Antenatal exposure to DES: lessons learned…future concerns

DES-exposed offspring : certain complications have no time limit and continued follow-up is necessary, 2007


The short- and long-term effects of the widespread use of diethylstilbestrol (DES) over 3 decades have become a distant memory for many clinicians. Others are too young to remember the flurry of activity in the early 1970s on the part of many medical centers to identify the offspring of women who were prescribed DES during their pregnancies.

This medication was given in an attempt to prevent multiple pregnancy-related problems such as miscarriage, premature birth, and abnormal bleeding.

The recognition of the association of DES with an increased incidence of cervical and vaginal cancers in very young women led the Food and Drug Administration to ban its use during pregnancy in 1971.

Other pregnancy-related problems for the daughters and genitourinary tract changes in the sons did not become apparent until years later.

Ongoing follow-up of these offspring has raised concerns for their future as well as their mothers’ future. Clinicians need to be up-to-date with current knowledge regarding risks for cancer and other health-related issues.

Abstract (Third-Generation Effects)

Animal studies have shown tumor growth in older third-generation mice (human equivalent to age 70).

Multigenerational studies in humans are currently underway.

Several small studies of teenage third-generation females have not shown the same type of changes as in their mothers. Sons of DES daughters are at increased risk for hypospadias.


  • Antenatal exposure to DES: lessons learned…future concerns, Obstetrical and gynecological survey, NCBI PubMed PMID: 17634156, 2007 Aug.
  • Image credit wise owl tea ‏.

Informatively empty clusters with application to multigenerational studies

Recently, Kioumourtzoglou examined the impact of in-utero DES exposure among nurses on attention-deficit/hyperactivity disorder (ADHD) in their children

2019 Summary

Exposures with multigenerational effects have profound implications for public health, affecting increasingly more people as the exposed population reproduces. Multigenerational studies, however, are susceptible to informative cluster size, occurring when the number of children to a mother (the cluster size) is related to their outcomes, given covariates. A natural question then arises: what if some women bear no children at all? The impact of these potentially informative empty clusters is currently unknown.

This article first evaluates the performance of standard methods for informative cluster size when cluster size is permitted to be zero. We find that if the informative cluster size mechanism induces empty clusters, standard methods lead to biased estimates of target parameters. Joint models of outcome and size are capable of valid conditional inference as long as empty clusters are explicitly included in the analysis, but in practice empty clusters regularly go unacknowledged. In contrast, estimating equation approaches necessarily omit empty clusters and therefore yield biased estimates of marginal effects.

To resolve this, we propose a joint marginalized approach that readily incorporates empty clusters and even in their absence permits more intuitive interpretations of population-averaged effects than do current methods. Competing methods are compared via simulation and in a study of the impact of in-utero exposure to diethylstilbestrol on the risk of attention-deficit/hyperactivity disorder (ADHD) among 106 198 children to 47 540 nurses from the Nurses Health Study.

Study population

The proposed methods are motivated by a study of the effect of diethylstilbestrol exposure on thirdgeneration ADHD diagnosis in the Nurses Health Study II. The data consist of K=61 485 female nurses aged 25–42 in 1989 who returned a series of questionnaires in subsequent years and had no multiple sameyear births. In 2005 and 2013, nurses reported whether their children had been diagnosed with ADHD and analysis is restricted to concordant responses. The data are hierarchical in nature, with N = 106 198 children clustered within families identified by their mothers (nurses).

A key feature of the data is that cluster size (number of children) is potentially informative, as seen in Table 3: ADHD prevalence ranged from 5.62% in only-children to 3.22% in children from families of five or more children. Some of this relationship may be due to diethylstilbestrol exposure, whose rate was highest for nurses with no children (2.79%) and decreased to 1.18% for those with five or more children. Critically, 23% of nurses reported no live births and were thus excluded from previous analyses. To explore the impact of this decision on the conclusions of the analysis, we now consider the full population of nurses that met the eligibility criteria, this time including those without children.


The primary aim of the study was to quantify the effect of diethylstilbestrol on third-generation ADHD diagnosis, and we compared results of each analysis approach considered in the simulations. Logistic outcome models were adjusted for nurse’s exposure to diethylstilbestrol, smoking status, and year of birth. For the joint models, we modeled cluster size using a zero-inflated Poisson model (where the Poisson component adjusted for the same covariates and the zero inflation adjusted for exposure) in order to permit informative and non-informative emptiness. For the joint model that ignores empty clusters, we assumed a Poisson distribution, with a minimum size of one. We adopted a random intercepts model with exposure-dependent variance (as in the simulations), permitting correlation to depend on diethylstilbestrol exposure.


Estimates of marginal parameters can be found in Table 4. Diethylstilbestrol had a moderate adverse population-averaged (marginal) effect onA DHD risk, and estimates varied only somewhat across analyses: the independence estimating equations odds ratio estimate was 1.46 [95% confidence interval (CI) (1.19–1.78)] and was slightly larger than the cluster size weighted estimating equations estimate of 1.39 (1.13–1.71). Because these estimates are consistent for distinct parameters only under informative cluster size, these results (in light of the large sample size) suggest weak informativeness. As such, emptiness did not seem to have a large impact here, and the joint marginalized estimate fell between those of the estimating equations [1.41; 95% CI (1.14–1.73)].

Conditional parameter estimates can be found in Tables 5. The cluster-specific (conditional) estimates of the exposure-ADHD odds ratio were naturally much larger, but still varied little across analyses, ranging from 2.39 (1.38–4.12) under the outcome-only GLMM to 2.33 (1.33–4.06) under the complete joint model. The other covariate-outcome associations varied negligibly across conditional analyses.

Despite discrepant levels of correlation by exposure level (σ0 and σ1 are estimated to be 2.03 and 1.66 under the joint model), the variation in exposure effects across analyses is modest. This is because although there was a strong potential for informative cluster size (see Table 3), the actual level of informativeness was low (the estimate of the scaling parameter for the unexposed was −0.01).



Pregnancy Drugs, Fetal Germline Epigenome, and Risks for Next-Generation Pathology

A Call to Action, Environmental and Molecular Mutagenesis,
Escher J, Robotti S, 19 March 2019.


Drugs taken during pregnancy can affect three generations at once:

  • the gestating woman (F0),
  • her exposed fetus (F1),
  • and the fetal germ cells that confer heritable information for the grandchildren (F2).

Unfortunately, despite growing evidence for connections between F0 drug exposures and F2 pathology, current approaches to risk assessment overlook this important dimension of risk.

In this commentary, we argue that the uniquemolecular vulnerabilities of the fetal germline, particularly with regard to global epigenomic reprogramming, combined withempirical evidence for F2 effects of F1 in utero drug and other exposures, should change the way we consider potential long-term consequences of pregnancy drugs and alter toxicology’s standard somatic paradigm.

Specifically, we

  1. suggest that pregnancy drugs common in the post-war decades should be investigated as potential contributorsto the “missing heritability” of many pathologies now surging in prevalence;
  2. call for inclusion of fetal germline risks in pregnancy drugsafety assessment;
  3. and highlight the need for intensified research to ascertain generational impacts of diethylstilbestrol (DES), a vanguard question of human germline toxicity.

Only by fully addressing this important dimension of transplacental exposure can we responsibly evaluate safety of drug exposures during pregnancy and convey the full scope of risks, while also retrospectively comprehending the generational legacy of recent history’s unprecedented glut of evolutionarily novel intrauterine exposures.

The imperative to intensify research on diethylstilbestrol F2s

“Finally, we ask that research to ascertain generational impacts of diethylstilbestrol (DES) should be intensified and broadened in scope. The DES disaster presents a paradigmatic question of human germline toxicity, and a unique opportunity to better understand generational impacts of this drug, and also the broader phenomenon of hormone disruption in humans. But while research on DES F2s has linked the exposure to a variety of pathologies, including increased risks of infertility, hypospadias, tumor growth, ovarian cancer, menstrual irregularities, and ADHD, by and large the research has been limited compared to the magnitude of exposure and the breadth of possible ensuing pathologies.

For example, the issue of neurodevelopmental outcomes and socio-sexual behavior strikes us as very important and mostly unexplored. Only one study has attempted to ascertain F2 neurodevelopmental impairments, and it indeed detected a link. Though a recent study found no evidence of same-sex orientation in female F2s borne of female DES exposed F1s, no other study has probed socio-sexual outcomes in F2s other either sex, whether through male or female F1s. We note that a recent animal study found these types of F2 effects when F0 dams were exposed to EDCs. In other words, research must think more broadly about F2 pathologies precipitated by DES exposure to also encompass the brain, cognitive ability,behavior, sexuality, and other crucial endpoints beyond the standard teratogenesis paradigm.

The urgency of more F2 research is critical not just for our affected families, but for the science of endocrine disruption generally and an entire population increasingly worried about generational effects of hormone-disrupting chemicals. DES is the bellwether of hormone disruption—given the combined existence of exposure records and possibilities for ascertainment of F2 outcomes, it uniquely shines a light on this crucial scientific question of germline and heritable effects of disrupted hormone signaling.

The primary research study looking at DES F2 outcomes in the United States is the National Cancer Institute (NCI) DES Combined Cohort Follow-Up Study formed in 1992. It consists of eight different cohort studies which totaled about 21,000 participants at inception and included DES-exposed F0 mothers, DES F1 daughters, and DES F1 sons, as well as unexposed mothers, daughters and sons. The oldest cohort started in 1971, the most recent in 1984, and two are British studies. Almost all the studies look at F2 outcomes via F1 exposed females, but not the F1 exposed males. In the initial decade, all participants of the largest of the eight cohorts, the Diethylstilbestrol Adenosis Project (DESAD), were physically examined yearly. Since that time the research inputs consist of questionnaires every five years.
While some subjects have been lost to the study due to location mobility and death, extensive attempts are made to seek all participants. Decreasing ability to do robust follow-up concerns us. We are just beginning to appreciate the effects on F2s, and indeed many effects on F1s as well, and while several other teams have published significant DES studies, many of those other efforts lack access to a database of people with confirmed medical histories of DES exposure, which presents a limitation.

A withering of efforts from what had been the primary source of research on the effects of DES represents a loss of an opportunity to learn critical lessons for understanding what may be at stake when early germ cells undergo programming in a foreign hormonal milieu.”



Grandmaternal DES and ADHD in Children

Dr Kioumourtzoglou‘s reply to comments in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

We appreciate the interest that Drs Ryan and Smith and Dr Costas have expressed in our article.

Drs Ryan and Smith raise the issue of exposure misclassification and the potential problem of overreporting of grandmaternal diethylstilbestrol (DES) exposure by mothers whose children have attention-deficit/hyperactivity disorder (ADHD).

First, we were not clear in the article, but the κ of 0.74 for nurse and grandmother reporting was specific for DES.

We also found that the DES-specific κ value did not vary by ADHD status.

Concern for biased recall does need to be considered, and our results should indeed be interpreted cautiously, while hopefully spurring more work in this area.

That said, some aspects of our study differ from the military study Drs Ryan and Smith reference.

When the nurses reported their mothers’ DES exposure (1993), there had been no human studies of any third-generation effects and possibly only 1 mouse study of tumors. Thus, the level of attention to potential anthrax effects on those directly exposed was not there for third-generation DES effects in 1993.

In addition, the nurses were not queried about ADHD in their children until, at earliest, 12 years after the question about their mothers’ DES exposure (2005). So the possibility of priming the nurse to think about a possible DES-ADHD association by asking about exposure and outcome at the same time was avoided.

Additionally, the specificity of elevated effects in the first trimester would argue against recall bias. In our models, we simultaneously included terms for unknown trimester DES exposure and exposure in other trimesters as a check for confounding, which also works as a check for recall bias in this case.

Indeed, one might expect that nurses falsely recalling grandmaternal DES exposure would be more likely to report that they did not know the trimester of exposure, yet that group did not exhibit increased odds of ADHD.

In addition to these points, in responding to this letter we conducted the same analysis restricted to the 18 792 F0 women (42 097 F2 children) who provided information themselves about DES use during pregnancy with F1, instead of F1 reporting. In these analyses, we found virtually the same result, although with wider 95% CI as expected with the smaller numbers: adjusted odds ratio, 1.31 (95% CI, 1.00-1.71).



Grandmaternal DES and ADHD in Children – Costas’ Question

Dr Costas editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“I read with interest the article “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits” by Kioumourtzoglou et al,  published in JAMA Pediatrics.

Using a valuable cohort of 47 450 women in the Nurses’ Health Study II, the authors found that exposure to the potent synthetic estrogen diethylstilbestrol during pregnancy was associated with increased risk of attention-deficit/hyperactivity disorder (ADHD) in the grandchildren.

They reasonably conclude that “diethylstilbestrol exposure is associated with multigenerational neurodevelopmental deficits.”

This result is a valuable contribution to the literature on etiology of neurodevelopmental disorders, adding further evidence for the role of endocrine-disrupting chemicals as risk factors for ADHD.

The etiology of ADHD is complex, involving a combination of genetic as well as environmental risk factors.

Heritability for ADHD has been estimated as 70% to 80%.

Ehe genetics basis of ADHD is partly owing to many variants of individual low effect scattered along the genome.” …



Grandmaternal DES and ADHD in Children – Ryan’s Question

Drs Ryan and Smith editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“We read with interest the publication by Kioumourtzoglou et al, identifying a link between grandmaternal diethylstilbesterol (DES) exposure and attention-deficit/hyperactivity disorder in children.

We certainly agree with the JAMA Pediatrics Editorial that this transgenerational association is novel and provocative.” …


  • Continue readingGrandmaternal Diethylstilbesterol and Attention-Deficit/Hyperactivity Disorder in Children, on JAMA Pediatrics doi:10.1001/jamapediatrics.2018.3737, December 2018.
  • Featured image by psycom.

DES side effects in the third generation, known in 2005

image of DES russian dolls

The history of DES, lessons to be learned

“Health care professionals have to know the (full) history of DES to prevent future disasters with drugs prescribed.”

Abstract – 2005

At this moment, little is known about the implications of DES exposure for the third generation. Research has been done in mice to investigate the possibility of negative effects of DES on the male offspring of in utero exposed mice. The fertility of these males appeared to be the same as the fertility of the reference group. However, an increase in lesions of the testis and in tumours of the reproductive tract was reported, thereby providing the suggestion that effects of DES exposure can be transmitted to the third generation in mice.

Female offspring of mice exposed to DES in utero was also examined. In a group of 40 third generation mice ten uterine adenocarcinomas and five ovarian cystadenocarcinomas were seen, while tumours were absent in the control group. The development of these forms of cancer seemed to be age-dependent.

To find out whether these results would also be seen in humans, a group of 28 third-generation daughters was observed. While 61.5% of their mothers demonstrated cervical or vaginal changes associated with DES exposure in utero, none of these daughters showed any abnormality in the lower genital tract. However, this study is too small to draw conclusions but it seems that the carryover of DES effects in the female third generation is not of great significance.

Because the effects of DES in the third generation might be caused by biologic mechanisms, a study focused on the age at menarche in DES granddaughters. Fifty-two girls of mothers exposed to DES in utero were compared with 71 girls of unexposed mothers. The age at menarche was unaffected by DES administration to the grandmothers of these girls.

Next to the consequences in DES granddaughters, DES effects on the third-generation sons were also examined. As a result of two cases of hypospadia in sons of women that were exposed to DES in utero, a cohort study was done to find out whether an association between hypospadia in third generation males and DES exists. Hypospadia is a defect of the urogenital system in which the urethral opening is located on the ventral surface of the penis or on the scrotum. Sons of women with fertility problems (N ¼ 8934) were included in this study; 205 boys were sons of women exposed to DES in utero and the mothers of the remaining 8729 were unexposed. Four cases of hypospadia were reported in sons of exposed women compared with eight in the sons of unexposed. The prevalence rate ratio for hypospadia following from these results is 21.3, thereby suggesting an association between the administration of DES and the risk of hypospadia in the male third generation.

These outcomes suggest that there are no effects of DES exposure in females of the third generation, but that an increased risk of hypospadias exists for male offspring of women exposed to DES in utero. However, these results are obtained from small studies, so more research has to be carried out to obtain more reliable information.


  • The history of DES, lessons to be learned, Pharmacy world & science : PWS, PMID: 16096877, 2005 Jun.
  • Image credit rawdonfox.

Six cases of women with diethylstilbestrol in utero demonstrating long-term manifestations and current evaluation guideline

Journal of lower genital tract disease, 2005


Diethylstilbestrol (DES), a nonsteroidal estrogen, was widely used in the United States from 1940 through 1971 to prevent pregnancy loss.

In the late 1960s, an association was made with an increased incidence of clear cell adenocarcinoma in young women exposed in utero to DES. Additional study of these women over the next 35 years has shown an increased risk of other health problems including intraepithelial neoplasia, ectopic pregnancy, first trimester spontaneous abortion and second trimester pregnancy loss.

The National Institutes of Health continues to fund studies to follow cohorts of DES-exposed mothers, daughters, sons and third generation children. The Centers for Disease Control have conducted a large DES Education Project and have established guidelines for management.

The following six cases studies illustrate common problems seen in DES exposed daughters and management of problems encountered.


  • Six cases of women with diethylstilbestrol in utero demonstrating long-term manifestations and current evaluation guidelines, Journal of lower genital tract disease, PMID: 15870516, 2005 Jan.
  • Image credit earlymenopause.

Findings in female offspring of women exposed in utero to diethylstilbestrol

Lower genital tract abnormalities in DES granddaughters

2002 Study Abstract

To examine a group of women (third-generation daughters) whose mothers were exposed in utero to diethylstilbestrol (DES) and compare their findings on pelvic examination with those noted in their mothers.

Letters were mailed to women documented to have been exposed in utero to DES who had given birth to a female offspring, inviting them to have their daughters come in for a detailed history and pelvic examination. Records of the mothers whose daughters appeared for examination were reviewed, and findings noted at the time of their initial examination were recorded. Detailed pelvic examination of the third-generation daughters included colposcopic examination and iodine staining of the vagina and cervix and Papanicolaou smear. The findings observed in these women were compared with those noted in their mothers at the time of their mothers’ first examination.

Twenty-eight third-generation daughters were examined. Three of the daughters were delivered from one mother. Review of the mothers’ records indicated that 16 (61.5%) of the mothers exposed to DES during their pregnancy demonstrated structural changes of the cervix, upper vagina, or vaginal epithelial changes consisting of adenosis, nonstaining vaginal epithelium after application of iodine solution, or white epithelium within the vagina. None of the daughters were found to have changes usually associated with DES exposure.

The absence of abnormalities in the lower genital tract in third-generation women compared with the high frequency of these abnormalities in their mothers suggests that third-generation carryover effects of in utero DES exposure are unlikely.


  • Findings in female offspring of women exposed in utero to diethylstilbestrol, Obstetrics and gynecology, PMID: 11814496, 2002 Feb.
  • 3D Animal Models featured image credit Paolo Bendandi.