Evidence from a French cohort of 1002 prenatally exposed children and the example of diethylstilbestrol (DES) as a model for PE study
2016 Study Abstract
Aim of the work In utero diethylstilbestrol (DES) exposure has been demonstrated to be associated with somatic abnormalities in adult men and women as well as shown for its trangenerationnal effect.
Endocrine disruptors and psychiatric disorders in children exposed in utero: evidence from a French cohort of 1002 prenatally exposed children and the example of diethylstilbestrol (DES) as a model for PE study, Conference Paper, Research Gate, publication/293333931, January 2016.
Researchers Marie-Odile Soyer-Gobillard and Charles Sultan, image credit lamarseillaise.
Conversely, the data are contradictory regarding the association with psychological or psychiatric disorders during adolescence and adulthood.
This work was designed to determine whether prenatal exposure to DES and/or Ethinyloestradiol affects brain development and whether it is associated with psychiatric disorders in male and female adolescents and young adults.
Methods HHORAGES Association, a national patient support group, has assembled a cohort of 1280 women (spontaneous testimonies communicated after various informations) who took DES and/or EE during pregnancy. We obtained responses to detailed questionnaire from 529 families, corresponding to 1182 children divided into three groups:
Group 1 (n=180): firstborn children without DES treatment,
Group 2 (n=740): exposed children,
and Group 3 (n=262): children born after a previous pregnancy treated by DES and/or EE.
No psychiatric disorders were reported in Group 1. In Group 2, the incidence of psychiatric disorders was drastically elevated (83.8%), and in Group 3, the incidence was still elevated (6.1%) compared with the general population.
Total number of psychological/psychiatric disorders among the 982 (1002-20 stillborns) DES-exposed and post-DES children
Among the 982 DES-exposed adolescents (1002-20 stillborns) (Group 2) and post-DES adolescents (Group 3):
This work demonstrates that prenatal exposure to DES and/or EE is associated with a high risk of psychiatric disorders in adolescence and adulthood. Molecular epigenetic mechanisms subtending these toxic effects are in progress.
Using a familial case control study, Marie-Odile Soyer-Gobillard – former director emeritus at the CNRS (French National Center for Scientific Research) – and Charles Sultan show that there are serious effects on the psychological and physical health of the descendants of women treated with synthetic hormones during their pregnancy: psychiatric illnesses are often found associated with somatic disorders which are well known to be the DES and EE signature.
Behavioral and Somatic Disorders in Children Exposed in Utero to Synthetic Hormones: A Testimony-Case Study in a French Family Troop, Endocrinology and Metabolism, intechopen, DOI: 10.5772/48637, October 3, 2012.
Synthetic hormones, acting as endocrine disturbers, are toxic for humans, especially for pregnant women and their children, probably partly in relation with their toxic degradation status.
In all cases girls suffered more than boys either of somatic and/or psychiatric disorders due to the estrogen receptor alpha or beta concentration higher in female fetus than in male. It is also clear that in all the families most of the exposed children are ill while quite the unexposed are not.
2012 Study Overview
Materials and methods: Gathering questionnaires and the evidence
Results / Data Analysis / Discussion
A multi-generational effect? By what mechanism?
A multi-generational effect? By what mechanism?
Multi-generational carcinogenesis studies were realized on mice after diethylstilbestrol impregnation with impressive and undisputable results. Our observations presented in this present work from the French HHORAGES troop raises the question of the mechanism through with synthetic hormones as DES cause either psychiatric disorders in exposed children and/or adverse effects in subsequent generations. Since Abdomaleky et al concluded that modulation of gene-environment interactions may be trough DNA methylation, authors put forward hypothesis that DES-induced changes in epigenetic background and alteration of DNA methylations could be significant factors. The pregnant mother’s exposure to DES at very early neurodevelopment time and/or at time of sex determination would appear to be sufficient to alter the remethylation of neuron precursors and/or of the fetus germ line. Only a few third-generation children suffering psychiatric illness are mentioned in testimonies. This is understandable because third generation exposed children are still too young (excepted in some cases) to present psychiatric disorders as schizophrenia which is not the case for hypospads that are detectable from birth in male children and grand-children. Work is already under way concerning the gene X environment DES impact hypothesis by comparing DES and EE exposed children, various genetic and epigenetic factors to those of mother and unexposed children of the same family as studied by the INSERM team U796 in collaboration with the HHORAGES families.
In the present familial case control study, we have shown that there are serious effects on the psychological and physical health of the descendants of women treated with synthetic hormones during their pregnancy: psychiatric illnesses are often found associated with somatic disorders which are well known to be the DES and EE signature. Synthetic hormones, acting as endocrine disturbers, are toxic for humans, especially for pregnant women and their children, probably partly in relation with their toxic degradation status. In all cases girls suffered more than boys either of somatic and/or psychiatric disorders due to the estrogen receptor alpha or beta concentration higher in female fetus than in male. It is also clear that in all the families most of the exposed children are ill while quite the unexposed are not.
So what now? As the precautionary principle was not applied in the past, and still is not in force today, and since the lessons of recent history were never taken into account , it is our common duty to repair the damage by supporting the devastated families, and by pursuing research work on the observation of trans-generational effects. Such effects are already highlighted by the demonstration that cancers are observed even in the fourth generation in mice . According to the Skinner’s mini review “the ability of an environmental compound (as DES or EE) to promote the reprogramming of the germ-line appears to be the causal factor in the epigenetic transgenerational phenotype,” we observed an increase of the genital malformations in the third generation in male infants whose mothers were treated with xenoestrogens. In the HHORAGES troop, DES and EEexposed infants are already pointed out as bodily and/or psychologically impaired after their mothers were treated with clomifene citrate (an ovulation stimulator previously used for IVF-type medically assisted procreation). Another concern is the putative future effect of ethinylestradiol containing oral estrogenic contraception on future generations due to its lipophily after its metabolization and its future release in fetus through the placenta. As for the demonstration of the causality link within the HHORAGES troop, will we have to wait for a large-scale epidemiological study, or are we allowed to think that the impressive figures that we are publishing in this work are not merely random? The only way now is to respect absolutely the precautionary principle and to delete completely or to give the less possible toxic (synthetic) hormone medication: for example Clavel Chapelon and her Endogenous Hormones and Breast Cancer Collaborative Group in Villejuif informed that natural hormone as micronized (natural) progestin associated with estrogens (synthetic alas!) is more often ordered for SHT (Substitutive Hormonal Treatment) in order to avoid breast cancer. Unfortunately, she said also that in the contrary the same SHT is not recommended to avoid the endometrium cancer …
As Newbold et al said after they reviewed the damages caused by DES ,
“only new advances in the knowledge of genetic and epigenetic mechanisms of the disruptions of fetal development will enable us to be aware of the risks entailed by the other estrogenic disruptors which are present around us and in ourselves, even at very low doses”
, whilst Theo Colborn insists on the fact that the foetus cannot be protected against endocrine disruptors, whatever they may be, except at zero level.
Executive Summary to EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, The Endocrine Society, dx.doi.org/10.1210/er.2015-1093, September 28, 2015. Full study: EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, The Endocrine Society, dx.doi.org/10.1210/er.2015-1010, November 06, 2015.
Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans—especially during development—may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, and epigenetic changes, thereby producing effects in exposed individuals as well as their descendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans.
In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest:
obesity and diabetes;
hormone-sensitive cancers in females;
and neurodevelopment and neuroendocrine systems.
Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in a range that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings can be much better translated to human health.
Armed with this information, researchers, physicians, and other healthcare providers can guide regulators and policymakers as they make responsible decisions.
Discussion (DES and Fertility-specific)
Diethylstilbestrol and beyond: perhaps the best-studied endocrine-based example is in utero exposure to diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen taken by pregnant women from the 1940s to 1975 to prevent miscarriage and other complications. DES was prescribed at doses from less than 100 mg (in most cases) upward to 47 000 mg, with a median dose of 3650 to 4000 mg in the United States (IARC 2012). Most women received low doses (ie, 5 mg) and increased their intake (up to 125 mg) as symptoms or pregnancy progressed, translating to doses of about 100 μg/kg to 2 mg/kg DES per day. In 1953, a study proved DES was ineffective. Its use was discontinued when a subset of exposed daughters presented with early-onset vaginal clear-cell adenocarcinoma, with a 40-fold increase in risk compared to unexposed individuals. A highly significant incidence ratio for clear-cell adenocarcinoma was also found in the Dutch DES cohort, a population that may have had lower exposures than US women. It was subsequently determined that exposed offspring of both sexes had increased risk for multiple reproductive disorders, certain cancers, cryptorchidism (boys), and other diseases, although the risk for sons is more controversial. New data are emerging to implicate increased disease risk in grandchildren. Not surprisingly, a plethora of examples is emerging for increased disease susceptibility later in life as a function of developmental exposures to EDCs that include BPA, phthalates, PCBs, pesticides, dioxins, and tributyltin (TBT), among others.
Epigenetics and transgenerational effects of EDCs: EDC-induced epigenetic changes are also influenced by dose of exposure, and they are tissue specific. Thus, it is important to consider both dose of EDC and the tissue before making firm conclusions about the epigenetic effects of EDCs. DNA methylation changes are the best-studied mechanism in this regard. For example, prenatal exposure to DES caused hypermethylation of the Hoxa10 gene in the uterus of mice and was linked to uterine hyperplasia and neoplasia later in life. Beyond the effects of prenatal exposure to DES on the daughters exposed in utero are suggestions that this leads to transgenerational effects of the chemical on the reproductive system, although whether this is linked to DNA methylation changes in humans is unknown.
Little is known about the ability of EDCs to cause histone modifications and whether this leads to transgenerational effects in animals or humans. The herbicides paraquat and dieldrin caused histone modifications in immortalized rat mesencephalic dopaminergic cells, and the insecticide propoxur causes histone modifications in gastric cells in vitro. DES caused histone deacetylation in the promoter region of the cytochrome P450 side chain cleavage (P450scc) gene. Further studies, however, need to be conducted to identify other EDCs causing histone modifications in animals and humans and to determine whether such modifications lead to transgenerational effects.
Female Reproductive Health: in the past 5 years, no new information became available on the effects of DES on the postnatal human ovary. Recent animal studies indicate that DES adversely affected the postnatal ovary. Neonatal exposure to DES inhibited germ cell nest breakdown (408) and caused the formation of polyovular follicles in mice, likely by interfering with the ERβ pathway and inhibiting programmed oocyte death and germ cell loss. It also reduced the primordial follicle pool and increased atresia in prepubertal lambs, and it caused polyovular ovaries in hamsters. Although these previous studies provide solid evidence that DES adversely affects ovarian structure in a variety of species, studies are needed to determine whether other synthetic estrogens adversely affect the ovary.
Effects of EDCs on uterine structure and function: synthetic estrogens are well known disruptors of uterine structure and function in humans and animals. Consistent with previous studies, recent data indicate that neonatal DES exposure caused endometrial hyperplasia/dysplasia in hamsters and increased uterine adenocarcinoma and uterine abnormalities in Donryu rats. Neonatal DES exposure also caused the differential expression of 900 genes in one or both layers of the uterus. Specifically, DES altered multiple factors in the PPARγ pathway that regulate adipogenesis and lipid metabolism, and it perturbed glucose homeostasis, suggesting that DES affects energy metabolism in the uterus. In the mouse uterus, DES altered the expression of chromatin-modifying proteins and Wnt signaling pathway members, caused epigenetic changes in the sine oculis homeobox 1 gene, and decreased the expression of angiogenic factors. DES also altered the expression of genes commonly involved in metabolism or endometrial cancer in mice, and it activated nongenomic signaling in uterine myometrial cells and increased the incidence of cystic glands in rats.
Effects of EDCs on the vagina: only a limited number of studies assessed the effects of EDCs on the vagina, and of these, all but one on phthalates focused on DES. A recent study of women showed an association between in utero exposure to DES and clear cell carcinoma of the vagina, confirming previous findings. Furthermore, DES disrupted the expression of transformation-related protein 63, which makes cell fate decisions of Müllerian duct epithelium and induces adenosis lesions in the cervix and vagina in women.
Studies in mice showed that DES induced vaginal adenosis by down-regulating RUNX1, which inhibits the BMP4/activin A-regulated vaginal cell fate decision; induced epithelial cell proliferation and inhibited stromal cell proliferation (520); and caused persistent down-regulation of basic-helix-loop-helix transcription factor expression (Hes1, Hey1, Heyl) in the vagina, leading to estrogen-independent epithelial cell proliferation. Neonatal exposure to DES caused persistent changes in expression of IGF-1 and its downstream signaling factors in mouse vaginas. It also up-regulated Wnt4, a factor correlated with the stratification of epithelial cells, in mouse vaginas. Interestingly, the simultaneous administration of vitamin D attenuated the ability of DES to cause hyperplasia of the vagina in neonatal mice.
In the one study in the previous 5 years that did not focus on DES, polypropylene and polyethylene terephthalate did not increase vaginal weight in Sprague-Dawley rats. Although a few studies have been conducted during the previous 5 years on the effects of EDCs on the vagina, such studies are very few in number, small in scope, and focused on DES. Thus, future studies are needed in this largely understudied area before we fully appreciate whether other EDCs impair the vagina.
Premature ovarian failure/early menopause: combined data from three studies on DES indicated that in utero exposure was associated with an increased lifetime risk of early menopause in women (602). However, animal studies have not determined whether DES exposure causes premature ovarian failure. Thus, future studies should focus on this issue.
Fibroids: a few recent studies confirmed the known association between DES exposure and fibroids. In the Sister Study, in utero exposure to DES was positively associated with early-onset fibroids. Similarly, in the Nurses’ Health Study II, prenatal DES exposure was associated with uterine fibroids, with the strongest risk being for women exposed to DES in the first trimester. Given the consistency in findings, future studies should be focused on determining the mechanism by which DES exposure increases the risk of fibroids.
There is growing interest in the possible health threat posed by endocrine-disrupting chemicals (EDCs), which are substances in our environment, food, and consumer products that interfere with hormone biosynthesis, metabolism, or action resulting in a deviation from normal homeostatic control or reproduction.
In this first Scientific Statement of The Endocrine Society, we present the evidence that endocrine disruptors have effects on male and female reproduction, breast development and cancer, prostate cancer, neuroendocrinology, thyroid, metabolism and obesity, and cardiovascular endocrinology.
Results from animal models, human clinical observations, and epidemiological studies converge to implicate EDCs as a significant concern to public health. The mechanisms of EDCs involve divergent pathways including (but not limited to) estrogenic, antiandrogenic, thyroid, peroxisome proliferator-activated receptor ?, retinoid, and actions through other nuclear receptors; steroidogenic enzymes; neurotransmitter receptors and systems; and many other pathways that are highly conserved in wildlife and humans, and which can be modeled in laboratory in vitro and in vivo models. Furthermore, EDCs represent a broad class of molecules such as organochlorinated pesticides and industrial chemicals, plastics and plasticizers, fuels, and many other chemicals that are present in the environment or are in widespread use.
We make a number of recommendations to increase understanding of effects of EDCs, including enhancing increased basic and clinical research, invoking the precautionary principle, and advocating involvement of individual and scientific society stakeholders in communicating and implementing changes in public policy and awareness.
Discussion (DES and Fertility-specific)
General Introduction: the group of molecules identified as endocrine disruptors is highly heterogeneous and includes synthetic chemicals used as industrial solvents/lubricants and their byproducts [polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), dioxins], plastics [bisphenol A (BPA)], plasticizers (phthalates), pesticides [methoxychlor, chlorpyrifos, dichlorodiphenyltrichloroethane (DDT)], fungicides (vinclozolin), and pharmaceutical agents [diethylstilbestrol (DES)].
Reproduction: in the adult female, the first evidence of endocrine disruption was provided almost 40 yr ago through observations of uncommon vaginal adenocarcinoma in daughters born 15–22 yr earlier to women treated with the potent synthetic estrogen DES during pregnancy. Subsequently, DES effects and mechanisms have been substantiated in animal models. Thus, robust clinical observations together with experimental data support the causal role of DES in female reproductive disorders. However, the link between disorders such as premature pubarche and EDCs is so far indirect and weak, based on epidemiological association with both IUGR and ovulatory disorders. The implications of EDCs have been proposed in other disorders of the female reproductive system, including disorders of ovulation and lactation, benign breast disease, breast cancer, endometriosis, and uterine fibroids.
In the case of DES, there are both human and experimental observations indicating heritability.
Premature ovarian failure, decreased ovarian reserve, aneuploidy, granulosa steroidogenesis: interestingly, mice exposed in utero to DES, between d 9–16 gestation, have a dose-dependent decrease in reproductive capacity, including decreased numbers of litters and litter size and decreased numbers of oocytes (30%) ovulated in response to gonadotropin stimulation with all oocytes degenerating in the DES-exposed group, as well as numerous reproductive tract anatomic abnormalities. In women with in utero exposure to DES, Hatch et al reported an earlier age of menopause between the 43–55 yr olds, and the average age of menopause was 52.2 yr in unexposed women and 51.5 yr in exposed women. The effect of DES increased with cumulative doses and was highest in a cohort of highest in utero exposure during the 1950s. These observations are consistent with a smaller follicle pool and fewer oocytes ovulated, as in DES-exposed mice after ovulation induction.
Reproductive tract anomalies: disruption of female reproductive tract development by the EDC DES is well documented. A characteristic T-shaped uterus, abnormal oviductal anatomy and function, and abnormal cervical anatomy are characteristic of thisin utero exposure, observed in adulthood, as well as in female fetuses and neonates exposed in utero to DES. Some of these effects are believed to occur through ER? and abnormal regulation of Hox genes. Clinically, an increased risk of ectopic pregnancy, preterm delivery, miscarriage, and infertility all point to the devastating effect an endocrine disruptor may have on female fertility and reproductive health. It is certainly plausible that other EDCs with similar actions as DES could result in some cases of unexplained infertility, ectopic pregnancies, miscarriages, and premature deliveries. Although another major health consequence of DES exposurein utero was development of rare vaginal cancer in DES daughters, this may be an extreme response to the dosage of DES or specific to pathways activated by DES itself. Other EDCs may not result in these effects, although they may contribute to the fertility and pregnancy compromises cited above. Of utmost importance clinically is the awareness of DES exposure (and perhaps other EDC exposures) and appropriate physical exam, possible colposcopy of the vagina/cervix, cervical and vaginal cytology annually, and careful monitoring for fertility potential and during pregnancy for ectopic gestation and preterm delivery.
Endometriosis is an estrogen-dependent gynecological disorder associated with pelvic pain and infertility. There are suggestive animal data of adult exposure to EDCs and development of or exacerbation of existing disease, and there is evidence that in utero exposure in humans to DES results in an increased relative risk = 1.9 (95% confidence interval, 1.2–2.8).
Environmental estrogens effects on the prostate: DES exposure is an important model of endocrine disruption and provides proof-of-principle for exogenous estrogenic agents altering the function and pathology of various end-organs. Maternal usage of DES during pregnancy resulted in more extensive prostatic squamous metaplasia in human male offspring than observed with maternal estradiol alone. Although this prostatic metaplasia eventually resolved during postnatal life, ectasia and persistent distortion of ductal architecture remained. These findings have led to the postulation that men exposed in utero to DES may be at increased risk for prostatic disease later in life, although the limited population studies conducted to date have not identified an association. Nonetheless, several studies with DES in mouse and rat models have demonstrated significant abnormalities in the adult prostate, including increased susceptibility to adult-onset carcinogenesis after early DES exposures. It is important to note that developmental exposure to DES, as with other environmental estrogens, has been shown to exhibit a biphasic dose- response curve with regard to several end-organ responses, and this has been shown to be true for prostatic responses as well. Low-dose fetal exposure to DES or BPA (see full study) resulted in larger prostate size in adulthood compared with controls, an effect associated with increased levels of prostatic ARs. This contrasts with smaller prostate sizes, dysplasia, and aging- associated increases in carcinogenesis found after perinatal high-dose DES exposures as noted above. This differential prostatic response to low vs. high doses of DES and other EDCs must be kept in mind when evaluating human exposures to EDCs because the lack of a response at high doses may not translate into a lack of negative effects at low, environmentally relevant doses of EDCs.
Linking basic research to clinical practice: it should be clear from this Scientific Statement that there is considerable work to be done. A reconciliation of the basic experimental data with observations in humans needs to be achieved through translation in both directions, from bench to bedside and from bedside (and populations) to bench. An example of how human observation and basic research have successfully converged was provided by DES exposure in humans, which revealed that the human syndrome is faithfully replicated in rodent models. Furthermore, we now know that DES exposure in key developmental life stages can have a spectrum of effects spanning female reproduction, male reproduction, obesity, and breast cancer. It is interesting that in the case of breast cancer, an increased incidence is being reported now that the DES human cohort is reaching the age of breast cancer prevalence. The mouse model predicted this outcome 25 yr before the human data became available. In the case of reproductive cancers, the human and mouse data have since been confirmed in rats, hamsters, and monkeys. This is a compelling story from the perspective of both animal models and human exposures on the developmental basis of adult endocrine disease.
Prevention and the “precautionary principle”: although more experiments are being performed to find the hows and whys, what should be done to protect humans? The key to minimizing morbidity is preventing the disorders in the first place. However, recommendations for prevention are difficult to make because exposure to one chemical at a given time rarely reflects the current exposure history or ongoing risks of humans during development or at other life stages, and we usually do not know what exposures an individual has had in utero or in other life stages.
In the absence of direct information regarding cause and effect, the precautionary principle is critical to enhancing reproductive and endocrine health. As endocrinologists, we suggest that The Endocrine Society actively engages in lobbying for regulation seeking to decrease human exposure to the many endocrine-disrupting agents. Scientific societies should also partner to pool their intellectual resources and to increase the ranks of experts with knowledge about EDCs who can communicate to other researchers, clinicians, community advocates, and politicians.
2. In 1953, a study of 2000 women at the University of Chicago showed that DES did not prevent miscarriage; on the contrary, it was associated with increases in premature labor and a higher rate of abortions.
3. Despite this study, the drug continued to be used. It wasn’t until 1971 that American drug companies were legally obliged to label DES “unsuitable for pregnant women”. The FDA did not ban the drug but issued a contraindication which means that the drug DES continued to be prescribed to pregnant women even after the link between a rare form of vaginal cancer in young women and prenatal exposure to DES was established.
4. A whole generation of new medical students and doctors don’t know about Diethylstilbestrol, yet a study published in 2011 confirmed lifetime risk of adverse health effect in DES daughters (the youngest are in their mid 30’s early 40’s). DES is one of those cases where the patients often know more about its effects than the doctors.
5. DES is a multi-generational tragedy. Research by the Netherlands Cancer Institute in 2002 suggests that hypospadias a misplaced opening of the penis occurred 20 times more frequently among third-generation sons. In laboratory studies of elderly third-generation DES-exposed mice born to DES daughter mice, an increased risk of uterine cancers, benign ovarian tumors and lymphomas were found. Third-generation male mice were shown to be at risk for certain reproductive tract tumors.
Are we going to ignore these results like we did in 1939?
Third-generation children, the offspring of DES daughters and DES sons, are just beginning to reach the age when relevant health problems can be studied. Funding for more research is critically needed to continue to look for evidence of reproductive abnormalities and cancers among third-generation DES women and men to ensure they receive appropriate follow-up care.