A Prospective Cohort Study of Prenatal Diethylstilbestrol Exposure and Cardiovascular Disease Risk

A Prospective Cohort Study of Prenatal Diethylstilbestrol Exposure and Cardiovascular Disease Risk Abstract Purpose Prenatal exposure to diethylstilbestrol (DES), a prototype endocrine-disrupting chemical, is associated with risk for adverse reproductive outcomes and cancer in women. We investigated whether cardiovascular disease (CVD) risk might also be greater in women prenatally exposed to DES. Methods DES-exposed (n = 3941) and -unexposed (n = 1705) women participating in the Combined DES Cohort Follow-up Study were followed prospectively from 1994 to 2013. Prenatal DES exposure (or lack of exposure) was documented in the birth record or physician’s note. Participants reported by questionnaire any “serious medical conditions requiring hospitalization, surgery or long-term treatment,” including coronary artery disease (CAD), myocardial infarction (MI), and stroke. We sought physician’s verification of self-reports and identified CVD deaths from the National Death Index. Hazard ratios (HRs) with 95% confidence intervals (CIs) from Cox proportional hazard regression models estimated associations between DES exposure and CVD incidence, adjusted for birth year, original cohort, and potential confounders. Results In comparison of the exposed to the unexposed women, the HRs for reported conditions were 1.74 (95% CI, 1.03 to 2.93) for CAD, 2.20 (95% CI, 1.15 to 4.21) for MI, 1.01 (95% CI, 0.54 to 1.90) for stroke, and 1.31 (95% CI, 0.93 to 1.86) for the combined conditions (i.e., total CVD). The HRs were similar for verified outcomes (CAD, 1.72; MI, 2.67; stroke, 0.92; and total CVD, 1.25) and with additional adjustment for hypertension, diabetes, and high cholesterol (HRs: CAD, 1.67; MI, 2.04; stroke, 0.96; and total CVD, 1.24). Conclusions These data demonstrate associations in women who have prenatal DES exposure with CAD and MI, but not with stroke, which appear to be independent of established CVD risk factors. Diethylstilbestrol (DES), a potent synthetic estrogen and endocrine disrupter, was administered in the United States and Europe to several million pregnant women to prevent complications of pregnancy from the 1940s until 1971, when prenatal exposure was found to be strongly associated with clear cell adenocarcinoma of the vagina and cervix in young women (1). Subsequently, numerous anatomic anomalies, infertility, adverse reproductive outcomes, and grade 2 or higher cervical intraepithelial neoplasia were linked with in utero exposure in women (2, 3). Breast cancer incidence may also be greater in DES-exposed women (2). Data from animals prenatally exposed to DES (4), along with human studies of bisphenol A (BPA) (5), a chemically similar although weaker synthetic estrogen, have raised concern that exposure to endocrine disruptors may be positively linked with cardiovascular disease (CVD) risk (5). This issue is especially important because of BPA’s ubiquitous presence in the environment. Preliminary evidence from the National Cancer Institute (NCI) Combined DES Cohort Follow-up Study suggested a possible excess of CVD in women who were prenatally exposed to DES, but this was based on participants’ CVD report without verification (6). In this paper, we update findings with additional follow-up and include physician-verified CVD diagnoses in a study of documented human exposure to DES in utero. These data, based on high doses of DES first administered early in the pregnancy, provide a model to calibrate concerns about the influence of environmental estrogens on health. Participants and Methods Cohorts The US NCI Combined DES Cohort Follow-up Study consists of prenatally exposed and unexposed women who met one of the following criteria: (1) They participated in the National Cooperative Diethylstilbestrol Adenosis Project (DESAD) cohort) (7), (2) their mothers participated in a clinical trial of DES from 1951 to 1952 (Dieckmann cohort) (8), (3) their mothers were treated in a large, private infertility practice in Massachusetts (Horne), and (4) they were from Massachusetts, New Hampshire, and Maine and their mothers participated in the Women’s Health Study cohort (9). The follow-up of the combined cohorts began in 1994 with a mailed questionnaire, and subsequent questionnaires were mailed at ~5-year intervals in 1997, 2001, 2006, and 2011. Participants Women were eligible for analysis if they responded to the 2001, 2006, or 2011 questionnaire. Among 6084 (4236 exposed and 1848 unexposed) women who participated in the NCI follow-up of the combined cohort, roughly 6% were excluded because they responded only to the 1994 and/or 1997 questionnaire. An additional 1% of the exposed and unexposed died (of causes other than those being analyzed) without responding to the required questionnaires. The remaining 5646 (3941 exposed and 1705 unexposed) women were included in the analysis (detailed in Table 1). Table 1. Participants Included in the Analysis and Questionnaire Response by Prenatal DES Exposure Status Variable  DES-Exposed a  DES-Unexposed a  Total  4236 (100)  1848 (100)  Reason for exclusion       1994 and 1997 questionnaires only  256 (6.0)  118 (6.4)   Deceasedb  39 (0.92)  25 (1.4)  Included in analysis  3941 (93.0)  1705 (92.3)  Responded to 2001, 2006, and 2011 questionnaires  2980 (70.3)  1331 (72.0)  Variable  DES-Exposed a  DES-Unexposed a  Total  4236 (100)  1848 (100)  Reason for exclusion       1994 and 1997 questionnaires only  256 (6.0)  118 (6.4)   Deceasedb  39 (0.92)  25 (1.4)  Included in analysis  3941 (93.0)  1705 (92.3)  Responded to 2001, 2006, and 2011 questionnaires  2980 (70.3)  1331 (72.0)  Values are expressed as number (percentage) of participants. a Percentages are based on the total number of women in each column. b Women who died were excluded if they did not respond to a 2001, 2006, or 2011 questionnaire (in which women were queried about CVD), and their cause of death was not one of the outcomes being analyzed. View Large Ascertainment of medical conditions The 2001, 2006, and 2011 questionnaires included a checklist of “serious medical conditions requiring hospitalization, surgery or long-term treatment” and queried the date of diagnosis; therefore, only participants who completed at least one of these questionnaires were included in the analysis. The checklist included coronary artery disease (CAD), myocardial infarction (MI), and stroke, as well as adult-onset diabetes, high cholesterol, and hypertension. For the analysis, stroke, CAD, and MI were analyzed individually and combined to create a total CVD category. Also, an open-ended question was included to allow reporting of other, unlisted conditions, which were coded by a blinded nosologist using the International Classification of Diseases, Ninth Revision, with discrepancies arbitrated by a supervisor. We attempted to obtain physician verification of self-reported CVD diagnosed after 1 January 2001 (details presented in Table 2) because we thought that records would be difficult to obtain for earlier diagnoses and because of the relatively young age, and hence lower risk, of the cohort before that time (the mean age of the women in 2001 was 47 years). The proportions of DES-exposed and -unexposed women who provided consent to obtain records were 66% and 77%, respectively. Of 115 women reporting CVD on the questionnaire, we obtained 87 physician records for 67 participants (58%). Physician records confirmed that 52 of the 67 participants (78%) had a diagnosis of MI, CAD, and stroke, and an additional six indicated a probable CVD condition (increasing the verification to 87%). An additional 22 CVD cases (of which six were self-reported without confirmation) were identified from International Classification of Diseases coding of underlying and contributing causes of death from the National Death Index Plus or death certificate. Participants could contribute to multiple CVD events. In total, there were 40 cases of verified CAD, 23 cases of verified MI, 20 cases of verified stroke, and 6 cases of probable CVD. Table 2. Source of Diagnosis and Verification of CVD From 2001 to 2013, by DES Exposure Status Variable  DES-Exposed (n = 3941)  DES-Unexposed (n = 1705)  Total  Number of participants         Reported CVDa on questionnaire (n)  85  30  115   Physician records obtained, n (%)  49 (58)  18 (60)  67 (58)   Confirmed reported diagnosis, n (%)  37 (76)  15 (83)  52 (78)   Indicated probable CVD diagnosisb, n (%)  4 (84)  2 (94)  6 (87)   NDI or death certificate alone indicated CVD as cause of death (n)  13  9  22   Total with CVD outcomes confirmed by physician’s records or NDI/death certificate (n)  54  26  80  Number of eventsc        Reported CVDa on questionnaire (n)  102  35  137   Physician records obtained, n (%)  65 (64)  22 (63)  87 (64)   Confirmed reported diagnosis, n (%)  47 (72)  18 (82)  65 (75)  Indicated probable CVD diagnosisb, n (%)  4 (78)  2 (91)  6 (82)   Verified CVD including deaths (n)          CAD  28  12  40    MI  18  5  23    Stroke  12  8  20  Variable  DES-Exposed (n = 3941)  DES-Unexposed (n = 1705)  Total  Number of participants         Reported CVDa on questionnaire (n)  85  30  115   Physician records obtained, n (%)  49 (58)  18 (60)  67 (58)   Confirmed reported diagnosis, n (%)  37 (76)  15 (83)  52 (78)   Indicated probable CVD diagnosisb, n (%)  4 (84)  2 (94)  6 (87)   NDI or death certificate alone indicated CVD as cause of death (n)  13  9  22   Total with CVD outcomes confirmed by physician’s records or NDI/death certificate (n)  54  26  80  Number of eventsc        Reported CVDa on questionnaire (n)  102  35  137   Physician records obtained, n (%)  65 (64)  22 (63)  87 (64)   Confirmed reported diagnosis, n (%)  47 (72)  18 (82)  65 (75)  Indicated probable CVD diagnosisb, n (%)  4 (78)  2 (91)  6 (82)   Verified CVD including deaths (n)          CAD  28  12  40    MI  18  5  23    Stroke  12  8  20  a CVD events from the questionnaire included CAD, MI, and stroke. b Probable CVD was included only with total CVD, and not with individual CVD events in the data analysis. c Participants can have more than one type of event (i.e., CAD, MI, stroke), so number of events is larger than the number of participants. View Large We also evaluated two conditions (chronic fatigue syndrome and fibromyalgia) chosen a priori with no known associations with DES or other endocrine disruptors to gauge the extent of possible over-reporting of medical conditions by the DES exposed. DES exposure and covariate ascertainment For all combined cohort participants, prenatal exposure to DES, or the lack thereof, was documented by the birth record or physician’s note. Gestational week of first and last DES use, respectively, was available for 74% and 25% of exposed women; duration of use was calculated from these. Data on total cumulative DES dose were available for only 38% of the women; therefore, we classified the individual cohorts as high- or low-dose based on differences in prescribing practices by US region (unknown for a subgroup of the Women's Health Study). Agreement between the dose categories and individual doses was excellent among those with complete data (10). Participant’s birth weight and gestational age were available from birth records. Highest level of education completed, cigarette smoking (ever smoked cigarettes regularly for ≥6 months; current and former status with date of cessation in the latter), and ever use of alcohol (at least one alcoholic beverage per month for 6 months or longer), as well as information on body size (height and weight), age at menarche, and frequency of routine medical examinations in the last 5 years were collected on the 1994 questionnaire. Smoking, body weight, and routine medical screening were updated on subsequent questionnaires. Body mass index (BMI; weight in kilograms/height in meters squared) was calculated. Menopausal status and postmenopausal hormone use were ascertained on all five questionnaires and represented by time-dependent variables. Statistical analysis Two analyses were performed: one using self-reported conditions and one using verified diagnoses. For the analysis of self-reported conditions, follow-up began in 1994. If physician records disconfirmed a self-reported diagnosis, the participant was treated as a noncase. Person-years accrued until the earliest of the following dates: first reported diagnosis of CAD, stroke, or MI (or date of death) or return of the latest questionnaire. For the analysis of verified CVD diagnoses, follow-up began in 2001. If a self-reported diagnosis was not verified by the physician record, National Death Index Plus, or the death certificate, the participant was treated as a noncase and censored at their self-reported diagnosis date. Person-years accrued until the earliest of the following dates: first confirmed diagnosis of the CAD, stroke, or MI (or date of death) or return of the latest questionnaire. Associations of DES and CVD were estimated with hazard ratios (HRs) and 95% confidence intervals (CIs) from Cox proportional hazard regression models (11), with age as the underlying time parameter, by using SAS statistical software, version 9.4 (SAS Institute Inc., Cary, NC) (12). The models included terms for DES study cohort (n = 4) and birth year (continuous). Additional models also included terms for education, BMI, smoking status, alcohol use, age at menarche, menopausal status (time-dependent), postmenopausal hormone use (time-dependent), and number of physical examinations in the previous 5 years as of the start of follow-up. The categories for each of the covariates other than birth year (which was treated as continuous) are those shown in Table 3. Missing values were categorized separately and included in the models: Ninety-one percent of the participants had values for all five of the covariates (BMI, alcohol, smoking, physical examinations, and education), 4% were missing all five, 1.5% were missing education only, 1.5% were missing physical examinations only, and the remaining 2% were missing a mix of values. Results were similar when adjusted analyses were repeated excluding participants who had missing covariate values (complete case approach) compared with results from models that included categories for missing covariate values (data not shown). In separate models, we adjusted for hypertension, high cholesterol, and diabetes. Associations between DES and the conditions were stratified by menopausal status. In addition, associations were evaluated by DES dose, timing of first and last DES use, and duration of use (excluding the subcohort of the Women’s Health Study in which dose was unknown). Table 3. Characteristics of Study Participants by Prenatal DES Exposure Status Characteristic  DES-Exposed a  DES-Unexposed a  Total  3941 (100)  1705 (100)  Cohort       DESAD  3222 (81.8)  812 (47.6)   Dieckmann  251 (6.4)  219 (12.8)   Horne  204 (5.2)  142 (8.3)   Women’s Health Study  264 (6.7)  532 (31.2)  Year of birth       <1950  650 (16.5)  431 (25.3)   1950–1954  1666 (42.3)  705 (41.4)   1955–1959  996 (25.3)  403 (23.6)   1960+  629 (16.0)  166 (9.7)  Education (1994)       High school or less  510 (12.9)  341 (20.0)   Some college  863 (21.9)  415 (24.3)   4-y college  1332 (33.8)  518 (30.4)   Graduate school  1037 (26.3)  394 (23.1)   Missing  199 (5.1)  37 (2.2)  Parity       Nulliparous  1289 (32.7)  399 (23.4)   Parous  2637 (66.9)  1303 (76.4)   Missing  15 (0.38)  3 (0.18)  Smoking status (1994)       Never  2198 (55.8)  848 (49.7)   Ever  1534 (38.9)  812 (47.6)   Missing  209 (5.3)  45 (2.6)  BMI (1994)       <20 kg/m2  555 (14.1)  250 (14.7)   20–24 kg/m2  1978 (50.2)  839 (49.2)   25–29 kg/m2  727 (18.5)  363 (21.3)   ≥30 kg/m2  459 (11.7)  204 (12.0)   Missing  222 (5.6)  49 (2.9)  Alcohol intake (1994)       No  815 (20.7)  352 (20.7)   Yes  2892 (73.4)  1297 (76.1)   Missing  234 (5.9)  56 (3.3)  General physical exams (last 5 y; 1994)       0  573 (14.5)  212 (12.4)   1  922 (23.4)  381 (22.4)   2–3  1224 (31.1)  556 (32.6)   ≥4  951 (24.1)  483 (28.3)   Missing  271 (6.9)  73 (4.3)  Birth weight       <3000 g  1451 (36.8)  359 (21.1)   3000–3499 g  1360 (34.5)  570 (33.4)   ≥3500 g  847 (21.5)  438 (25.7)   Missing  283 (7.2)  338 (19.8)  Gestational age        <37 wk  446 (11.3)  59 (3.5)   37–39 wk  1416 (35.9)  549 (32.2)   ≥40 wk  1272 (32.3)  620 (36.4)   Missing  807 (20.5)  477 (28.0)  Age at menarche (1994)       <12 y  614 (15.6)  284 (16.7)   12–13 y  2368 (60.1)  987 (57.9)   ≥14 y  919 (23.3)  414 (24.3)   Missing  40 (1.0)  20 (1.2)  Menstrual status (at last follow-up)       Premenopausal  352 (8.9)  152 (8.9)   Postmenopausal  2637 (66.9)  1187 (69.6)   Censored  952 (24.2)  366 (21.5)  Postmenopausal hormone use (at last follow-up)       Never  2185 (55.4)  948 (55.6)   Ever  1714 (43.5)  744 (43.6)   Censored  42 (1.1)  13 (0.76)  Characteristic  DES-Exposed a  DES-Unexposed a  Total  3941 (100)  1705 (100)  Cohort       DESAD  3222 (81.8)  812 (47.6)   Dieckmann  251 (6.4)  219 (12.8)   Horne  204 (5.2)  142 (8.3)   Women’s Health Study  264 (6.7)  532 (31.2)  Year of birth       <1950  650 (16.5)  431 (25.3)   1950–1954  1666 (42.3)  705 (41.4)   1955–1959  996 (25.3)  403 (23.6)   1960+  629 (16.0)  166 (9.7)  Education (1994)       High school or less  510 (12.9)  341 (20.0)   Some college  863 (21.9)  415 (24.3)   4-y college  1332 (33.8)  518 (30.4)   Graduate school  1037 (26.3)  394 (23.1)   Missing  199 (5.1)  37 (2.2)  Parity       Nulliparous  1289 (32.7)  399 (23.4)   Parous  2637 (66.9)  1303 (76.4)   Missing  15 (0.38)  3 (0.18)  Smoking status (1994)       Never  2198 (55.8)  848 (49.7)   Ever  1534 (38.9)  812 (47.6)   Missing  209 (5.3)  45 (2.6)  BMI (1994)       <20 kg/m2  555 (14.1)  250 (14.7)   20–24 kg/m2  1978 (50.2)  839 (49.2)   25–29 kg/m2  727 (18.5)  363 (21.3)   ≥30 kg/m2  459 (11.7)  204 (12.0)   Missing  222 (5.6)  49 (2.9)  Alcohol intake (1994)       No  815 (20.7)  352 (20.7)   Yes  2892 (73.4)  1297 (76.1)   Missing  234 (5.9)  56 (3.3)  General physical exams (last 5 y; 1994)       0  573 (14.5)  212 (12.4)   1  922 (23.4)  381 (22.4)   2–3  1224 (31.1)  556 (32.6)   ≥4  951 (24.1)  483 (28.3)   Missing  271 (6.9)  73 (4.3)  Birth weight       <3000 g  1451 (36.8)  359 (21.1)   3000–3499 g  1360 (34.5)  570 (33.4)   ≥3500 g  847 (21.5)  438 (25.7)   Missing  283 (7.2)  338 (19.8)  Gestational age        <37 wk  446 (11.3)  59 (3.5)   37–39 wk  1416 (35.9)  549 (32.2)   ≥40 wk  1272 (32.3)  620 (36.4)   Missing  807 (20.5)  477 (28.0)  Age at menarche (1994)       <12 y  614 (15.6)  284 (16.7)   12–13 y  2368 (60.1)  987 (57.9)   ≥14 y  919 (23.3)  414 (24.3)   Missing  40 (1.0)  20 (1.2)  Menstrual status (at last follow-up)       Premenopausal  352 (8.9)  152 (8.9)   Postmenopausal  2637 (66.9)  1187 (69.6)   Censored  952 (24.2)  366 (21.5)  Postmenopausal hormone use (at last follow-up)       Never  2185 (55.4)  948 (55.6)   Ever  1714 (43.5)  744 (43.6)   Censored  42 (1.1)  13 (0.76)  Values are expressed as number (percentage) of participants. a Percentages in table do not always add to 100% because of rounding. View Large Institutional review board approval Approvals for the study were obtained from the human investigations committees at the field centers and the NCI. Participants indicated their informed consent by completion of a questionnaire or telephone interview and by signed consent for medical record retrieval. Results Characteristics of the DES-exposed and -unexposed women Most of the exposed and unexposed women were from the DESAD cohort (Table 3). Exposed women were slightly younger, completed more years of education, and were less likely to smoke than the unexposed but were roughly similar in BMI, alcohol intake, and use of postmenopausal hormones. Frequency of general physical examinations in the last 5 years was slightly lower in the exposed than unexposed women. As previously shown (13), mean birth weight and gestational age were lower in the DES-exposed women compared with the unexposed. Association of DES and self-reported CVD Women who were prenatally exposed to DES had about twice the risk for self-reported CAD (HR, 1.74; 95% CI, 1.03 to 2.93) and MI (HR, 2.20; 95% CI, 1.15 to 4.21) compared with those who were not exposed. The HR for stroke was not elevated (1.01; 95% CI, 0.54 to 1.90), and the HR for the combined CVD category was 1.31 (95% CI, 0.93 to 1.86) (Table 4). The associations were similar in the fully adjusted models, which contained terms for potential confounders (Table 4), and when time-dependent variables were used for smoking, BMI, and the updated variable for routine medical examinations (data not shown). Further adjustment for the participant’s own birth weight, gestational length, or small-for-gestational-age status did not change the HRs for DES or any of the conditions (data not shown). The results were also similar when we included hypertension, high cholesterol, and diabetes in the fully adjusted models to explore whether any of these risk factors mediated the association of DES and CVD; the HRs were 1.67 (95% CI, 0.99 to 1.83) for CAD, 2.04 (95% CI, 1.06 to 3.91) for MI, 0.96 (95% CI, 0.51 to 1.81) for stroke, and 1.24 (95% CI, 0.87 to 1.75) for total CVD. Table 4. HRs and 95% CIs for Prenatal DES Exposure and Self-Reported CVD Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n  Person-Years  Total  3941 (100)    1705 (100)        CAD  64 (1.6)  64,149  23 (1.4)  28,002  1.74 (1.03–2.93)  1.81 (1.07–3.04)  MI  44 (1.1)  64,264  14 (0.8)  27,992  2.20 (1.15–4.21)  2.21 (1.15–4.25)  Stroke  34 (0.9)  64,174  19 (1.1)  28,078  1.01 (0.54–1.90)  1.10 (0.58–2.09)  Total CVDc  126 (3.2)  63,737  55 (3.3)  27,799  1.31 (0.93–1.86)  1.36 (0.96–1.92)  Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n  Person-Years  Total  3941 (100)    1705 (100)        CAD  64 (1.6)  64,149  23 (1.4)  28,002  1.74 (1.03–2.93)  1.81 (1.07–3.04)  MI  44 (1.1)  64,264  14 (0.8)  27,992  2.20 (1.15–4.21)  2.21 (1.15–4.25)  Stroke  34 (0.9)  64,174  19 (1.1)  28,078  1.01 (0.54–1.90)  1.10 (0.58–2.09)  Total CVDc  126 (3.2)  63,737  55 (3.3)  27,799  1.31 (0.93–1.86)  1.36 (0.96–1.92)  a Adjusted for birth year and DES cohort. b Adjusted for birth year, cohort, BMI, smoking status, alcohol use, education, number of general physical examinations, age at menarche, menopausal status, and postmenopausal hormone use. c Includes women with CAD, MI, and/or stroke; the number of cases for the CVD category does not match the sum of the individual conditions because participants could have multiple conditions. View Large The HRs for DES and CAD, MI, stroke, and CVD were, respectively, 1.42, 1.93, 0.90, and 1.16 in the combined observational cohorts and 8.69, 4.63, 4.25, and 3.03 in the Dieckmann clinical trial cohort. However, the latter were based on only 21 CVD reports and the CIs were wide. Most of the cases were in the postmenopausal women. When premenopausal women were excluded, the results were similar to the overall findings (data not shown). There were no consistent patterns in the associations by DES dose and timing of first gestational exposure in the subset of women for whom this information was available (Supplemental Table 1). However, the number of cases was limited, and risks were higher, in the subset of women with information on when DES administration ended compared with the sample used for the main analysis. Association of DES and verified CVD The fully adjusted HRs for DES and CAD (1.74), MI (2.63), and total CVD (1.31) were similar for verified outcomes compared with the self-reported outcomes, but the CIs were wider owing to smaller sample sizes (Table 5). Table 5. HRs and 95% CIs for Prenatal DES Exposure and Verified CVD Outcomes Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n (%)  Person-Years  CAD  28 (0.7)  36,737  12 (0.7)  16,203  1.72 (0.82–3.64)  1.74 (0.82–3.68)  MI  18 (0.5)  36,828  5 (0.3)  16,211  2.67 (0.93–7.66)  2.63 (0.90–7.74)  Stroke  12 (0.3)  36,789  8 (0.5)  16,254  0.92 (0.34–2.50)  1.06 (0.37–3.04)  Total CVDc  54 (1.4)  36,412  26 (1.6)  16,030  1.25 (0.75–2.09)  1.31 (0.78–2.20)  Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n (%)  Person-Years  CAD  28 (0.7)  36,737  12 (0.7)  16,203  1.72 (0.82–3.64)  1.74 (0.82–3.68)  MI  18 (0.5)  36,828  5 (0.3)  16,211  2.67 (0.93–7.66)  2.63 (0.90–7.74)  Stroke  12 (0.3)  36,789  8 (0.5)  16,254  0.92 (0.34–2.50)  1.06 (0.37–3.04)  Total CVDc  54 (1.4)  36,412  26 (1.6)  16,030  1.25 (0.75–2.09)  1.31 (0.78–2.20)  a Adjusted for birth year and DES cohort. b Adjusted for birth year, cohort, BMI, smoking status, alcohol use, education, number of general physical examinations, age at menarche, menopausal status, and postmenopausal hormone use. c Includes women with CAD, MI, and/or stroke; the number of cases for the CVD category does not match the sum of the individual conditions because participants could have multiple conditions. View Large Analyses addressing possible biases The percentage of participants who reported a serious medical condition in the open-ended questions was similar for those exposed (48.7%) and unexposed (45.6%) to DES, after exclusion of conditions related to DES (infertility, pregnancy complications, cervical dysplasia, and breast cancer). Among women who reported a condition in the open-ended questions, the average number of reports was the same in the exposed and unexposed (2.0 for both groups). The HRs were also unchanged after exclusion of CVD cases identified through the open-ended question or those categorized as probable CVD (data not shown). Adjusted for birth year and cohort, the HR for fibromyalgia was 1.08 (95% CI, 0.65 to 1.80; based on 61 exposed and 26 unexposed) and for chronic fatigue syndrome was 0.98 (95% CI, 0.52-1.83; 36 exposed and 19 unexposed); the HRs for these conditions were essentially unchanged with adjustment for all potentially confounding factors (HRs, 1.09 and 1.01, respectively). As reported previously (6), for 52% of the mothers of exposed women in the DESAD cohort, the medical record did not specify a reason for DES treatment. For the remaining DESAD mothers, the most common reason stated in the medical record was spotting/bleeding (43.4%), followed by history of miscarriage, stillbirth, and abortion (24.1%); cramping or uterine irritability (20.1%); and threatened miscarriage (14.4%). Other reasons included headache (5.3%), history of infertility (3.2%), edema (1.6%), nausea/vomiting (1.3%), diabetes (0.6%), high blood pressure (0.2%), other (8.4%), and "routinely given" (1.0%). Discussion The risks for CAD and MI were higher among women who were exposed in utero to DES than in those who were not, independent of several established CAD risk factors, including hypertension, high cholesterol, diabetes, BMI, and smoking, as well as low birth weight. These findings confirm preliminary data from the NCI DES cohort that were based on a smaller number of unverified outcomes (6). Animal studies provide some support for a direct effect of DES on the heart. Limited data from the rodent model, which has been useful in predicting and replicating other adverse health outcomes in DES-exposed humans, show alterations in cardiac structure and function in female offspring (4) whose mothers were administered DES. Other animal data provide evidence of effects of in utero exposure to DES and other endocrine-disrupting chemicals that could indirectly influence CVD through metabolic disease in adulthood, including increases in body weight (14), abdominal fat, and elevated circulating levels of leptin, adiponectin, and insulin. Prenatal BPA exposure is associated with alterations in glucose homeostasis and endocrine pancreatic function in offspring (15). The effects of prenatal DES exposure on cardiac morphology and function in humans have not been studied. The known associations between DES and anomalies of the female reproductive tract suggest that tissues with estrogen receptors are most susceptible to the adverse effects of prenatal DES exposure. Whereas cardiomyocytes have estrogen receptors (16, 17), effects of DES on CAD may be more likely to be mediated through effects on glucose and insulin metabolism, which are altered in humans with exposure to endocrine-disrupting chemicals in adulthood (18–21). Adult concentrations of persistent organic pollutants, such as dioxin, dichlorodiphenyltrichloroethane, and polychlorinated biphenyls, are associated with diabetes, insulin resistance, and an altered lipid profile in healthy adults, as well as with the metabolic syndrome (18–21). In addition, urinary concentrations of BPA, a less potent endocrine-disrupting chemical than DES, have been positively associated with diagnosed diabetes mellitus and CVD (22). Our data, however, showed positive associations of prenatal DES exposure with CAD and MI despite adjustment for these possible mediators (hypertension, high cholesterol, and diabetes), suggesting that other mechanisms, such as changes in hormones or epigenetic markers, may be involved. We observed no evidence that the associations in the current study varied by DES dose, which might cast doubt on the possibility that the observations with CVD are causal. However, laboratory evidence challenges the role of dose-response models in the setting of endocrine disruption (23, 24). Systematic overreporting of CVD by exposed participants or overdiagnosis by their physicians could result in elevated risks. However, DES is not widely suspected of being associated with CVD, and the percentage of participants who reported any medical conditions in the open-ended question was similar for the exposed and unexposed women, as was the average number of conditions reported. Also, the lack of associations between DES and conditions chosen a priori with less rigid diagnostic criteria (chronic fatigue syndrome, fibromyalgia) suggests that the associations observed with CAD and MI are not due to systematic reporting bias. Differences in risk factors for these conditions that are not due to effects of prenatal DES exposure could explain some of our findings, but differences by DES exposure status were generally small for measured covariates and tended to be in the direction of higher risk for the unexposed. For example, cigarette smoking, a strong risk factor for CVD, was less common in the DES-exposed women. Although we cannot exclude the possibility of bias by DES indication, our data show that most women’s mothers had no documented reason for DES administration [consistent with contemporary advertisements promoting its use in uncomplicated pregnancy (25)]. Also, we observed elevated risks in the Dieckmann cohort, a clinical trial of DES, in which DES exposure was not associated with the mother’s pregnancy history. In summary, these data provide evidence of effects of prenatal DES exposure on CAD and MI in women. Further research should address possible underlying biological mechanisms, including differences in hormones and other biomarkers, as well as epigenetic patterns in adult offspring. Abbreviations: BMI body mass index BPA bisphenol A CAD coronary artery disease CI confidence interval CVD cardiovascular disease DES diethylstilbestrol DESAD National Cooperative Diethylstilbestrol Adenosis Project HR hazard ratio MI myocardial infarction NCI National Cancer Institute. Acknowledgments The authors thank the study managers at the field centers and Westat, Inc., for study-wide coordination efforts, and the DES-exposed and unexposed daughters and sons who participated in this study for their longstanding cooperation. Financial Support: Funding was provided through contracts from the National Cancer Institute. Author Contributions: R.T., L.T., E.E.H., J.R.P., D.H., W.C.S., E.A., and R.N.H. designed the study. R.T., L.T., E.E.H., J.R.P., W.C.S., and R.N.H. developed the statistical analysis plan, and R.T., W.R., and M.H. analyzed the data. R.T. wrote the paper and L.T., E.E.H., J.R.P., D.H., W.C.S., and R.N.H. edited the paper. R.T. is the guarantor of this work and, as such, had full access to all the study data and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women. N Engl J Med . 1971; 284( 15): 878– 881. Google Scholar CrossRef Search ADS PubMed  2. Hoover RN, Hyer M, Pfeiffer RM, Adam E, Bond B, Cheville AL, Colton T, Hartge P, Hatch EE, Herbst AL, Karlan BY, Kaufman R, Noller KL, Palmer JR, Robboy SJ, Saal RC, Strohsnitter W, Titus-Ernstoff L, Troisi R. Adverse health outcomes in women exposed in utero to diethylstilbestrol. N Engl J Med . 2011; 365( 14): 1304– 1314. Google Scholar CrossRef Search ADS PubMed  3. Herbst AL, Kurman RJ, Scully RE. Vaginal and cervical abnormalities after exposure to stilbestrol in utero. Obstet Gynecol . 1972; 40( 3): 287– 298. Google Scholar PubMed  4. Haddad R, Kasneci A, Sebag IA, Chalifour LE. Cardiac structure/function, protein expression, and DNA methylation are changed in adult female mice exposed to diethylstilbestrol in utero. Can J Physiol Pharmacol . 2013; 91( 9): 741– 749. Google Scholar CrossRef Search ADS PubMed  5. Han C, Hong YC. Bisphenol A, hypertension, and cardiovascular diseases: epidemiological, laboratory, and clinical trial evidence. Curr Hypertens Rep . 2016; 18( 2): 11– 15. Google Scholar CrossRef Search ADS PubMed  6. Troisi R, Hyer M, Hatch EE, Titus-Ernstoff L, Palmer JR, Strohsnitter WC, Herbst AL, Adam E, Hoover RN. Medical conditions among adult offspring prenatally exposed to diethylstilbestrol. Epidemiology . 2013; 24( 3): 430– 438. Google Scholar CrossRef Search ADS PubMed  7. Labarthe D, Adam E, Noller KL, O’Brien PC, Robboy SJ, Tilley BC, Townsend D, Barnes AB, Kaufman RH, Decker DG, Fish CR, Herbst AL, Gundersen J, Kurland LT. Design and preliminary observations of National Cooperative Diethylstilbestrol Adenosis (DESAD) Project. Obstet Gynecol . 1978; 51( 4): 453– 458. Google Scholar CrossRef Search ADS PubMed  8. Bibbo M, Gill WB, Azizi F, Blough R, Fang VS, Rosenfield RL, Schumacher GF, Sleeper K, Sonek MG, Wied GL. Follow-up study of male and female offspring of DES-exposed mothers. Obstet Gynecol . 1977; 49( 1): 1– 8. Google Scholar PubMed  9. Greenberg ER, Barnes AB, Resseguie L, Barrett JA, Burnside S, Lanza LL, Neff RK, Stevens M, Young RH, Colton T. Breast cancer in mothers given diethylstilbestrol in pregnancy. N Engl J Med . 1984; 311( 22): 1393– 1398. Google Scholar CrossRef Search ADS PubMed  10. Palmer JR, Wise LA, Hatch EE, Troisi R, Titus-Ernstoff L, Strohsnitter W, Kaufman R, Herbst AL, Noller KL, Hyer M, Hoover RN. Prenatal diethylstilbestrol exposure and risk of breast cancer. Cancer Epidemiol Biomarkers Prev . 2006; 15( 8): 1509– 1514. Google Scholar CrossRef Search ADS PubMed  11. Cox DR, Oakes RA. Analysis of Survival Data . London: Chapman and Hall; 1984. 12. SAS/STAT 13.2 [software]. Version 9.4 of the SAS System for Linux . Cary, NC: SAS Institute Inc.; 2013. 13. Hatch EE, Troisi R, Wise LA, Titus-Ernstoff L, Hyer M, Palmer JR, Strohsnitter WC, Robboy SJ, Anderson D, Kaufman R, Adam E, Hoover RN. Preterm birth, fetal growth, and age at menarche among women exposed prenatally to diethylstilbestrol (DES). 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Lee DH, Lee IK, Song K, Steffes M, Toscano W, Baker BA, Jacobs DR, Jr. A strong dose-response relation between serum concentrations of persistent organic pollutants and diabetes: results from the National Health and Examination Survey 1999-2002. Diabetes Care . 2006; 29( 7): 1638– 1644. Google Scholar CrossRef Search ADS PubMed  19. Lee DH, Lee IK, Jin SH, Steffes M, Jacobs DR, Jr. Association between serum concentrations of persistent organic pollutants and insulin resistance among nondiabetic adults: results from the National Health and Nutrition Examination Survey 1999-2002. Diabetes Care . 2007; 30( 3): 622– 628. Google Scholar CrossRef Search ADS PubMed  20. Lee DH, Lee IK, Porta M, Steffes M, Jacobs DR, Jr. Relationship between serum concentrations of persistent organic pollutants and the prevalence of metabolic syndrome among non-diabetic adults: results from the National Health and Nutrition Examination Survey 1999-2002. Diabetologia . 2007; 50( 9): 1841– 1851. 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Google Scholar CrossRef Search ADS PubMed  23. vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA, Eriksen M, Farabollini F, Guillette LJ Jr, Hauser R, Heindel JJ, Ho SM, Hunt PA, Iguchi T, Jobling S, Kanno J, Keri RA, Knudsen KE, Laufer H, LeBlanc GA, Marcus M, McLachlan JA, Myers JP, Nadal A, Newbold RR, Olea N, Prins GS, Richter CA, Rubin BS, Sonnenschein C, Soto AM, Talsness CE, Vandenbergh JG, Vandenberg LN, Walser-Kuntz DR, Watson CS, Welshons WV, Wetherill Y, Zoeller RT. Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reprod Toxicol . 2007; 24( 2): 131– 138. Google Scholar CrossRef Search ADS PubMed  24. Vandenberg LN. Non-monotonic dose responses in studies of endocrine disrupting chemicals: bisphenol a as a case study. Dose Response . 2013; 12( 2): 259– 276. Google Scholar PubMed  25. Troisi R, Hatch EE, Titus L. The diethylstilbestrol legacy: a powerful case against intervention in uncomplicated pregnancy. Pediatrics . 2016; 138( Suppl 1): S42– S44. Google Scholar CrossRef Search ADS PubMed  This work is written by US Government employees and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

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Abstract

Abstract Purpose Prenatal exposure to diethylstilbestrol (DES), a prototype endocrine-disrupting chemical, is associated with risk for adverse reproductive outcomes and cancer in women. We investigated whether cardiovascular disease (CVD) risk might also be greater in women prenatally exposed to DES. Methods DES-exposed (n = 3941) and -unexposed (n = 1705) women participating in the Combined DES Cohort Follow-up Study were followed prospectively from 1994 to 2013. Prenatal DES exposure (or lack of exposure) was documented in the birth record or physician’s note. Participants reported by questionnaire any “serious medical conditions requiring hospitalization, surgery or long-term treatment,” including coronary artery disease (CAD), myocardial infarction (MI), and stroke. We sought physician’s verification of self-reports and identified CVD deaths from the National Death Index. Hazard ratios (HRs) with 95% confidence intervals (CIs) from Cox proportional hazard regression models estimated associations between DES exposure and CVD incidence, adjusted for birth year, original cohort, and potential confounders. Results In comparison of the exposed to the unexposed women, the HRs for reported conditions were 1.74 (95% CI, 1.03 to 2.93) for CAD, 2.20 (95% CI, 1.15 to 4.21) for MI, 1.01 (95% CI, 0.54 to 1.90) for stroke, and 1.31 (95% CI, 0.93 to 1.86) for the combined conditions (i.e., total CVD). The HRs were similar for verified outcomes (CAD, 1.72; MI, 2.67; stroke, 0.92; and total CVD, 1.25) and with additional adjustment for hypertension, diabetes, and high cholesterol (HRs: CAD, 1.67; MI, 2.04; stroke, 0.96; and total CVD, 1.24). Conclusions These data demonstrate associations in women who have prenatal DES exposure with CAD and MI, but not with stroke, which appear to be independent of established CVD risk factors. Diethylstilbestrol (DES), a potent synthetic estrogen and endocrine disrupter, was administered in the United States and Europe to several million pregnant women to prevent complications of pregnancy from the 1940s until 1971, when prenatal exposure was found to be strongly associated with clear cell adenocarcinoma of the vagina and cervix in young women (1). Subsequently, numerous anatomic anomalies, infertility, adverse reproductive outcomes, and grade 2 or higher cervical intraepithelial neoplasia were linked with in utero exposure in women (2, 3). Breast cancer incidence may also be greater in DES-exposed women (2). Data from animals prenatally exposed to DES (4), along with human studies of bisphenol A (BPA) (5), a chemically similar although weaker synthetic estrogen, have raised concern that exposure to endocrine disruptors may be positively linked with cardiovascular disease (CVD) risk (5). This issue is especially important because of BPA’s ubiquitous presence in the environment. Preliminary evidence from the National Cancer Institute (NCI) Combined DES Cohort Follow-up Study suggested a possible excess of CVD in women who were prenatally exposed to DES, but this was based on participants’ CVD report without verification (6). In this paper, we update findings with additional follow-up and include physician-verified CVD diagnoses in a study of documented human exposure to DES in utero. These data, based on high doses of DES first administered early in the pregnancy, provide a model to calibrate concerns about the influence of environmental estrogens on health. Participants and Methods Cohorts The US NCI Combined DES Cohort Follow-up Study consists of prenatally exposed and unexposed women who met one of the following criteria: (1) They participated in the National Cooperative Diethylstilbestrol Adenosis Project (DESAD) cohort) (7), (2) their mothers participated in a clinical trial of DES from 1951 to 1952 (Dieckmann cohort) (8), (3) their mothers were treated in a large, private infertility practice in Massachusetts (Horne), and (4) they were from Massachusetts, New Hampshire, and Maine and their mothers participated in the Women’s Health Study cohort (9). The follow-up of the combined cohorts began in 1994 with a mailed questionnaire, and subsequent questionnaires were mailed at ~5-year intervals in 1997, 2001, 2006, and 2011. Participants Women were eligible for analysis if they responded to the 2001, 2006, or 2011 questionnaire. Among 6084 (4236 exposed and 1848 unexposed) women who participated in the NCI follow-up of the combined cohort, roughly 6% were excluded because they responded only to the 1994 and/or 1997 questionnaire. An additional 1% of the exposed and unexposed died (of causes other than those being analyzed) without responding to the required questionnaires. The remaining 5646 (3941 exposed and 1705 unexposed) women were included in the analysis (detailed in Table 1). Table 1. Participants Included in the Analysis and Questionnaire Response by Prenatal DES Exposure Status Variable  DES-Exposed a  DES-Unexposed a  Total  4236 (100)  1848 (100)  Reason for exclusion       1994 and 1997 questionnaires only  256 (6.0)  118 (6.4)   Deceasedb  39 (0.92)  25 (1.4)  Included in analysis  3941 (93.0)  1705 (92.3)  Responded to 2001, 2006, and 2011 questionnaires  2980 (70.3)  1331 (72.0)  Variable  DES-Exposed a  DES-Unexposed a  Total  4236 (100)  1848 (100)  Reason for exclusion       1994 and 1997 questionnaires only  256 (6.0)  118 (6.4)   Deceasedb  39 (0.92)  25 (1.4)  Included in analysis  3941 (93.0)  1705 (92.3)  Responded to 2001, 2006, and 2011 questionnaires  2980 (70.3)  1331 (72.0)  Values are expressed as number (percentage) of participants. a Percentages are based on the total number of women in each column. b Women who died were excluded if they did not respond to a 2001, 2006, or 2011 questionnaire (in which women were queried about CVD), and their cause of death was not one of the outcomes being analyzed. View Large Ascertainment of medical conditions The 2001, 2006, and 2011 questionnaires included a checklist of “serious medical conditions requiring hospitalization, surgery or long-term treatment” and queried the date of diagnosis; therefore, only participants who completed at least one of these questionnaires were included in the analysis. The checklist included coronary artery disease (CAD), myocardial infarction (MI), and stroke, as well as adult-onset diabetes, high cholesterol, and hypertension. For the analysis, stroke, CAD, and MI were analyzed individually and combined to create a total CVD category. Also, an open-ended question was included to allow reporting of other, unlisted conditions, which were coded by a blinded nosologist using the International Classification of Diseases, Ninth Revision, with discrepancies arbitrated by a supervisor. We attempted to obtain physician verification of self-reported CVD diagnosed after 1 January 2001 (details presented in Table 2) because we thought that records would be difficult to obtain for earlier diagnoses and because of the relatively young age, and hence lower risk, of the cohort before that time (the mean age of the women in 2001 was 47 years). The proportions of DES-exposed and -unexposed women who provided consent to obtain records were 66% and 77%, respectively. Of 115 women reporting CVD on the questionnaire, we obtained 87 physician records for 67 participants (58%). Physician records confirmed that 52 of the 67 participants (78%) had a diagnosis of MI, CAD, and stroke, and an additional six indicated a probable CVD condition (increasing the verification to 87%). An additional 22 CVD cases (of which six were self-reported without confirmation) were identified from International Classification of Diseases coding of underlying and contributing causes of death from the National Death Index Plus or death certificate. Participants could contribute to multiple CVD events. In total, there were 40 cases of verified CAD, 23 cases of verified MI, 20 cases of verified stroke, and 6 cases of probable CVD. Table 2. Source of Diagnosis and Verification of CVD From 2001 to 2013, by DES Exposure Status Variable  DES-Exposed (n = 3941)  DES-Unexposed (n = 1705)  Total  Number of participants         Reported CVDa on questionnaire (n)  85  30  115   Physician records obtained, n (%)  49 (58)  18 (60)  67 (58)   Confirmed reported diagnosis, n (%)  37 (76)  15 (83)  52 (78)   Indicated probable CVD diagnosisb, n (%)  4 (84)  2 (94)  6 (87)   NDI or death certificate alone indicated CVD as cause of death (n)  13  9  22   Total with CVD outcomes confirmed by physician’s records or NDI/death certificate (n)  54  26  80  Number of eventsc        Reported CVDa on questionnaire (n)  102  35  137   Physician records obtained, n (%)  65 (64)  22 (63)  87 (64)   Confirmed reported diagnosis, n (%)  47 (72)  18 (82)  65 (75)  Indicated probable CVD diagnosisb, n (%)  4 (78)  2 (91)  6 (82)   Verified CVD including deaths (n)          CAD  28  12  40    MI  18  5  23    Stroke  12  8  20  Variable  DES-Exposed (n = 3941)  DES-Unexposed (n = 1705)  Total  Number of participants         Reported CVDa on questionnaire (n)  85  30  115   Physician records obtained, n (%)  49 (58)  18 (60)  67 (58)   Confirmed reported diagnosis, n (%)  37 (76)  15 (83)  52 (78)   Indicated probable CVD diagnosisb, n (%)  4 (84)  2 (94)  6 (87)   NDI or death certificate alone indicated CVD as cause of death (n)  13  9  22   Total with CVD outcomes confirmed by physician’s records or NDI/death certificate (n)  54  26  80  Number of eventsc        Reported CVDa on questionnaire (n)  102  35  137   Physician records obtained, n (%)  65 (64)  22 (63)  87 (64)   Confirmed reported diagnosis, n (%)  47 (72)  18 (82)  65 (75)  Indicated probable CVD diagnosisb, n (%)  4 (78)  2 (91)  6 (82)   Verified CVD including deaths (n)          CAD  28  12  40    MI  18  5  23    Stroke  12  8  20  a CVD events from the questionnaire included CAD, MI, and stroke. b Probable CVD was included only with total CVD, and not with individual CVD events in the data analysis. c Participants can have more than one type of event (i.e., CAD, MI, stroke), so number of events is larger than the number of participants. View Large We also evaluated two conditions (chronic fatigue syndrome and fibromyalgia) chosen a priori with no known associations with DES or other endocrine disruptors to gauge the extent of possible over-reporting of medical conditions by the DES exposed. DES exposure and covariate ascertainment For all combined cohort participants, prenatal exposure to DES, or the lack thereof, was documented by the birth record or physician’s note. Gestational week of first and last DES use, respectively, was available for 74% and 25% of exposed women; duration of use was calculated from these. Data on total cumulative DES dose were available for only 38% of the women; therefore, we classified the individual cohorts as high- or low-dose based on differences in prescribing practices by US region (unknown for a subgroup of the Women's Health Study). Agreement between the dose categories and individual doses was excellent among those with complete data (10). Participant’s birth weight and gestational age were available from birth records. Highest level of education completed, cigarette smoking (ever smoked cigarettes regularly for ≥6 months; current and former status with date of cessation in the latter), and ever use of alcohol (at least one alcoholic beverage per month for 6 months or longer), as well as information on body size (height and weight), age at menarche, and frequency of routine medical examinations in the last 5 years were collected on the 1994 questionnaire. Smoking, body weight, and routine medical screening were updated on subsequent questionnaires. Body mass index (BMI; weight in kilograms/height in meters squared) was calculated. Menopausal status and postmenopausal hormone use were ascertained on all five questionnaires and represented by time-dependent variables. Statistical analysis Two analyses were performed: one using self-reported conditions and one using verified diagnoses. For the analysis of self-reported conditions, follow-up began in 1994. If physician records disconfirmed a self-reported diagnosis, the participant was treated as a noncase. Person-years accrued until the earliest of the following dates: first reported diagnosis of CAD, stroke, or MI (or date of death) or return of the latest questionnaire. For the analysis of verified CVD diagnoses, follow-up began in 2001. If a self-reported diagnosis was not verified by the physician record, National Death Index Plus, or the death certificate, the participant was treated as a noncase and censored at their self-reported diagnosis date. Person-years accrued until the earliest of the following dates: first confirmed diagnosis of the CAD, stroke, or MI (or date of death) or return of the latest questionnaire. Associations of DES and CVD were estimated with hazard ratios (HRs) and 95% confidence intervals (CIs) from Cox proportional hazard regression models (11), with age as the underlying time parameter, by using SAS statistical software, version 9.4 (SAS Institute Inc., Cary, NC) (12). The models included terms for DES study cohort (n = 4) and birth year (continuous). Additional models also included terms for education, BMI, smoking status, alcohol use, age at menarche, menopausal status (time-dependent), postmenopausal hormone use (time-dependent), and number of physical examinations in the previous 5 years as of the start of follow-up. The categories for each of the covariates other than birth year (which was treated as continuous) are those shown in Table 3. Missing values were categorized separately and included in the models: Ninety-one percent of the participants had values for all five of the covariates (BMI, alcohol, smoking, physical examinations, and education), 4% were missing all five, 1.5% were missing education only, 1.5% were missing physical examinations only, and the remaining 2% were missing a mix of values. Results were similar when adjusted analyses were repeated excluding participants who had missing covariate values (complete case approach) compared with results from models that included categories for missing covariate values (data not shown). In separate models, we adjusted for hypertension, high cholesterol, and diabetes. Associations between DES and the conditions were stratified by menopausal status. In addition, associations were evaluated by DES dose, timing of first and last DES use, and duration of use (excluding the subcohort of the Women’s Health Study in which dose was unknown). Table 3. Characteristics of Study Participants by Prenatal DES Exposure Status Characteristic  DES-Exposed a  DES-Unexposed a  Total  3941 (100)  1705 (100)  Cohort       DESAD  3222 (81.8)  812 (47.6)   Dieckmann  251 (6.4)  219 (12.8)   Horne  204 (5.2)  142 (8.3)   Women’s Health Study  264 (6.7)  532 (31.2)  Year of birth       <1950  650 (16.5)  431 (25.3)   1950–1954  1666 (42.3)  705 (41.4)   1955–1959  996 (25.3)  403 (23.6)   1960+  629 (16.0)  166 (9.7)  Education (1994)       High school or less  510 (12.9)  341 (20.0)   Some college  863 (21.9)  415 (24.3)   4-y college  1332 (33.8)  518 (30.4)   Graduate school  1037 (26.3)  394 (23.1)   Missing  199 (5.1)  37 (2.2)  Parity       Nulliparous  1289 (32.7)  399 (23.4)   Parous  2637 (66.9)  1303 (76.4)   Missing  15 (0.38)  3 (0.18)  Smoking status (1994)       Never  2198 (55.8)  848 (49.7)   Ever  1534 (38.9)  812 (47.6)   Missing  209 (5.3)  45 (2.6)  BMI (1994)       <20 kg/m2  555 (14.1)  250 (14.7)   20–24 kg/m2  1978 (50.2)  839 (49.2)   25–29 kg/m2  727 (18.5)  363 (21.3)   ≥30 kg/m2  459 (11.7)  204 (12.0)   Missing  222 (5.6)  49 (2.9)  Alcohol intake (1994)       No  815 (20.7)  352 (20.7)   Yes  2892 (73.4)  1297 (76.1)   Missing  234 (5.9)  56 (3.3)  General physical exams (last 5 y; 1994)       0  573 (14.5)  212 (12.4)   1  922 (23.4)  381 (22.4)   2–3  1224 (31.1)  556 (32.6)   ≥4  951 (24.1)  483 (28.3)   Missing  271 (6.9)  73 (4.3)  Birth weight       <3000 g  1451 (36.8)  359 (21.1)   3000–3499 g  1360 (34.5)  570 (33.4)   ≥3500 g  847 (21.5)  438 (25.7)   Missing  283 (7.2)  338 (19.8)  Gestational age        <37 wk  446 (11.3)  59 (3.5)   37–39 wk  1416 (35.9)  549 (32.2)   ≥40 wk  1272 (32.3)  620 (36.4)   Missing  807 (20.5)  477 (28.0)  Age at menarche (1994)       <12 y  614 (15.6)  284 (16.7)   12–13 y  2368 (60.1)  987 (57.9)   ≥14 y  919 (23.3)  414 (24.3)   Missing  40 (1.0)  20 (1.2)  Menstrual status (at last follow-up)       Premenopausal  352 (8.9)  152 (8.9)   Postmenopausal  2637 (66.9)  1187 (69.6)   Censored  952 (24.2)  366 (21.5)  Postmenopausal hormone use (at last follow-up)       Never  2185 (55.4)  948 (55.6)   Ever  1714 (43.5)  744 (43.6)   Censored  42 (1.1)  13 (0.76)  Characteristic  DES-Exposed a  DES-Unexposed a  Total  3941 (100)  1705 (100)  Cohort       DESAD  3222 (81.8)  812 (47.6)   Dieckmann  251 (6.4)  219 (12.8)   Horne  204 (5.2)  142 (8.3)   Women’s Health Study  264 (6.7)  532 (31.2)  Year of birth       <1950  650 (16.5)  431 (25.3)   1950–1954  1666 (42.3)  705 (41.4)   1955–1959  996 (25.3)  403 (23.6)   1960+  629 (16.0)  166 (9.7)  Education (1994)       High school or less  510 (12.9)  341 (20.0)   Some college  863 (21.9)  415 (24.3)   4-y college  1332 (33.8)  518 (30.4)   Graduate school  1037 (26.3)  394 (23.1)   Missing  199 (5.1)  37 (2.2)  Parity       Nulliparous  1289 (32.7)  399 (23.4)   Parous  2637 (66.9)  1303 (76.4)   Missing  15 (0.38)  3 (0.18)  Smoking status (1994)       Never  2198 (55.8)  848 (49.7)   Ever  1534 (38.9)  812 (47.6)   Missing  209 (5.3)  45 (2.6)  BMI (1994)       <20 kg/m2  555 (14.1)  250 (14.7)   20–24 kg/m2  1978 (50.2)  839 (49.2)   25–29 kg/m2  727 (18.5)  363 (21.3)   ≥30 kg/m2  459 (11.7)  204 (12.0)   Missing  222 (5.6)  49 (2.9)  Alcohol intake (1994)       No  815 (20.7)  352 (20.7)   Yes  2892 (73.4)  1297 (76.1)   Missing  234 (5.9)  56 (3.3)  General physical exams (last 5 y; 1994)       0  573 (14.5)  212 (12.4)   1  922 (23.4)  381 (22.4)   2–3  1224 (31.1)  556 (32.6)   ≥4  951 (24.1)  483 (28.3)   Missing  271 (6.9)  73 (4.3)  Birth weight       <3000 g  1451 (36.8)  359 (21.1)   3000–3499 g  1360 (34.5)  570 (33.4)   ≥3500 g  847 (21.5)  438 (25.7)   Missing  283 (7.2)  338 (19.8)  Gestational age        <37 wk  446 (11.3)  59 (3.5)   37–39 wk  1416 (35.9)  549 (32.2)   ≥40 wk  1272 (32.3)  620 (36.4)   Missing  807 (20.5)  477 (28.0)  Age at menarche (1994)       <12 y  614 (15.6)  284 (16.7)   12–13 y  2368 (60.1)  987 (57.9)   ≥14 y  919 (23.3)  414 (24.3)   Missing  40 (1.0)  20 (1.2)  Menstrual status (at last follow-up)       Premenopausal  352 (8.9)  152 (8.9)   Postmenopausal  2637 (66.9)  1187 (69.6)   Censored  952 (24.2)  366 (21.5)  Postmenopausal hormone use (at last follow-up)       Never  2185 (55.4)  948 (55.6)   Ever  1714 (43.5)  744 (43.6)   Censored  42 (1.1)  13 (0.76)  Values are expressed as number (percentage) of participants. a Percentages in table do not always add to 100% because of rounding. View Large Institutional review board approval Approvals for the study were obtained from the human investigations committees at the field centers and the NCI. Participants indicated their informed consent by completion of a questionnaire or telephone interview and by signed consent for medical record retrieval. Results Characteristics of the DES-exposed and -unexposed women Most of the exposed and unexposed women were from the DESAD cohort (Table 3). Exposed women were slightly younger, completed more years of education, and were less likely to smoke than the unexposed but were roughly similar in BMI, alcohol intake, and use of postmenopausal hormones. Frequency of general physical examinations in the last 5 years was slightly lower in the exposed than unexposed women. As previously shown (13), mean birth weight and gestational age were lower in the DES-exposed women compared with the unexposed. Association of DES and self-reported CVD Women who were prenatally exposed to DES had about twice the risk for self-reported CAD (HR, 1.74; 95% CI, 1.03 to 2.93) and MI (HR, 2.20; 95% CI, 1.15 to 4.21) compared with those who were not exposed. The HR for stroke was not elevated (1.01; 95% CI, 0.54 to 1.90), and the HR for the combined CVD category was 1.31 (95% CI, 0.93 to 1.86) (Table 4). The associations were similar in the fully adjusted models, which contained terms for potential confounders (Table 4), and when time-dependent variables were used for smoking, BMI, and the updated variable for routine medical examinations (data not shown). Further adjustment for the participant’s own birth weight, gestational length, or small-for-gestational-age status did not change the HRs for DES or any of the conditions (data not shown). The results were also similar when we included hypertension, high cholesterol, and diabetes in the fully adjusted models to explore whether any of these risk factors mediated the association of DES and CVD; the HRs were 1.67 (95% CI, 0.99 to 1.83) for CAD, 2.04 (95% CI, 1.06 to 3.91) for MI, 0.96 (95% CI, 0.51 to 1.81) for stroke, and 1.24 (95% CI, 0.87 to 1.75) for total CVD. Table 4. HRs and 95% CIs for Prenatal DES Exposure and Self-Reported CVD Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n  Person-Years  Total  3941 (100)    1705 (100)        CAD  64 (1.6)  64,149  23 (1.4)  28,002  1.74 (1.03–2.93)  1.81 (1.07–3.04)  MI  44 (1.1)  64,264  14 (0.8)  27,992  2.20 (1.15–4.21)  2.21 (1.15–4.25)  Stroke  34 (0.9)  64,174  19 (1.1)  28,078  1.01 (0.54–1.90)  1.10 (0.58–2.09)  Total CVDc  126 (3.2)  63,737  55 (3.3)  27,799  1.31 (0.93–1.86)  1.36 (0.96–1.92)  Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n  Person-Years  Total  3941 (100)    1705 (100)        CAD  64 (1.6)  64,149  23 (1.4)  28,002  1.74 (1.03–2.93)  1.81 (1.07–3.04)  MI  44 (1.1)  64,264  14 (0.8)  27,992  2.20 (1.15–4.21)  2.21 (1.15–4.25)  Stroke  34 (0.9)  64,174  19 (1.1)  28,078  1.01 (0.54–1.90)  1.10 (0.58–2.09)  Total CVDc  126 (3.2)  63,737  55 (3.3)  27,799  1.31 (0.93–1.86)  1.36 (0.96–1.92)  a Adjusted for birth year and DES cohort. b Adjusted for birth year, cohort, BMI, smoking status, alcohol use, education, number of general physical examinations, age at menarche, menopausal status, and postmenopausal hormone use. c Includes women with CAD, MI, and/or stroke; the number of cases for the CVD category does not match the sum of the individual conditions because participants could have multiple conditions. View Large The HRs for DES and CAD, MI, stroke, and CVD were, respectively, 1.42, 1.93, 0.90, and 1.16 in the combined observational cohorts and 8.69, 4.63, 4.25, and 3.03 in the Dieckmann clinical trial cohort. However, the latter were based on only 21 CVD reports and the CIs were wide. Most of the cases were in the postmenopausal women. When premenopausal women were excluded, the results were similar to the overall findings (data not shown). There were no consistent patterns in the associations by DES dose and timing of first gestational exposure in the subset of women for whom this information was available (Supplemental Table 1). However, the number of cases was limited, and risks were higher, in the subset of women with information on when DES administration ended compared with the sample used for the main analysis. Association of DES and verified CVD The fully adjusted HRs for DES and CAD (1.74), MI (2.63), and total CVD (1.31) were similar for verified outcomes compared with the self-reported outcomes, but the CIs were wider owing to smaller sample sizes (Table 5). Table 5. HRs and 95% CIs for Prenatal DES Exposure and Verified CVD Outcomes Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n (%)  Person-Years  CAD  28 (0.7)  36,737  12 (0.7)  16,203  1.72 (0.82–3.64)  1.74 (0.82–3.68)  MI  18 (0.5)  36,828  5 (0.3)  16,211  2.67 (0.93–7.66)  2.63 (0.90–7.74)  Stroke  12 (0.3)  36,789  8 (0.5)  16,254  0.92 (0.34–2.50)  1.06 (0.37–3.04)  Total CVDc  54 (1.4)  36,412  26 (1.6)  16,030  1.25 (0.75–2.09)  1.31 (0.78–2.20)  Condition  DES-Exposed  DES-Unexposed  HR a (95% CI)  HR b (95% CI)  Participants, n (%)  Person-Years  Participants, n (%)  Person-Years  CAD  28 (0.7)  36,737  12 (0.7)  16,203  1.72 (0.82–3.64)  1.74 (0.82–3.68)  MI  18 (0.5)  36,828  5 (0.3)  16,211  2.67 (0.93–7.66)  2.63 (0.90–7.74)  Stroke  12 (0.3)  36,789  8 (0.5)  16,254  0.92 (0.34–2.50)  1.06 (0.37–3.04)  Total CVDc  54 (1.4)  36,412  26 (1.6)  16,030  1.25 (0.75–2.09)  1.31 (0.78–2.20)  a Adjusted for birth year and DES cohort. b Adjusted for birth year, cohort, BMI, smoking status, alcohol use, education, number of general physical examinations, age at menarche, menopausal status, and postmenopausal hormone use. c Includes women with CAD, MI, and/or stroke; the number of cases for the CVD category does not match the sum of the individual conditions because participants could have multiple conditions. View Large Analyses addressing possible biases The percentage of participants who reported a serious medical condition in the open-ended questions was similar for those exposed (48.7%) and unexposed (45.6%) to DES, after exclusion of conditions related to DES (infertility, pregnancy complications, cervical dysplasia, and breast cancer). Among women who reported a condition in the open-ended questions, the average number of reports was the same in the exposed and unexposed (2.0 for both groups). The HRs were also unchanged after exclusion of CVD cases identified through the open-ended question or those categorized as probable CVD (data not shown). Adjusted for birth year and cohort, the HR for fibromyalgia was 1.08 (95% CI, 0.65 to 1.80; based on 61 exposed and 26 unexposed) and for chronic fatigue syndrome was 0.98 (95% CI, 0.52-1.83; 36 exposed and 19 unexposed); the HRs for these conditions were essentially unchanged with adjustment for all potentially confounding factors (HRs, 1.09 and 1.01, respectively). As reported previously (6), for 52% of the mothers of exposed women in the DESAD cohort, the medical record did not specify a reason for DES treatment. For the remaining DESAD mothers, the most common reason stated in the medical record was spotting/bleeding (43.4%), followed by history of miscarriage, stillbirth, and abortion (24.1%); cramping or uterine irritability (20.1%); and threatened miscarriage (14.4%). Other reasons included headache (5.3%), history of infertility (3.2%), edema (1.6%), nausea/vomiting (1.3%), diabetes (0.6%), high blood pressure (0.2%), other (8.4%), and "routinely given" (1.0%). Discussion The risks for CAD and MI were higher among women who were exposed in utero to DES than in those who were not, independent of several established CAD risk factors, including hypertension, high cholesterol, diabetes, BMI, and smoking, as well as low birth weight. These findings confirm preliminary data from the NCI DES cohort that were based on a smaller number of unverified outcomes (6). Animal studies provide some support for a direct effect of DES on the heart. Limited data from the rodent model, which has been useful in predicting and replicating other adverse health outcomes in DES-exposed humans, show alterations in cardiac structure and function in female offspring (4) whose mothers were administered DES. Other animal data provide evidence of effects of in utero exposure to DES and other endocrine-disrupting chemicals that could indirectly influence CVD through metabolic disease in adulthood, including increases in body weight (14), abdominal fat, and elevated circulating levels of leptin, adiponectin, and insulin. Prenatal BPA exposure is associated with alterations in glucose homeostasis and endocrine pancreatic function in offspring (15). The effects of prenatal DES exposure on cardiac morphology and function in humans have not been studied. The known associations between DES and anomalies of the female reproductive tract suggest that tissues with estrogen receptors are most susceptible to the adverse effects of prenatal DES exposure. Whereas cardiomyocytes have estrogen receptors (16, 17), effects of DES on CAD may be more likely to be mediated through effects on glucose and insulin metabolism, which are altered in humans with exposure to endocrine-disrupting chemicals in adulthood (18–21). Adult concentrations of persistent organic pollutants, such as dioxin, dichlorodiphenyltrichloroethane, and polychlorinated biphenyls, are associated with diabetes, insulin resistance, and an altered lipid profile in healthy adults, as well as with the metabolic syndrome (18–21). In addition, urinary concentrations of BPA, a less potent endocrine-disrupting chemical than DES, have been positively associated with diagnosed diabetes mellitus and CVD (22). Our data, however, showed positive associations of prenatal DES exposure with CAD and MI despite adjustment for these possible mediators (hypertension, high cholesterol, and diabetes), suggesting that other mechanisms, such as changes in hormones or epigenetic markers, may be involved. We observed no evidence that the associations in the current study varied by DES dose, which might cast doubt on the possibility that the observations with CVD are causal. However, laboratory evidence challenges the role of dose-response models in the setting of endocrine disruption (23, 24). Systematic overreporting of CVD by exposed participants or overdiagnosis by their physicians could result in elevated risks. However, DES is not widely suspected of being associated with CVD, and the percentage of participants who reported any medical conditions in the open-ended question was similar for the exposed and unexposed women, as was the average number of conditions reported. Also, the lack of associations between DES and conditions chosen a priori with less rigid diagnostic criteria (chronic fatigue syndrome, fibromyalgia) suggests that the associations observed with CAD and MI are not due to systematic reporting bias. Differences in risk factors for these conditions that are not due to effects of prenatal DES exposure could explain some of our findings, but differences by DES exposure status were generally small for measured covariates and tended to be in the direction of higher risk for the unexposed. For example, cigarette smoking, a strong risk factor for CVD, was less common in the DES-exposed women. Although we cannot exclude the possibility of bias by DES indication, our data show that most women’s mothers had no documented reason for DES administration [consistent with contemporary advertisements promoting its use in uncomplicated pregnancy (25)]. Also, we observed elevated risks in the Dieckmann cohort, a clinical trial of DES, in which DES exposure was not associated with the mother’s pregnancy history. In summary, these data provide evidence of effects of prenatal DES exposure on CAD and MI in women. Further research should address possible underlying biological mechanisms, including differences in hormones and other biomarkers, as well as epigenetic patterns in adult offspring. Abbreviations: BMI body mass index BPA bisphenol A CAD coronary artery disease CI confidence interval CVD cardiovascular disease DES diethylstilbestrol DESAD National Cooperative Diethylstilbestrol Adenosis Project HR hazard ratio MI myocardial infarction NCI National Cancer Institute. Acknowledgments The authors thank the study managers at the field centers and Westat, Inc., for study-wide coordination efforts, and the DES-exposed and unexposed daughters and sons who participated in this study for their longstanding cooperation. Financial Support: Funding was provided through contracts from the National Cancer Institute. Author Contributions: R.T., L.T., E.E.H., J.R.P., D.H., W.C.S., E.A., and R.N.H. designed the study. R.T., L.T., E.E.H., J.R.P., W.C.S., and R.N.H. developed the statistical analysis plan, and R.T., W.R., and M.H. analyzed the data. R.T. wrote the paper and L.T., E.E.H., J.R.P., D.H., W.C.S., and R.N.H. edited the paper. R.T. is the guarantor of this work and, as such, had full access to all the study data and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women. N Engl J Med . 1971; 284( 15): 878– 881. Google Scholar CrossRef Search ADS PubMed  2. 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Journal

Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Jan 1, 2018

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