Transgenerational neuroendocrine disruption of reproduction

2011 summary of epigenetic and transgenerational effects of DES

Key points

  • The hypothalamic neuroendocrine systems develop in a sexually dimorphic manner, largely because of differences in levels of gonadal steroids
  • Environmental endocrine disrupting chemicals (EDCs) impair the function of the neuroendocrine systems that control reproduction
  • Developmental exposure to EDCs, particularly during embryonic and early postnatal periods, permanently impairs functions and predisposes individuals to disease later in life owing to altered epigenetic programming
  • The mechanisms of EDC action include effects on the epigenetic molecular machinery that controls gene expression in hypothalamic and reproductive tissues
  • Effects of EDCs may be transmitted transgenerationally through molecular changes to the germline or through context-dependent modifications to somatic cells by continued exposures to EDCs or the individual’s social or environmental context

DES and epigenetic transmission

Transgenerational epigenetic effects of the estrogenic EDC diethylstilbesterol (DES) began with observations of rare vaginal clear-cell carcinomas and reproductive tract abnormalities in young women whose mothers had been prescribed DES in a misguided effort to avert miscarriage. These observations provided the first evidence for developmental programming or the fetal basis of adult disease caused by exogenous estrogens in humans. A potent estrogenic pharmaceutical, DES not only failed to reduce miscarriage risk, but it also exposed the developing daughters and sons to high levels of prenatal estrogens and predisposed them to adult diseases. Animal studies have replicated many of these effects of prenatal DES treatment and have begun to reveal the molecular mechanisms by which DES programs developing tissues. The case of DES is important because it is one of the first to link fetal exposure to a hormonally active compound with the latent development of disease years or even decades after the insult. This example in humans has laid the groundwork for much of the work on the effect of other EDC exposures to the fetus.

Epidemiological studies have also found that DES is associated with a small but significant increase in birth defects in grandchildren of women given DES during pregnancy. The granddaughters reported an increase in heart conditions,  slight but significant differences in their menstrual cycles and a reduction in live births when compared with women whose grandmothers were not exposed to DES.  Another study found an increase in the risk of hypospadias in the sons of women who were exposed to DES in utero,  and there was the single clinical observation that the 15-year-old granddaughter of a woman who took DES while she was pregnant developed a small-cell carcinoma of the ovary  Although the sample sizes in the last two studies were exceedingly small (28 and 1, respectively), they provide preliminary evidence for the potential of transgenerational effects of EDCs in the human population.

Rodent studies have revealed transgenerational epigenetic effects of DES. DES exposure of the F1 generation during embryonic and early postnatal development at a range of dosages had adverse consequences in the F2 generations. In F2 males, the incidence of proliferative lesions of the testes was increased, and serum estradiol concentration was reduced. In F2 females, the incidence of uterine adenocarcinomas and other tumors of the reproductive tract was increased in a subset of the dosage groups. Exposure of newborn (postnatal day 1–5) F1 female mice to 2 μg of DES led to demethylation of the estrogen-sensitive gene lactotransferrin promoter at two CpG dinucleotides (−464 and −454) in the uterus, and to upregulation of uterine expression of lactotransferrin in both F1 female mice and their F2 female offspring.  Female F1 animals exposed to the same dosing paradigm had decreased methylation of exon 4 of the Fos gene on postnatal day 5 and increased Fos mRNA expression in the uterus in adulthood.  Additionally, mice exposed gestationally to 10 μg/kg of DES from embryonic days 9–16 resulted in increased methylation of the homeobox A10 promoter and intron and increased Hox-A10 protein expression in the postnatal uterus (day 14).

Treatment of newborn, female and male mice on post-natal days 1–5 with 3 μg per pup per day of DES changed total DNA methylation in reproductive tissues. DES also altered expression of the enzymes that regulate DNA methylation. For example, expression of DNA methyltransferases 1, 3a and 3b was increased in the epididymis, and DNA methyltransferases 1 and 3b were increased in the uterus of mice exposed to DES compared with animals exposed to the vehicle control. Finally, in rats treated with 1 mg/kg DES on postnatal days 10–12, the exposure decreased global histone trimethylation at lysine 27 in the uterus on postnatal day 12 and altered the expression of several estrogen-sensitive genes in the uterus of the neonatal and adult exposed animals. Although most of these studies have only been conducted in tissues from F1 individuals, they provide insight into the potential targets for molecular epigenetic modifications that merit further study in subsequent generations in rodents.

Sources

  • Transgenerational neuroendocrine disruption of reproduction, Endocrinology, NCBI PubMed PMC3976559, 2011 Jan 25.
  • Summary of epigenetic and transgenerational effects of DES featured image PMC3976559/table/T1.
DES DIETHYLSTILBESTROL RESOURCES

Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes

image of Environmental Epigenetics

2012 Study Abstracts

Introduction

This review focuses on how environmental factors through epigenetics modify disease risk and health outcomes. Major epigenetic events, such as histone modifications, DNA methylation, and microRNA expression, are described. The function of dose, duration, composition, and window of exposure in remodeling the individual’s epigenetic terrain and disease susceptibility are addressed. The ideas of lifelong editing of early-life epigenetic memories, transgenerational effects through germline transmission, and the potential role of hydroxylmethylation of cytosine in developmental reprogramming are discussed. Finally, the epigenetic effects of several major classes of environmental factors are reviewed in the context of pathogenesis of disease. These include endocrine disruptors, tobacco smoke, polycyclic aromatic hydrocarbons, infectious pathogens, particulate matter, diesel exhaust particles, dust mites, fungi, heavy metals, and other indoor and outdoor pollutants. We conclude that the summation of epigenetic modifications induced by multiple environmental exposures, accumulated over time, represented as broad or narrow, acute or chronic, developmental or lifelong, may provide a more precise assessment of risk and consequences. Future investigations may focus on their use as readouts or biomarkers of the totality of past exposure for the prediction of future disease risk and the prescription of effective countermeasures.

Implications of Lifelong Editing of Early-Life Epigenetic Memories

The concept of continued editing of early-life epigenetic markings or memories during adult life has been proposed on the basis of evidence from limited experimental studies. Exposure of mice to diethylstilbestrol (DES, a xenoestrogen) or genistein (a phytoestrogen) during the perinatal period induced specific epigenetic markings in their uteri. However, some of these epigenetic markings (hypomethylation of Nsbp1) remained “hidden” during prepuberty life and appeared in adulthood only in the exposed intact females but not in their ovariectomized counterparts, suggesting that adult exposure to ovarian steroids may cause these markings to “surface.” Coincidentally, the prevalence of uterine cancer was higher in neonatally exposed intact mice, but not in mice ovariectormized before puberty.

Epigenetic Factors Shown to Trigger Epigenetic Events and Affect Disease States

Exposure to EDCs during early developmental periods is a major health concern because it can cause persistent changes in gene expression through epigenetic reprogramming in somatic cells, as well as germ-line cells, and subsequently promote transgenerational inheritance. The xenoestrogen DES was widely used in cattle and other livestock industries and is still an EDC in many populations. Early-life exposure of mice to DES increases risk of uterine cancer that is accompanied by demethylation of an estrogen-responsive gene, lactoferrin, in the mouse uterus. In utero exposure of mice to DES triggered hypermethylation of the homeobox A10 with attended uterine hyperplasia and neoplasia in later life. A more recent report documented hypermethylation of nucleosome binding protein 1 (Nsbp1 or Hmgn5) as a hidden uterine epigenetic mark after neonatal DES exposure that only appeared upon sexual maturation of the exposed mice but failed to manifest if the animals were ovarietomized before puberty. Of significant interest is the transgenerational effect of developmental exposure of mice to DES that promoted c-fos expression, hypomethylation of specific exon CpGs, and increased susceptibility to tumorigenesis in the F2 generation. These experimental data support the hypothesis that epigenetic reprogramming is responsible for the devastating consequences observed in the offspring of women who took DES during pregnancy. The DES effects include female genital abnormalities, vaginal cancer, and male urogenital disorders. The adverse effects may be reverberating in the grandchildren of these women.

Sources

  • Full study (free access) : Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes, ILAR Journal, NCBI PubMed PMC4021822, 2012 Dec.
  • Featured image by h heyerlein.
DES DIETHYLSTILBESTROL RESOURCES

Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds

image of transgenerational-epigenetic-inheritance

2014 Study Abstract

The classic case of an EDC is diethylstilbestrol (DES), an estrogen agonist and androgen receptor antagonist synthesized first in the 1930s and prescribed to at least 5 million women at risk for miscarriage or experiencing other reproductive problems, from 1938 up to 1975. Instead of the desired effects, use of this compound lead to increased incidence of breast, vaginal, and cervical cancers.

In addition, maternal exposure has documented adverse affects on daughters. These include the same types of cancers as well as a variety of difficulties conceiving and maintaining pregnancies, reproductive tract malformations, abnormal menstrual cycles, early puberty, and behavioral issues. The vast variety of effects is probably related to complexities introduced by the timing of DES treatment and doses. For example, a recent large study of women exposed prenatally to DES revealed a strong correlation between DES, particularly in the first trimester, and noncancerous uterine fibroids. Offspring of rodents exposed to DES during pregnancy recapitulate many of these effects.

Although the initial clinical studies were limited to female offspring, correlations between DES exposure and hypospadias, cryptorchidism, and testicular cancer have been reported in F1 and F2 sons and grandsons of women given DES. Analyses of the clinical studies suggest that the male reproductive illnesses are related to but not necessarily caused by estrogen actions. An alternative hypothesis is that DES produces low-birth-weight babies, and these infants are more prone to testicular dysgenesis syndrome. Multigenerational work in mice has demonstrated that high, but clinically relevant, doses of DES increase the incidence of uterine and other reproductive tract tumors in females and lesions in the male rete testes in F2 offspring. Few data on the critical F3 generation in humans are available nor are there experimental data from rodent models.

EDCs, such as DES, share many properties with steroid hormones: they act at low doses (picograms) and can act in a nonmonotonic manne. Like hormones, they are particularly effective during development, at which time they can modify the course of reproductive tract and brain development. Importantly, the EDCs are more promiscuous than steroids and bind to a larger variety of receptors than normal ligands, albeit with reduced affinities.

Sources

  • Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds, Endocrinology, NCBI PubMed PMC4098001, 2014 Aug.
  • Featured image Matt Artz.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias in DES grandsons : a cohort study

image of grandsons

Hypospadias: a transgenerational effect of diethylstilbestrol ?

2002 Study Abstract

BACKGROUND
Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero.

METHODS
We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero.

FINDINGS
16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7).

INTERPRETATION
Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.

Sources

  • Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study, Lancet, NCBI PubMed PMID: 11943257, 2002 Mar 30.
  • Featured image Donna Borzyskowski.
DES DIETHYLSTILBESTROL RESOURCES

Transgenerational Inheritance of DES Exposure

Mechanisms of the Maternal Exposome and Implications for Health Outcomes

2016 Study Abstract

It is well established that the environment contributes to health. However, few studies have evaluated environmental exposures in women that may influence future health of their offspring. Knowledge gained may inform nursing how to better advocate for patients and families; and provide individualized interventions and education. Therefore, a more comprehensive investigation of the maternal exposome to uncover mechanistic insight into complex disease in offspring is warranted. To advance understanding of biological mechanisms that contribute to high-risk birth outcomes and offspring predisposition to disease, it will be necessary to measure a range of exposures and biomarkers before and during pregnancy.

Evidence for Trans-generational Inheritance of Exposures

Exposures of the gametes, including those that occur during the prenatal period and early development, have been found to play a large role in risk of disease over the lifespan. Studies in animal models have documented epigenetic changes that persist from generation to generation. For example, in murine models, the methylation changes associated with DES exposure persist into the third generation suggesting in utero exposures may have a lasting impact on the health of future generations. The same chemical clearly demonstrates a link between environment and germ-line mutations in humans as a trans-generational alteration in DNA methylation patterns of the fetus caused by exposure to DES in utero. DES is a synthetic estrogen that was administered to pregnant women to prevent spontaneous abortions prior to the mid 1970’s. DES causes hypermethylation of the homeobox protein Hox-A10 (HOXA10), a gene that controls uterine organ development, resulting in reproductive tract anomalies that persist into adulthood. Hypermethylation of HOXA10 was specific to the fetus and did not occur in laboratory experiments using cell lines or the somatic cells of pregnant women who received DES, suggesting that this molecular alteration is unique to in utero exposure.

Investigations are ongoing in grandchildren of women given DES. Current clinical evidence suggests DES granddaughters have more menstrual irregularities, infertility, and stillbirths; and both granddaughters and grandsons exhibit more birth defects. This accumulating evidence supports that epimutations occur from exposure during gonadal sex determination, a highly vulnerable period when the germ line DNA is demethylated and remethylated in a sex specific manner. The chemically-induced modifications in epigenetic programming of the germ line results in differentially altered epigenomes and transcriptomes in all tissues propagated from the affected sperm or ovum, which can influence development of disease in later life. Although data is sparse related to trans-generational effects of environmental exposures in human studies, there is a need to better inform clinicians and patients about this critically vulnerable period during, and prior to, pregnancy. Confirmation of animal models of trans-generational effects will require the enrollment at least 3 generations of participants (grandparent, parent, and child).

Sources

  • Full study (free access) : Mechanisms of the Maternal Exposome and Implications for Health Outcomes, ANS Adv Nurs Sci, NCBI PubMed, PMC4860277, 2016 Apr-Jun.
  • Exposome Penetrance featured image PMC4860277/figure/F2.
DES DIETHYLSTILBESTROL RESOURCES

DES transgenerational transmission of defects

image of transgenerational transmission

Adverse health effects in children of women exposed in utero to diethylstilbestrol (DES)

2016 Abstract

OBJECTIVE
Exposure to diethylstilbestrol (DES) in utero is associated with adverse health effects, including genital anomalies in women and men, and cancers in women. Animal studies showed birth defects and tumors in the offspring of DES exposed mice, revealing transgenerational transmission of DES effects. In humans, birth defects, such as hypospadias were observed in children of prenatally exposed women. The aim of this research was to further assess the health effects in children of prenatally exposed women.

METHODS
In a retrospective cohort study, the reports of women exposed to DES in utero on their 4409 children were compared with those of unexposed women on their 6203 children. Comparisons used odd ratios (OR) between children of exposed and unexposed women and standardized incidence rate (SIR) with the general population. These cohorts were recruited on a voluntary basis to answer questionnaires.

RESULTS
There was a global increase of defects in children born to exposed women when compared with those born to unexposed (OR 2.29, 95% CI: 1.80-2.79, P<0.001) and with the general population (SIR 2.39, 95% CI: 2.11-2.68). Increased defects were observed in male genital tract, esophagus, lip or palate, musculoskeletal and circulatory systems. For female genital tract anomalies, there was no significant increase. However, this cohort being relatively young, further follow-up is needed. An increase of cerebral palsy was revealed. The incidence of cancers was not increased, in particular for breast, uterus and ovary.

CONCLUSION
Our results confirmed a transgenerational transmission of defects in male genital tract. With caution due to possible bias associated with this method, our data suggest an increase of defects for esophagus, lip or palate, musculoskeletal and circulatory system in children of exposed women.

Sources

  • Adverse health effects in children of women exposed in utero to diethylstilbestrol (DES), Therapie, NCBI PubMed, PMID: 27203157, 2016 Feb 5.
  • Featured image Thought Catalog.
DES DIETHYLSTILBESTROL RESOURCES

Transgenerational transmission of environmental effects through epigenetic modifications

The history of Distilbène® (Diethylstilbestrol) told to grandchildren – the transgenerational effect

2015 Study Abstract

The Distilbène® story is a dramatic episode which belongs to the history of medicine. It provided several useful lessons such as the importance of evidence-based medicine and the hazard to develop treatments during pregnancy without careful animal verifications. However, this experience has also provided unexpected progress by suggesting new pathophysiological concepts: fetal programming of adult diseases and/or transgenerational transmission of environmental effects through epigenetic modifications.

Overview

  • Introduction
  • History
  • Diseases reported in second generation after foetal exposure to DES
    • Developmental and reproductive anomalies
    • Non-reproductive anomalies
  • Diseases reported in the third generation after the grandmother was treated with DES during pregnancy
    • Human abnormalities
    • Concordance with rodent model
  • Which molecular mechanisms?
    • Exposure window
    • Chemical nature of estrogens
    • The mechanisms involved in foetal programming of adult diseases – epigenetic modifications
    • Transgenerational transmission mechanisms
  • Conclusion
  • Disclosure of interest

Sources

  • Full paper (free access) : The history of Distilbène® (Diethylstilbestrol) told to grandchildren–the transgenerational effect, ANNALES D’ENDOCRINOLOGIE, EM Consulte, article/990390, 22/07/15.
  • Featured image Isaiah Rustad.
DES DIETHYLSTILBESTROL RESOURCES

The trans-generational effects of DES exposure may pose a risk for future generations

Endocrine disruption of oestrogen action and female reproductive tract cancers

2014 Study Abstract

Endocrine disrupting chemicals (EDC) are ubiquitous and persistent compounds that have the capacity to interfere with normal endocrine homoeostasis. The female reproductive tract is exquisitely sensitive to the action of sex steroids, and oestrogens play a key role in normal reproductive function. Malignancies of the female reproductive tract are the fourth most common cancer in women, with endometrial cancer accounting for most cases. Established risk factors for development of endometrial cancer include high BMI and exposure to oestrogens or synthetic compounds such as tamoxifen. Studies on cell and animal models have provided evidence that many EDC can bind oestrogen receptors and highlighted early life exposure as a window of risk for adverse lifelong effects on the reproductive system. The most robust evidence for a link between early life exposure to EDC and adverse reproductive health has come from studies on women who were exposed in utero to diethylstilbestrol. Demonstration that EDC can alter expression of members of the HOX gene cluster highlights one pathway that might be vulnerable to their actions. In summary, evidence for a direct link between EDC exposure and cancers of the reproductive system is currently incomplete. It will be challenging to attribute causality to any single EDC when exposure and development of malignancy may be separated by many years and influenced by lifestyle factors such as diet (a source of phytoestrogens) and adiposity. This review considers some of the evidence collected to date.

  • ER signalling
  • ER expression and actions of oestrogens in reproductive tract tissues
  • Oestrogen biosynthesis and ER expression in benign and malignant endometrial tract disorders
  • Evidence that oestrogens increase the risk of developing a reproductive cancer
  • Endocrine disruptors implicated in reproductive tract disorders and cancer
Diethylstilbestrol

DES, a synthetic non-steroidal oestrogen, is often regarded as the archetypal endocrine disruptor. From about 1940 to 1970, DES was given to pregnant women in the mistaken belief that it would reduce the risk of pregnancy complications. Herbst et al. reported a probable link between DES and vaginal clear cell adenocarcinoma in girls and young women who had been exposed to this drug. It is estimated that five to ten million people were exposed to DES, including the pregnant mothers who received treatment and their offspring. Of the several million women exposed to DES in utero, a cohort of 4653 DES-exposed women have been followed up to investigate the long-term consequences of exposure (Hoover et al. 2011). Patient data stratified to account for the extent of exposure or dose effects of DES identified an association between treatment of mothers earlier during their pregnancy and adverse vaginal epithelial changes at a younger age in their offspring. While DES exposure is associated with increased risk of breast and cervical/vaginal clear cell adenocarcinoma, several studies have indicated that there is no associated risk of endometrial or ovarian cancer. As endometrial cancer is most likely to present after menopause, many of the DES-exposed women may not yet be old enough to determine whether they are at excess risk, as in the 2011 report only 27% were older than 50 years. Although to date epidemiological data indicate that DES-exposed women may not be at increased risk of developing endometrial cancer, studies in animal models provide evidence to the contrary.

In the early 1990s, Newbold et al. developed a mouse model for investigating hormonal carcinogenesis in mice by investigating the effects of neonatal exposure to oestrogens on cancer development. Treatment of CD1 neonatal mice with DES on postnatal days 1–5, which correspond to late prenatal human development, resulted in 90% of DES-exposed mice developing uterine adenocarcinomas after 18 months while none of the control animals had neoplastic lesions. Crucially, while administration of DES increased the risk of uterine adenocarcinoma, endogenous oestrogen was required for tumour development with prepubertal ovariectomy preventing tumour development. In DES-exposed women, vaginal and cervical carcinomas were only detected post-menarche consistent with a requirement for endogenous oestrogen in tumour development. ERα knockout mice (ERKO) did not develop tumours following neonatal DES exposure (Couse & Korach 2004); transgenic mice overexpressing ERα displayed accelerated tumour development but mice with a dominant negative isoform of ERα (ERΔ3) were not protected, highlighting the complexity of the molecular signalling mechanisms involved.

Interestingly, gene expression analysis indicates that developmental DES exposure results in persistent altered gene expression of oestrogen-responsive genes in the uterus that may explain the increased susceptibility to tumour development. Gene ontology analysis of microarray data revealed altered expression of genes involved in cell growth, differentiation and adhesion. Kabbarah et al. collected uterine cancer tissue RNA from DES-exposed mice by laser capture microdissection to minimise contamination with other cell types and performed targeted transcriptional profiling. Interestingly, the tumour suppressor PTEN was down-regulated in the majority of tumours, analogous to loss of PTEN expression in human tumours. In addition, genes associated with cell adhesion, such as Decorin, were down-regulated in DES-induced tumours while suppressor of cytokine signalling 3 (Socs3) was over-expressed. Other studies have also identified molecular similarities between DES-induced tumours in mice and endometrial cancer in humans, such as microsatellite instability brought about by defects in expression of DNA mismatch repair genes such as MSH2 and MSH6.

It could be argued that the apparent trans-generational effect of endocrine disruption is of greater significance. Following neonatal DES exposure in mice, the F1 generation of DES daughters have an increased incidence of uterine adenocarcinoma. Newbold et al.  found that 31% of F1 females from the maternal germ cell lineage developed tumours after 18 months despite there being no exogenous endocrine exposure in these animals, highlighting the potential for future risk to the daughters of DES-exposed women. DES is reported to induce epigenetic changes. Altered methylation patterns have been reported for several uterine genes that are permanently dysregulated after developmental DES exposure; lactoferrin and c-Fos are permanently up-regulated following neonatal DES exposure to due to hypomethylation of the promoter region.

DES has been reported to promote hypermethylation of the homeobox gene Hoxa10 in mice exposed in utero to DES. DES exposure was also associated with increased expression of DNA methyltransferases 1 and 3b leading to long-term altered expression of Hoxa10. Contrary to the reported action of DES on Hoxa10, exposure to BPA in mice in utero results in hypomethylation of the Hoxa10 promoter, which leads to enhanced binding of ERα to EREs in the promoter region and an increase in an ERE-driven reporter gene in vitro.

Thus, epigenetic changes in uterine genes may indicate a possible mechanism for trans-generational effects of DES because altered expression of genes is reported to persist in DES-lineage females.

  • Evidence that expression of HOX genes can be altered by exposure to EDC
  • Evidence that early life exposure to EDC can alter onset of puberty or timing of menopause
  • Evidence that lifestyle factors can increase the influence of EDC on lifetime risk of developing reproductive tract cancers
  • Summary and future perspectives

Sources

  • Endocrine disruption of oestrogen action and female reproductive tract cancers, Endocrine-Related Cancer, endocrinology-journals, doi: 10.1530/ERC-13-0342, April 1, 2014.
  • Featured image chuttersnap.
DES DIETHYLSTILBESTROL RESOURCES

DES and third-generation hypospadias

image of third-generation hypospadias

Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

2011 Study Abstract

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

Although the role of fetal androgens is crucial to male genital development during the first trimester of pregnancy, defects in the synthesis or molecular action of testosterone are rare in isolated hypospadias (Hypospadias may be a multifactorial defect arising from genetic, hormonal, and environmental factors). It has been hypothesized that changes in androgen/estrogen balance due to endogenous or exogenous hormonal factors during the critical period of penile and urethral development contribute to this malformation.

Men who were exposed in utero to diethylstilbestrol (DES), a synthetic estrogen, may exemplify the effects of environmental chemicals with endocrine-disrupting activity on genital development (DES was prescribed for pregnant women from the late 1930s to the 1970s in the mistaken belief that it would prevent miscarriage or premature birth). Unfortunately, DES was found to be not only ineffective but also harmful. Daughters born from DES-related pregnancies often show abnormalities in the Müllerian structures and have elevated risks of peripubertal vaginal and cervical clear-cell adenocarcinoma, fertility problems, ectopic pregnancies, miscarriages, and premature births (The risk of reproductive tract abnormalities also appears to be increased for DES sons, who may present hypoplastic testis, epididymal cysts, cryptorchidism, or hypospadias).

After several studies in animals, a question emerged as to whether the harmful effects of DES can be “transmitted” to subsequent generations. Newbold and colleagues reported an increased incidence of reproductive tract tumors in male and female descendants of mice developmentally exposed to DES. In the human, Klip et al. reported an increased risk of hypospadias in sons of DES daughters in a cohort of women diagnosed with fertility problems. Other studies either confirmed or questioned these results. However, the direct implication of DES in the occurrence of hypospadias remains debatable, since many other uncontrolled factors, especially environmental and genetic, are implicated in this malformation. We studied the prevalence of hypospadias in the grandsons of DES-treated and -untreated women and ruled out other environmental and genetic factors that could have been associated with the malformation in these patients.

A nationwide cohort study was conducted in collaboration with a French association of DES-treated women (HHORAGES Association). The reason the women joined this association was not the presence of hypospadias in the second or third generation but rather psychological disturbances, vaginal and cervical clear-cell adenocarcinomas, miscarriages, and other abnormalities. Five hundred twenty-nine families were included. All of the second- and third-generation offspring were accounted for and included in the study. No one declined to participate. DES exposure was reported in 1,000 out of 1,180 pregnancies. The featured image details the patient groups. The clinical diagnosis of hypospadias was standardized and based on a detailed operative report or direct clinical examination by a pediatrician and/or urologist. The malformation was characterized as severe (proximal, penoscrotal) or nonsevere (glandular, subcoronal, distal, midshaft). Each mother with a hypospadiac son was contacted and responded to a short questionnaire validated in Europe for data collection (no. QLK4-1999-01422) to determine whether other occupational exposure had occurred during the pregnancy. To exclude a defect of the androgen pathway, we performed molecular analysis of the genes known to be associated with hypospadias such as androgen receptor (AR), 5α reductase (srd5A2), and MAMLD1 genes in DNA from peripheral blood. The local university hospital ethics committee approved this study (ID RCB No. 2008-A00781-54), and each patient gave informed consent through the Hhorage Association.

The prevalence of hypospadias was low in boys unexposed to DES in utero (0/180), whereas it was high in the in utero–exposed boys (3.57%, 16/448, P=.02). In the third generation, the prevalence of hypospadias in boys born to DES daughters was high when compared with boys born to unexposed parents (8.2%, n = 8/97 vs. n = 0/360; P<.001). The hypospadiac patients of the second and third generations were not related. The results are summarized in the featured image.

Neither mutations nor polymorphisms of the AR and MAMLD1 genes were found among hypospadiac boys of the third generation. Only one polymorphism of the srd5A2 gene was detected (A49T) in a boy. The mothers of the third-generation affected boys indicated little environmental or occupational exposure to endocrine-disrupting chemicals during pregnancy (no professional activity, n = 2; sales clerk in a food or clothing shop, n = 3; office worker, n = 3), and such exposure was therefore unlikely to have contributed to the occurrence of hypospadias. Two mothers of hypospadiac sons exhibited hypoplastic or bifid uterus.

The main effect of DES is profound disturbance in the androgenic/estrogenic balance of animal and human fetuses since it has both estrogenic and antiandrogenic actions by competing with natural androgens for the ligand-binding domain of the androgen receptor. In utero exposure to DES during the critical period of reproductive tract development is known to induce genital malformation in mice. In utero–exposed sons show greater risks of structural urogenital abnormalities like hypospadias, epididymal cysts, micropenis, and cryptorchidism. The present study reinforces these data with a prevalence of hypospadias greater than 3%, although it should be noted that the second-generation population included only 180 controls from the same families since this series was specifically designed to study the third-generation boys.

More interesting is the hypothesis of a transgenerational effect of DES. Animal studies first suggested that DES might increase transgenerational susceptibility to malignant tumors of the female reproductive tract, presumably by damage to germ cells and abnormal imprinting. In human beings, DES exposure may also lead to permanently altered germ cells. The suggestion of a transgenerational effect of DES in human beings was based on the observation of a high prevalence of hypospadias, particularly with severe phenotypes, in the sons of women exposed to DES in utero.

But variations in the definition of the control population may explain the wide range of odds ratios reported in the literature to date. Palmer et al. reported a prevalence 6 times higher than that of Klip et al. The present study, which shows a high prevalence of hypospadias of various severities in the third generation, tried to limit this bias and included DES-free pregnancies and DES-exposed pregnancies from the same families. Nevertheless, two limitations should be noted:

  1. DES-exposed women without problems were not included;
  2. and the fertility status of the exposed and non-exposed couples, the age at pregnancy, and the parity for each women were variable, and this may have hidden fertility problems or greater use of contraception.

The low fertility rates of the DES sons may also be explained by other findings, such as severe psychotic disorders or oligospermia in cases of hypospadias with additional defects.

We did not identify any genetic or environmental factors that would have explained the hypospadias in DES grandsons. Our results thus raise the question of the mechanism through which DES causes adverse effects in subsequent generations. The frequency of transmission both observed in our series of hypospadiac grandsons and previously reported in generations examined for various disease states secondary to DES exposure is particularly high. This frequency of a transgenerational phenotype is such that a mutational event involving the DNA sequence could not be implicated. DES-induced changes in epigenetic background and alteration of DNA methylation could be significant factors in the susceptibility to disease development. The primordial germ cells undergo demethylation during migration and early colonization of the embryonic gonad, followed by remethylation starting at the time of sex determination in a sex-specific manner. The pregnant mother’s exposure to DES at the time of fetal sex determination appears to be sufficient to alter the remethylation of the germ line in the male fetus and permanently reprogram the imprinted pattern of DNA methylation. The transmission of multigenerational DES effects would thus occur through the paternal lineage. But our findings indicated that most of the third-generation hypospadiac boys were born to DES daughters. This agreed with previous studies (although paternal transmission of DES effects is not excluded). Epigenetic changes in the AR gene, transmitted through the DES daughter, could explain such a finding since the antiandrogen effect of DES is known to modify the phosphorylation level of AR.

The association between hypospadias in grandsons and uterine abnormalities in their mothers suggests other hypotheses for the transgenerational mechanisms of DES. First, DES daughters may have displayed disturbed hormonal balance during their reproductive life or placental malfunction that might have interfered with the genital development of a male fetus. Second, the estrogen receptor gene ERα and estrogen-responsive genes that contribute to the development of both female internal genitalia and hypospadias may also be involved since ERα is implicated in the induction of abnormalities after DES exposure. Last, the genes involved in the structural differentiation of both the female and male reproductive tracts may be altered by DES exposure. DES has been reported to delay expression of HOXa family genes during Müllerian duct development. DES could also interfere with HOX gene expression during penile formation.

For many authors, DES is an experimental environmental xenoestrogen. Despite the bias that could not be fully eliminated and the difficulty of extrapolating the risks of exposure (no monotonic dose-response relationship, varying effects depending on the timing of exposure in the developing organism, manifestations delayed until later in life), this clinical study strengthens the suspicion of the transgenerational effects of environmental endocrine disruptors.

Sources

  • Full text (free access) : Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study, CHU Montpellier and Universite Montpellier, France, hhorages, February 23, 2011.
  • Featured image : Detailed patient groups included in the study. In the second generation, the phenotype of affected boys was isolated hypospadias in all cases, severe in 12 cases (posterior or penoscrotal), and not severe in four cases (mild or anterior). In the third generation, the hypospadias was severe in five cases and not severe in three cases. Bilateral cryptorchidism was present in one case. *The size of the population was under 1,000, and the prevalence of hypospadias of about 1/1,000, as seen in the general population, could not be represented.
DES DIETHYLSTILBESTROL RESOURCES