In the 1960s, 7 cases of vaginal cancer were diagnosed in women between the ages of 15 and 22 at the Vincent Memorial Hospital in Boston. Since no diagnosis had ever been made in this age group and the 7 cases were diagnosed within 3 years, a retrospective study of the patients and their families was conducted. This study highlighted the use of DES in the mothers of these women during pregnancy. Nevertheless, DES was prescribed to 675 other patients in the hospital during this period and only 7 cases of vaginal cancer were diagnosed [
106]. A few years later, a larger study confirmed an increased risk of vaginal cancer with in utero exposure to DES [
107]. The diagnosis of vaginal cancer in several women exposed in utero to DES led to the follow-up of all persons exposed to DES in many countries. Five major cohorts including the National Cooperative Diethylstilbestrol Adenosis Project (DESAD) cohort, the Women’s Health Study cohort, the Mayo Clinic cohort, Dieckmann’s clinical study conducted at the University of Chicago (1951–1952), and Horne’s study conducted in a private clinic in Massachusetts were analyzed. The main studies are summarized in
Table 2.
The effect of DES was first evaluated in mothers exposed during pregnancy, known as “DES mothers”. A first study published in 1978 compared the risk of BC between the cohort of 693 mothers who had participated in Dieckmann’s clinical study and 668 control mothers. This study showed a non-significant increase in the number of BC in the exposed women. However, this study was conducted soon after exposure to DES relative to the time it generally takes for the effects of EDCs to become clinically evident. Although no information is given on age at diagnosis in the study, it is possible that the majority of the cohort was younger than the median age of BC diagnosis since only a maximum of 25 years elapsed between the time of DES exposure and the study [
108]. A study of 2885 DES mothers and 2816 controls found that DES exposure during pregnancy increased the risk of BC by 40–50% compared to unexposed women. Interestingly, the increase was statistically significant for women exposed 30 years prior the study. This study had a longer follow-up (up to 40 years after DES exposure), a larger cohort, and an auto-questionnaire that took into account other risk factors in the women of both cohorts [
109]. Another study of 2590 DES mothers and 2471 controls investigated the effect of DES more than 40 years after exposure. After adjustment for various risk factors (age, age at menarche, age at first pregnancy, history of miscarriage), DES exposure during pregnancy increased the risk of BC by 47% in women of 60 years of age or more. This study has the same strengths as the previous study, with an even longer follow-up (beyond 40 years) [
110]. Finally, a study involving 2434 DES mothers and 2402 controls from the Dieckmann and Mayo Clinic cohorts confirmed that women exposed to DES have an increased risk of BC, independent of other risk factors [
111].
The effect of the drug was then evaluated in the children of DES mothers (i.e., those exposed in utero) called “DES daughters or sons”. In DES sons, an increased risk of genital malformations such as hypospadias (malformation of the urethra) or cryptorchidism were observed [
119]. In addition to clear cell vaginal adenocarcinomas, several studies have reported an increase in cervical and uterine malformations, as well as problems during pregnancy (miscarriage, ectopic pregnancy, pre-eclampsia, premature birth, perinatal death), and infertility in DES daughters [
120]. Concerning cancer risk, a first study of 3650 DES daughters versus 1202 control girls found no increased risk for all types of cancers including BC. This study was conducted on a large cohort of DES girls from the DESAD, Dieckmann and Horne cohorts, and different variables were taken into account such as year of birth, level of education, age at menarche, at menopause and at first birth, parity, oral contraceptive or HRT use, and family history of breast or ovarian cancer. Nevertheless, the average age of women was only 38 years, which is much younger than the median age of diagnosis of BC (around 67 years) [
112]. Since this first study, others were conducted on large cohorts and showed an association between in utero exposure to DES and BC risk. A study including 3812 DES daughters and 1637 unexposed girls from different cohorts (DESAD, Women’s Health Study, Dieckmann and Horne) found a significant increase in BC risk in DES daughters but with age adjustment only. The increased risk of BC is greater in DES daughters than in the mothers. Indeed, women under the age of 40 had no increased risk, whereas the risk increased by more than 90% in women over 40 years of age. This risk is almost 4-times higher in postmenopausal women over the age of 50. Interestingly, the risk differed according to the characteristics of the tumor, as there was an increased risk only for ERα+ tumors. These results highlight the importance of long-term follow-up of women exposed to EDCs. This study took into account many risk factors, making it a statistically powerful multivariate analysis (year of birth, marital status, level of education, tabaco consumption (mother and daughter), oral contraceptive and HRT use, family history of BC, BMI and birth weight, age at menarche and menopause, parity, age at first birth, and number of mammograms in the last 5 years). This study also took into account gestational age at first exposure to DES as well as dose (low or high) [
113]. One year later, a study regrouping 3813 DES daughters and 1642 controls from the same 4 cohorts and also taking into account numerous BC risk factors and gestational age at first DES exposure and dose, confirmed the increased risk in women over 40 years of age only. Indeed, DES daughters over 40 years of age had a BC risk increased by 83%, whereas no increase was found in younger women [
62]. In 2011, a study combining the DESAD, Women’s Health Study and Dieckmann cohorts of 3796 DES daughters and 1659 controls also found a 40% increased risk of BC in women over 40 years of age in women exposed in utero to DES compared to control. Interestingly, this study also found an increase in adverse health outcomes such as infertility, spontaneous abortion, preterm delivery and pre-eclampsia [
114]. More recently, a study analyzed the risk of BC in 4822 DES daughters compared to 2083 unexposed women from the DESAD, Women’s Health Study, Dieckmann and Horne cohorts. This study confirmed again an increased risk in DES daughters after age 40, but the increase in risk was less significant compared to data from the 2007 Troisi study. Indeed, only women between 40 and 49 years of age had a significant increase in BC risk of 33%. This study took into account many risk factors for BC as well as gestational age at first DES exposure and dose [
115].
Recently, a study of 796 children of women exposed in utero to DES, called “DES granddaughters or grandsons”, and 469 controls was published. The third generation presented an increase in genital malformations and other health problems similar to those of DES daughters or sons. Thereby, the impact of exposure of pregnant women to DES appears to be multigenerational, affecting the outcome of the third generation (e.g., hypospadias, miscarriage, ectopic pregnancy, premature birth). However, studies on the multigenerational effect of DES on adult diseases such as cancer are limited because the third generation is still young (average age 24 years in this study). Although this study was conducted on a relatively small cohort and statistical power was limited, it highlights the multigenerational impact of DES [
118].