex ratio, and adjustment for these variables made little difference in the association of DES with sex ratio (data not shown). The odds ratios did not vary significantly by cohort (pinteraction = 0.94) or paternal age (pinteraction = 0.52). A reduced sex ratio was found among men first exposed at 11 or more gestational weeks in the Mayo cohort but not the Dieckmann cohort and could not be evaluated in the other cohorts because of limited data (table 5). No association was observed between DES dose and sex ratio (data not shown).


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  		TABLE 4. Prenatal DES* exposure and offspring sex ratio, overall and by selected factors, DES Combined Cohort Follow-up Study, 2001

 

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  		TABLE 5. Offspring sex ratio according to gestational timing of first DES* exposure, DES Combined Cohort Follow-up Study, 2001{dagger}

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Only one prior study, based on the Dieckmann cohort (7), has examined DES in relation to time to pregnancy. Using the most recent pregnancy, Wilcox et al. (7) noted an inverse but nonsignificant fecundability ratio comparing exposed with unexposed men (FR = 0.9, 95 percent CI: 0.7, 1.1). Their finding is generally consistent with fecundability ratios in the present study for all pregnancies (FR = 0.95, 95 percent CI: 0.86, 1.06), the first pregnancy (FR = 0.93, 95 percent CI: 0.80, 1.09), and the most recent pregnancy (FR = 0.96, 95 percent CI: 0.83, 1.12).

Although the fecundability ratio in the present study was stronger among men who tried to conceive for 6 or more months relative to less than 6 months, these differences might be explained by digit preference at time to pregnancy of 9 months among the unexposed. Such error could arise if a portion of the men misread the question about time to pregnancy and wrote "9" to reflect gestational months of pregnancy. If this occurred to a larger extent among the unexposed, who tend to report time to pregnancy less accurately (68) and are less concerned about the reproductive effects of DES, then in the 6- or more-months subgroup they would appear to have higher fecundability than the exposed, who may have answered the question more carefully. The digit preference had little effect on the overall fecundability ratio because the number of events at 9 months was relatively small.

There are no prior studies of DES in relation to secondary sex ratio in humans. The sex ratio of 0.517 among unexposed men agreed with worldwide population estimates of 0.514 (69). Although we found no statistically significant association between prenatal DES exposure and sex ratio, the direction of the association was consistent with that found for fathers exposed to other endocrine disruptors such as dioxin (27–29), polychlorinated biphenyls (35), mercury (40), and lead (38). Our study may have been underpowered to detect a difference in sex ratio if the magnitude of the true association is small. A decreased proportion of male infants was noted among men first exposed to DES later in gestation, but the association was found only among Mayo participants and was most likely due to chance.

Study strengths include a large sample and the verification of exposure by medical record. Compared with previous reports on fertility in DES-exposed men (7, 16), the extended follow-up in this study allowed more men to complete their reproductive lives. Men were asked to provide a detailed account of their reproductive histories, including the date and outcome of all pregnancies fathered, the sex of their offspring, and time to pregnancy for planned pregnancies. Time to pregnancy is easily obtained by self-administered questionnaire, and men are shown to have good recall (68, 70, 71). Although most retrospective time-to-pregnancy surveys cannot measure sterility because they include only couples that achieve pregnancy, the present study was able to include pregnancy attempts from men who tried to conceive for 12 or more months without success. Finally, we were able to incorporate all pregnancies reported by each man, accounting for the correlation of waiting times with frailty models (60–62). This approach has greater validity and efficiency relative to those confined to a single pregnancy (21).

Study limitations include retrospectively assessed reproductive history, limited data on accidental pregnancies and determinants of time to pregnancy, and incomplete follow-up. Given that DES-exposed men report time-to-pregnancy data with greater accuracy than do unexposed men (68), differential reporting may explain the nonsignificantly reduced fecundability among exposed men. Although time-to-pregnancy data were limited to planned pregnancies only, bias is unlikely because there were similar proportions of accidental pregnancies among exposed and unexposed men. We did not ask men about their pregnancy intentions, sexual behaviors, or other determinants of time to pregnancy (e.g., age of female partner, change in partner, persistence in trying, trying for <12 months without success, or timing of intercourse during the fertile period), and uncontrolled differences by exposure may have introduced confounding. However, control for education and paternal age, which are highly correlated with time-to-pregnancy determinants such as maternal age (72) and persistence in trying (73), should minimize the extent of confounding. Finally, selection bias is unlikely given that similar proportions of exposed and unexposed men completed the 2001 questionnaire.

In conclusion, DES exposure was not associated with a significant decrease in fecundability or the proportion of male offspring overall. These results, based on the largest study of documented exposure to DES, support several prior studies showing no effect of prenatal DES exposure on reproductive outcomes in men.


   ACKNOWLEDGMENTS
 
This work was supported by National Cancer Institute contracts N01-CP-21168, N01-CP-51017, N01-CP-01289, and N01-CP-01290.

The authors acknowledge the technical assistance of Marianne Hyer and the helpful feedback of Allen Wilcox, Clarice Weinberg, Donna Day Baird, and Stanley J. Robboy.

Conflict of interest: none declared.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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