Developmental exposure to estrogenic compounds and obesity

DES fetal life influence on body weight

2005 Study Abstract

For >20 years, research in our laboratory has focused on the effects of estrogenic compounds on development and differentiation. Our working premise has been that the developing organism is extremely sensitive to perturbation by chemicals with estrogenic or endocrine disrupting activity and that exposure to these chemicals during critical stages of differentiation may have permanent long-lasting consequences, some of which may not be expressed or detected until later in life. Diethylstilbestrol (DES) is a well-known example of such a chemical; thus, we have used DES as a model chemical to study environmental estrogens.

DES, a synthetic estrogen, was widely prescribed from the 1940s through the 1970s for the prevention of threatened miscarriage. A range of 2–8 million treated pregnancies worldwide has been estimated. Today it is well recognized that prenatal DES treatment results in a low incidence of neoplasia in the female offspring and a high incidence of benign abnormalities in both the male and female offspring.

To study the mechanisms involved in the toxicity of DES, we developed an animal model using outbred CD-1 mice treated with DES by subcutaneous injections on GD 9–16 (the period of major organogenesis in the mouse) or days 1–5 of neonatal life (a period of cellular differentiation of the reproductive tract and a critical period of immune and behavioral differentiation). The prenatal DES animal model has successfully duplicated and, in some cases, predicted many of the alterations (structural, function, cellular, and molecular) observed in similarly DES-exposed humans.

Although our major focus has been on reproductive tract abnormalities, we also examined the effects of DES on body weight over a wide dose range of exposure.

  • High prenatal DES doses (10–100 μg/kg of maternal body weight) caused a decrease in the offspring’s adult body weight;
  • likewise, high neonatal DES doses (1000 μg/kg/day on days 1–5 [1 mg/kg/day]) caused a decrease in body weight later in life.
  • However, low doses of DES (either prenatal or neonatal) caused an increase in body weight; Figure 1 illustrates control and neonatal DES 0.001 mg/kg/day treatment (DES-0.001).
  • Note that body weight was not different between DES-exposed and unexposed controls during the time of treatment and shortly thereafter, but it gradually reached significance by 6 weeks of age.
  • Further, data from our laboratory indicate that this increase in body weight in DES-exposed mice is associated with an increase in the percentage of body fat. Using Lunar PIXImus mouse densitometry (Lunar PIXImus, GE Healthcare, Waukesha, WI), we measured the percentage of fat in untreated controls and neonatal DES-treated mice at 16 weeks of age. As seen in the image, mice treated neonatally with DES are markedly larger than controls. Measurements obtained from densitometry show a significant increase in the estimated body weight, estimated fat weight, and percent fat compared to controls.
  • Neonatal exposure to other estrogens such as 2OH estradiol (20 mg/kg/day) and 4OH estradiol (0.1 mg/kg/day), which are approximately equal estrogenic doses to DES-0.001, also caused an increase in body weight at 4 months of age, suggesting that DES is not a unique estrogenic chemical in causing this increased obesity.
  • Further, neonatal exposure to the naturally occurring phytoestrogen genistein at 50 mg/kg/day, an approximately equal estrogenic dose to DES, caused a significant increase in body weight at 3 and 4 months of age compared to untreated controls.

We are currently comparing the weight of fat depots from mice exposed neonatally to various environmental estrogens to determine possible alterations in adipose tissue, including size of specific fat pads and hormone levels (e.g., leptin, adiponectin). By 18 months age, differences in body weight between genistein-treated and untreated controls are difficult to determine due to large individual animal variability within groups.

Taken together, our data support the idea that brief exposure to low levels of environmental estrogens early in life increases body weight as the mice age. Whether our results can be extrapolated to humans, as in the reproductive abnormalities from the DES mouse model, remains to be determined, but this is a fruitful area for further research. In addition, the use of this mouse model to study mechanisms involved in altered weight homeostasis (direct and/or endocrine feedback loops, e.g., ghrelin, leptin) by environmental endocrine disrupting chemicals is an important basic research area that may shed light on the future prevention and treatment of obesity.

References

DES DIETHYLSTILBESTROL RESOURCES

Ability to produce offspring significantly diminished by DES exposure

Avian transgenerational reproductive toxicity test with in ovo exposure

2006 Study Abstract

Ecological risk assessment of environmental pollutants requires effective laboratory assays and extrapolation of the resultant data to wild species. Because avian reproductive disorder and accumulation of persistent compounds in wild birds and their eggs have long been observed in polluted regions, we have developed an assay for investigating whether pollutants accumulated in eggs impair the reproduction of the exposed birds and the survival of the next generation using the Japanese quail.

A typical estrogenic compound, diethylstilbestrol (DES), dissolved in olive oil was injected into the air-chamber of fertilized eggs on day 10 of incubation.

After sexual maturation of hatched chicks, we mated pairs of male and female quails following an observation period of egg production and collected their eggs. The collected eggs were incubated and checked for the fertility and hatchability, and then the hatchlings were raised and observed in growth for 3 weeks.

A dosage of 5 ng/g per egg of DES caused eggshell thinning in eggs laid by exposed females and reduction in eggshell strength. DES also induced shortening of the left oviduct and unexpected development of the right oviduct, while testis weight was reduced symmetrically.

The ability of quail pairs to produce offspring was significantly diminished by exposure of females to DES independently of exposure of males, which mainly arose from production of abnormal and inviable eggs. Fertility of normal-shelled eggs and hatchability of fertilized eggs were unchanged regardless of treatments.

External morphological abnormalities, which were mostly unopened toes of the foot, were frequently observed in hatchlings from exposed males independently of exposure of females.

Additionally, we attempted to extrapolate the experimental results to the northern bobwhite and to predict population trends for quails in a polluted habitat using a population projection model composed of a combination of a Leslie matrix and the logistic equation.

In the event of accumulation of an estrogenic compound equivalent to a dosage of 5 ng/g DES in quail eggs,

  • the average population size was predicted to decrease by 20.2% after 1 year,
  • to approximately half after 4 years,
  • and to a fifth after 14 years.

When observed weakening of individuals and the risk of egg breakage are taken into consideration, the decline in population was further accelerated. The proposed assay appears to be suitable not only for assessing adverse effects of chemicals on avian reproduction but for population projection of affected wild birds.

References

  • Avian transgenerational reproductive toxicity test with in ovo exposure, Archives of toxicology, NCBI PubMed PMID: 16758213, 2006.
  • Featured image credit jiangxulei1990.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetics, brain, behavior, and the environment

Some studies looked at whether DNA methylation (a process at the DNA level which affects which genes get turned on and turned off) has a link to increased risk for schizophrenia or schizophrenia-like conditions among the DES-exposed

Both men and women exposed to diethylstilbestrol (DES) in utero (hence having a body burden of the chemical) are more prone to depression compared to their unexposed siblings. Indeed, an issue of national concern is the significant environmental exposure to common-use chemicals in the household, a factor suggested as contributing to the increased incidence of affective disorders in the general population.

2010 Review Abstract

Early experiences can modify regulatory factors affecting gene expression in such a way that, although the DNA sequence itself is not changed, the individual’s physiology and behavior is substantially influenced.

In some instances these epigenetic effects are exerted upon exposure, while in other instances they are transmitted across generations via incorporation into the germline. Examples of both types of epigenetic effects are presented.

First, experience with siblings (littermates) organizes behaviors and their underlying neural substrates in such a way that, as adults, rats and knockout mice behave differently. Second, exposure to the fungicide vinclozolin early in pregnancy imprints the male lineage in such a manner that rats exhibit distinct behavioral profiles as well as unique patterns of gene expression in relevant brain regions.

Taken together, this work demonstrates that present and past environments alike modify both social and affiliative related behaviors and their related metabolic activity in specific brain nuclei as well as influencing the abundance of specific genes altering the epigenome in the target brain areas.

References

DES DIETHYLSTILBESTROL RESOURCES

Family History is Underestimated in Children with Isolated Hypospadias

A French Multicenter Report of 88 Families, 2018

In humans massive exposure to strong EDCs (DES) has effects through several generations and may contribute to some familial expression of hypospadias.

Abstract

Purpose
While familial forms of complex disorders/differences of sex development have been widely reported, data regarding isolated hypospadias are sparse and a family history is thought to be less frequent. We aimed to determine the frequency of hypospadias in families of boys with hypospadias, to establish whether these familial forms exhibit a particular phenotype and to evaluate the prevalence of genetic defects of the main candidate genes.

Materials and methods
A total of 395 boys with hypospadias were prospectively screened for a family history with a standardized questionnaire, extensive clinical description, family tree and sequencing of AR, SF1, SRD5A2 and MAMLD1.

Results
Family history of hypospadias was more frequent than expected (88 patients, 22.3%). In 17 instances (19.3%) familial hypospadias cases were multiple. Familial hypospadias was related to the paternal side in 59.1% of cases, consisting of the father himself (30.7%) as well as paternal uncles and cousins. Premature birth, assisted reproductive techniques, other congenital abnormalities and growth retardation were not more frequent in familial hypospadias than in sporadic cases. The severity of phenotype was similar in both groups. The results of genetic analysis combined with previous data on androgen receptor sequencing revealed that familial cases more frequently tend to demonstrate genetic defects than sporadic cases (5.68% vs 1.63%, p = 0.048).

Conclusions
Familial forms of hypospadias are far more frequent than previously reported. Even minor and isolated forms justify a full clinical investigation of the family history. Detecting these hereditary forms may help to determine the underlying genetic defects, and may improve followup and counseling of these patients.

References

DES DIETHYLSTILBESTROL RESOURCES

Neurodevelopmental disorders in children exposed in utero to synthetic progestins

Analysis from the national cohort of the Hhorages Association, 2018

Abstract

The medical and scientific communities have not yet fully acknowledged the undesirable effects of the synthetic hormones that have been administered to pregnant women for decades.

The somatic effects of in utero exposure to diethylstilbestrol (DES), such as genital malformations, infertility, and cancer, have long been recognized but this has not been the case concerning psychiatric disorders.

The progestins used in contraception and hormone replacement therapy are known to affect the adult brain, but no data exist on their effects due to in utero exposure of children.

The Hhorages Association, a national patient support group, has assembled a cohort of 1200 women who took synthetic hormones during pregnancy.

These women had a combined 1934 children. We obtained full questionnaire responses from 46 women treated with progestins only – and not an estrogenic cocktail – who gave birth to 115 children.

Three groups were observed:

  1. Group 1 (n = 18): firstborn unexposed children,
  2. Group 2 (n = 62): children exposed in utero to synthetic progestins,
  3. and Group 3 (n = 35): children born after a previous pregnancy treated with progestins.

No psychiatric disorders were reported in Group 1 and the incidence of psychiatric disorders was drastically elevated in Group 2.

Our work shows a striking increase in psychiatric disorders among children exposed in utero to progestins and strongly suggests that prenatal exposure is associated with a high risk of psychiatric disorders in adolescence and adulthood, whether accompanied or not by disorders of sex development.

References

  • Neurodevelopmental disorders in children exposed in utero to synthetic progestins: analysis from the national cohort of the Hhorages Association, Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology, DOI: 10.5772/intechopen.80969, November 5th 2018.
  • Image credit Alexander Krivitskiy.
DES DIETHYLSTILBESTROL RESOURCES

Evidence for Link Between Mental Disorders and in Utero Exposure to Synthetic Hormones

A Long and Crucial History, IntechOpen, 2018

Abstract

Somatic effects of diethylstilbestrol on children exposed in utero have long been recognized. This is not the case for psychiatric disorders, although animal studies provide evidence of somatic and behavioral disorders.

Recent studies have reported psychiatric effects of synthetic estrogens on the brain of children exposed in utero as schizophrenia, bipolar disorders, depression, eating disorders, suicides, suicide attempts. Recently, a team of St. Anne’s Hospital, Paris (Prof. Krebs, Dr. Kebir) demonstrated the epigenetic mechanism of DES effect on the brain, a specific methylation of two genes playing important roles in neurodevelopment: the ADAM TS9 (control of the formation of reproductive organs and of the fetus’s CNS) and the ZFP 57 gene suggested to be associated with psychosis.

Progestins used in contraception and in hormone replacement therapy are known to affect the adult brain, but no data on children existed before our recent paper on their effects after in utero exposure. Clinical data were collected from 1934 children of the Association of Patients HHORAGES cohort.

Our data show the presence of somatic disorders and a drastic increase of psychiatric disorders among children in utero exposed to progestins. These mental disorders are the same as pathologies provoked by exposure to synthetic estrogens.

Introduction

At the end of the 1990s, somewhere in France, an agricultural engineer, Mr. RA, made the observation that his three children were suffering from various psychic and somatic pathologies, the elder suffering from bilateral cryptorchidism, micropenis, infertility due to azoospermia (no spermatozoa), and schizotypal character; the second from anxiety, depression, and eating disorders (anorexia) coupled with small uterus and ovaries; and the third suffering from schizophrenia and severe depression associated with suicide attempts. He observed also the relationship with the fact that in all three cases, his wife received medical treatment consisting of a synthetic hormone cocktail: diethylstilbestrol (DES), ethinyl estradiol (EE), plus synthetic progestin delay during her three pregnancies after a previous miscarriage. He conducted research in the world literature on the subject and came to the conclusion that not only was one of these products, diethylstilbestrol (DES), already known for its misdeeds but that it continued to be administered in France until 1977/1982. The product, inexpensive to make, was not patented and was manufactured and distributed by many pharmaceutical laboratories. The same goes for EE, which was banned for pregnant women in 1980 but remains the best-selling estrogen in the world because it is part of the contraceptive pill.

In 1998, one of us (MOS-G), concerned by the same kind of problems in her two children who were exposed in utero to the same cocktail of synthetic estrogens, lost her two children after psychiatric illnesses. Following the reading of a “Call to Families” printed in a newspaper, she met Mr. RA, who had gathered around him about 20 French families concerned with their in utero-exposed children suffering from psychiatric illnesses. He wanted to expose his observations and the results of his bibliographic researches at a meeting of patient families collecting observations on the genital malformations of girls exposed in utero to DES. Alas, he was condemned, rejected, and disclaimed by doctors, mostly gynecologists and psychiatrists as well as by associative members. They denied the existence of psychiatric disorders in exposed boys and girls. Discouraged, Mr. RA died shortly afterward. In order to continue his work and regroup the families concerned by the origin of the heavy psychiatric pathologies of their children and despite the taboo surrounding such diseases as psychoses, we gathered several mothers concerned and created the Association of Patients Halt to Artificial Hormones for Pregnancies (HHORAGES), in 2002. This Association which collected more than 1300 French spontaneous testimonies is now registered with the Epidemiology Portal of French National Institute for Medical Research (INSERM) as a French Health Database.

Despite various alerts published in the 1940s, after work on animals proving in particular its carcinogenic effect, and despite the work of Dieckman et al., initiated as early as 1953, demonstrating in a large cohort of pregnant women given diethylstilbetrol (DES), a synthetic estrogen, versus placebo that the drug was inefficient in preventing miscarriages or premature births, this product has been widely distributed around the world, sowing a long list of misdeeds. After the discovery of cervicovaginal cancers called “clear cells adenoma (CCAD)” in the “DES girls,” DES was banned in the United States for pregnant women in 1971 but only in 1977 in France, where this recommendation disappeared from the “French Vidal book,” but DES continued to be prescribed sporadically until 1982. Meanwhile another synthetic estrogen, steroidal, also synthesized on 1938, 17-alpha-ethinyl estradiol (EE), was often added to DES as a cocktail or later as a replacement, sometimes with the addition of synthetic-delay progestin. The idea that prevailed at the time was that women had a hormonal deficit that triggered a miscarriage, whereas now we know that the miscarriage itself causes this deficiency. These products were prescribed not only to women who had miscarriages but also in comfort (“to have beautiful babies,” according to an advertising) or even as a “morning after pill” or to cut milk after childbirth. DES and 17-alpha-EE, although belonging to different estrogenic and degrading categories, are, however, bound to the same ER beta-estrogen receptors.

Behavioral disorders demonstrated in animals (rats) exposed in utero

Animal studies (on mice and rats) have demonstrated the toxicity of these synthetic estrogens on the offspring, including the cause of behavioral disorders. Palanza et al. demonstrated in particular that prenatal exposure to three different synthetic chemicals, DES and two pesticides, DDT and methoxychlor, and its analog, affects the behavior of young suckling mice, showing increased aggression in males (increased numbers of attacks and decreased reaction time before the attack). Doses of DES were 1000 times less than those of DDT and caused much larger aggression responses, demonstrating the considerable effect of DES at very low doses. The treatment period for rodent mothers from day 11 to day 17 of pregnancy was also critical because it represents a key period in the differentiation of the reproductive system and brain development in these rodents in the early stages of pregnancy.

Moreover, injection of 17-alpha-estradiol (EE) in pregnant rats causes not only many abortions in mothers but also anxiety and depression disorders in offspring, the synthetic hormone having been administered at the same relative doses as in humans (15 g/kg, 1 per day, versus 19 g/kg, 1 per day). At the cytological level of the brain, an alteration of the anterior part of the hippocampus in young rats exposed to EE in utero has been demonstrated in 2004. The hippocampus is indeed a part of the brain that contains many estrogen receptors during the prenatal period. Ogiue-Ikeda et al. showed in 2008 that synaptic plasticity can be upset by estrogens or other endocrine disruptors (EDs). Later and unequivocally, Newbold demonstrated the validity of the rodent model transposed to humans.

Behavioral disorders, psychoses, and depression demonstrated in humans after in utero exposure to DES/EE

In humans, the work concerning the appearance of behavioral disorders in children after in utero exposure to synthetic hormones is less numerous, but as early as 1977, June Reinisch published in Nature that prenatal exposure to estrogen and/or synthetic progestins could affect the personality of exposed children. More recently, in 2012, Kebir and Krebs have analyzed several epidemiological studies concerning the effects of DES on exposed children in utero and the occurrence of psychiatric disorders in these children. Their analysis shows that only three large epidemiological studies on the effects of DES were performed in 1952–1953 (followed up in 1983), in 2007, and in 2010. The first study (double-blinded) that supports the hypothesis of a link between psychiatric disorders and prenatal exposure to DES was performed by Vessey et al., in 1983, from a clinical trial that had been performed 30 years earlier in 1953 in London by Dieckman et al., on 700 women treated with DES versus 700 subjected to a placebo. A doubling of depression and anxiety disorders has been demonstrated in the population exposed in utero. The second, published in 2007 and conducted by Verdoux et al., from a cohort of women from the Mutuelle (Health) de l’Education Nationale (MGEN) concludes that there are no significant links between exposure to DES, suicides, and/or psychiatric consultations or hospitalizations. A detailed analysis later, however, revealed a number of biases in this study. The most recent Nurses’ Health Study was conducted by O’Reilly et al., 2010, from 76,240 American women among whom 1612 women were exposed to DES in utero. The statistical analysis shows that the latter experienced an increase in depressive and anxiety disorders by a factor of 1.3. Kebir and Krebs emphasized the limitations of such epidemiological studies and noted in particular that, apart from depression and anxiety, other psychiatric disorders have not been studied. Postadolescence behavioral disturbances reported for these two estrogens in exposed children were depression, anxiety, schizophrenia-like behavior, anorexia, and bulimia nervosa. All these observations were synthesized by Pillard et al. and Giusti. On 1987, Katz et al. described the case of four male adults prenatally exposed to DES. It is in late adolescence that they develop psychotic disorders requiring neuroleptic treatment even though they have no family history of this type. He then hypothesizes that there may be a causal relationship between disruptions in neurodevelopment related to DES and the subsequent onset of psychotic disorders. Pillard et al. showed that the frequency of recurrent major depressive episodes is significantly higher in the DES-exposed than in their unexposed siblings, which was confirmed in 2010 in the large cohort of DES girls by O’Reilly et al.

Investigation of the causal link between exposure to these synthetic hormones in utero and severe psychotic disorders such as schizophrenia, bipolar disorders with or without eating disorders, and schizoaffective disorders, occurring in postadolescence in exposed children, was made possible thanks to the families of the Association HHORAGES. Our database, constituted by these spontaneous families’ testimonies, is based on responses to a detailed questionnaire, written by doctors and researchers and accepted by the CNIL (French Center for the Protection of Data Processing and Freedom). Our first global analysis (2004–2005 data) was based on 967 pregnancies from 470 mothers in collaboration between 2 of us (MOS-G/CS) of us (CS). The first results as well as the family questionnaires were detailed in 2012 in the chapter published in 2012 by InTech “Behavioral and Somatic Disorders in Children Exposed In Utero to Synthetic Hormones” in which we detailed somatic and psychiatric disorders, associated or not, in the exposed children of our cohort.

We have conducted a more recent analysis (2016) based on 1182 pregnancies from 529 mothers. Among the 740 (20 stillborn) exposed children, 603 (exposed) +16 post-DES (born without exposure but after a previous exposed pregnancy) are suffering from psychiatric disorders. The prevalence of the psychiatric disorders in comparison with the general population shows a dramatic increase.

We were also interested by the effects on the brain of synthetic progestins on in utero-exposed children of our cohort. Currently, there is no research on these effects of in utero exposure of children to progestins given alone during pregnancy. Our recent observations were collected from 1200 families of the HHORAGES cohort, that is, 1934 children using always the same detailed questionnaire. As previously shown, most families of our cohort had children exposed to estrogens or to estro-progestins, but only 46 families (115 children) had at least 1 child exposed to 1 or more progestins prescribed alone and representing 62 in utero-exposed children. Thirty-five children were post-exposed. The prescribed progestins were 17-α-hydroxyprogesterone caproate (synthetic progestin, SP) against total indication in 2000 but reauthorized in 2011, 17-α-hydroxyprogesterone heptanoate (SP) against total indication in 2002, and chlormadinone acetate (SP), derived from hydroxyprogesterone, against total indication in 1970.

Among the 62 exposed children (22 girls and 40 boys), 49 presented psychiatric disorders, 6 presented somatic disorders only, and 7 did not present any disorder. Only 1 post-exposed presented psychiatric disorder, while 34 other post-exposed did not present any disorder.

Among the 49 children affected by psychiatric disorders, 3 boys and 7 girls presented both somatic disorders in addition to psychiatric ones: boys (3), hypospadias (1), no urinary meatus (1), bilateral cryptorchidia, and sexual ambiguity (1) and girls (7), hormonal sterility (2), hirsutism and enuresis (1), enuresis (1), hirsutism (1), hermaphrodism (1, operated), and sexual ambiguity and tight urethra (1). Among the six exposed children suffering from somatic disorders “only,” we observed for boys (four) mega bilateral ureter (one grandchild), unilateral cryptorchidia (one), hypospadias with numerous interventions of reconstruction (one grandchild), and sexual ambiguity (one) and for girls (two) hormonal sterility (two).

A comparison of synthetic estrogen and progestin exposures for girls and boys demonstrates that psychiatric disorders were of the same nature for progestin exposure as those observed after exposure to synthetic estrogens, that is, behavioral disorders = 2, eating disorders = 2, schizophrenia = 29, depression, bipolar disorders = 16, suicides attempts = 7 series, and death = 1. The percentage of suicide attempts (11.29%) and death after suicide (1.6%) is proportionately lower after exposure to progestins than after exposure to synthetic estrogens.

An epigenetic mechanism

Search for the molecular basis of the causal link between in utero, exposure to synthetic hormones and the appearance of psychoses as schizophrenia or bipolar disorder in children exposed in utero, has been achieved thanks to the partnership that unites HHORAGES Patients’ Association with the INSERM team of molecular psychiatrist Pr. MO Krebs (St. Anne’s Hospital, Paris, France, UMR S 894), which began in 2007.

“It would have been ‘crazy’ to miss the problem posed by the Association Hhorages to establish a causal link between taking artificial hormone(s) during pregnancy and appearance of psychiatric disorders of the type psychotic in exposed children, because diethylstilbestrol (Distilbene® or DES) has been given over a limited period of time and people who have taken this molecule are still there to testify. This is a case study that should not be missed”

said Dr. Kebir (Center for Psychiatry and Neuroscience, UMR S 894), manager of these researches as part of the Krebs’ team.

First, to document in utero exposure to synthetic estrogens, Kebir and Krebs were able to analyze from our data a small number of family records that occurred in HHORAGES testimonies and studied a cohort of 472 exposed subjects. They account for 46.7% of mood disorders, 22.9% of psychotic disorders, 6.6% of anxiety disorders, 11% of eating disorders, and 12.7% of others, which confirms their previous observations published on 2009 and 2010 at the seventh and eighth Congress of the Encephalon in Paris on 43 exposed children highlighting clinical pictures with atypical associations.

Second, genetic and epigenetic analyses of HHORAGES siblings have shown in patients suffering from psychotic disorders and exposed in utero to DES and/or EE that this prenatal exposure is associated with epigenetic processes. Starting from the fact that psychiatric diseases develop from a brain dysfunction during neurodevelopment, and knowing that DES and EE are synthetic hormones (estrogens), endocrine disruptors, and confirming from the HHORAGES data numerous cases of heavy psychiatric disorders in children exposed, the Krebs’team in association with HHORAGES designed 10 years ago, in 2007, a research project Partnership Citizen Institution for Research and Innovation (PICRI), funded by the Ile de France Region, net by the French National REsearch Agency (ANR), that developed the hypothesis that the DES administered during pregnancies could be an environmental risk factor for the development of psychiatric disorders in impregnated children: the epigenome of the foetus could have been modified by in utero exposure to synthetic estrogens. The PICRI project was titled “Influence of hormonal treatments on brain development during pregnancy: study of phenotypic, behavioral and biological changes in informative families.” The families of HHORAGES were called to perform peripheral blood sampling after thorough questioning. Many families volunteered to participate in the research: 31 families were selected, satisfying the rigorous inclusion criteria desired. Many more families had come forward during this study, but they could not be included because the psychotic patient refused to come to St. Anne’s Hospital for blood sampling. In the selected families, total siblings were composed of first-born unexposed children, exposed children, and post-exposed children, with first-born unexposed serving as intrafamilial control. For the exploration of their epigenome, 485,000 cytosines by genome were studied and analyzed, representing an immense work. To complete this study, a cohort of young adolescents with relational, emotional, and social difficulties was followed for 6 months, some of whom had developed schizophrenic-type psychosis in these 6 months, although not exposed to DES. A comparison of their methylome, analyzed before and after the onset of the disease, was performed. In this study, authors reported a global methylation of the psychotic patient genome.

After the analysis of the whole methylome of the selected HHORAGES cohort, the team of Krebs-Kebir highlighted differential specific methylated regions (DMR): in the zinc finger protein 57 gene ZFP57 and in the ADAM TS9gene and in young psychotic patients exposed in utero to DES/EE. In this work, the authors observed that in exposed individuals, ZFP57 gene methylation may be associated with their psychosis. The ZFP57 gene (located on chromosome 6) is expressed very early in development. It is a transcription regulator, directly related to the phenomenon of methylation and neurodevelopment. The ADAM TS9 gene is implicated in the control of organ shape, especially in the development and function of the uterus and reproductive organs which are often abnormal after in utero DES exposure as well as in the control of the CNS development and in several kinds of cancers.

Discussion and conclusion

Very few studies have investigated the impact of prenatal exposure to DES and EE on psychiatric outcome. Animal studies on rats or mice allowed us to hypothesize that estrogenic hormones induce neurodevelopmental disturbances in exposed human subjects and may potentially mediate an increased risk of behavioral and psychiatric disorders. Our data therefore strongly suggest that DES/EE exposure during pregnancy is associated with high incidence of behavioral and/or psychiatric disorders. They illustrate a higher risk of schizophrenia, as this disease was 17 times more prevalent than in the general population, with sons being more affected than DES daughters. Regarding the existence of eating disorders (bulimia, anorexia), it should be noted that girls are much more affected than boys, and we often observed the association of eating disorders with bipolarity (manic-depressive disorders), anxiety, and depression. With regard to suicides, our work clearly demonstrates a drastically increased risk of suicide attempts (65.4% versus 0 in the unexposed controls and 0.25% in the general population) and suicides (3.4% versus 0 in the unexposed controls and 0.02% in the general population). It could be noted that, as in the general population, DES sons commit more suicides than DES daughters and the inverse for suicide attempts. Moreover, our data reveal that 50% of the sons who committed suicide suffered from schizophrenia. Psychiatric studies in general have shown that the percentage of suicides is generally higher in individuals with psychiatric disorders than in the general population. But to our knowledge, there is no information or specific studies concerning this association in the context of DES exposure. Sixteen subjects in Group 3 (post-DES children) had diagnosed psychiatric disorders. An explanation for this finding might be that DES, being a very lipophilic synthetic estrogen, remains in the mothers’ fat after estrogenic impregnation in a previous pregnancy and is then released through the placental barrier during the next gestation.

No work had been reported on the impact of in utero exposure to synthetic progestin hormones administered alone on the occurrence of psychiatric disorders in exposed children before our first presentation in the European Congress of Gynecology in 2017. For the first time, we described psychiatric disorders that can affect children exposed in utero to progestins. Previously, and during many years, synthetic progestogens were not considered as dangerous during pregnancy or during replacement or contraceptive treatment. Moreover, they have been suggested to exert neuroprotective effects in several animal models of neurological disease. Negative mood symptoms have been reported by Andreen et al. in some women as a result of progesterone during the luteal phase of menstrual cycles. This is believed to be mediated via the action of allopregnanolone on the GABA-A system. A reduction of allopregnanolone circulating levels that correlates to depressive symptoms has been recently reported, and conversely, healthy women reported increased anxiety and mood disorders after long-acting subdermal implant of progestogens. In a group of 236 schizophrenic patients at onset, an elevated concentration of progesterone has been found, and authors suggested that steroid hormones may influence brain function, underlying schizophrenia, and major depressive disorders. Moreover, Buoli et al. (2016) found high DHAS levels in patients with a history of psychotic symptoms, suggesting a role of steroids in the etiology of psychosis and mood disorders. Progestins are known to induce GABA receptor activity/neural activation before birth; it is likely that a GABAergic system could contribute to schizophrenia, anxiety, depression, panic disorders, epilepsy, autism, and others. Although some progestins have been banned from the market, others are not: our data demonstrated that caution should be taken with regard to the use of these progestins during pregnancy and even outside these periods (contraception or hormone replacement therapy).

The brain is a very vulnerable organ because its development covers a very broad period extending from the early prenatal stage (third week of pregnancy) to end around the age of 20. During its development, there are times when its vulnerability is even greater than others; these periods are called “shooting windows,” during which the environment can impact the normal process of development. Abdolmaleky et al. as early as 2005 had developed the hypothesis that gene-environment modulations could be performed via DNA methylations. Krebs’ team put forward the hypothesis that DES-induced changes in epigenetic background and alteration of DNA functioning (methylations) could be significant factors to demonstrate a possible origin of psychotic disorders and a link with in utero DES exposure of the children suffering from these illnesses.

Numerous studies have shown that, for example, in the rat, early maternal separation or the fact of causing significant stress to the mother changes the methylation signals of certain genes of the rat directly related to the regulation of anxiety. It has also been discovered that the proper environment for changing the methylation signals may be chemical. This is the case of DES recognized by the scientific community as an endocrine disruptor and banned for pregnant women. This change in the level of methylation caused in utero by DES has been demonstrated for urogenital malformations of girls and boys as well as for cancers. On 2015, Harlid et al. published in a pioneer work the first study for evaluation of possible effects of in utero DES exposure on genome-wide DNA methylation in humans. They studied whole blood DNA methylation in 100 40–59-year-old women reporting in utero exposure, compared to 100 unexposed women. They did not find any differential methylation, but the DMR approach was not used in their recent work (2015). On the other hand, in 2017 Rivollier et al. described specific differential methylated regions (DMR) on two genes implicated in neurodevelopment (ZFP57 and ADAMTS9). Surprisingly, they cautiously claim that these DMR are “supposedly” associated with prenatal exposure to DES in young psychotic patients in utero exposed to DES/EE. Nevertheless, these modifications of methylation are really specific because they do not exist in the methylome of young psychotic patients not exposed to DES in which global methylation of the genome was observed. Moreover authors have compared exposed subjects to their unexposed siblings which do not present these specific methylations although they shared environmental and genetic factors.

The citizen work carried out between the French HHORAGES Patient Association and two major medical research laboratories has provided convincing scientific results: (1) on the detection and confirmation of the existence of psychiatric disorders (accompanied or not of somatic disorders) in children exposed in utero to synthetic hormones and (2) on the mechanisms of action of these synthetic hormones administered to pregnant mothers on the brain of their offspring. The effects of these endocrine disruptors in humans through what is becoming a public health scandal, denied for a long time by doctors, especially psychiatrists, scientists, and specialized journalists, are thus better known. The fact that these synthetic products do not degrade in the human body in the same way as the natural hormones and act on the functioning of genes implicated in neurodevelopment during the fetal life, following an epigenetic mechanism, is a real time bomb. Indeed, this mechanism induces a transgenerational effect already partially demonstrated in the HHORAGES cohort at the third-generation level for hypospadias, a specific genital malformation. So far, only a few third-generation children suffering psychiatric illness are documented in the HHORAGES 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 hypospadias that are detectable from birth in male children and grandchildren. In contrast, psychiatric disorders usually appear in postadolescence, 18–20 years, and sometimes later.

By Marie-Odile Soyer-Gobillard, Laura Gaspari and Charles Sultan,
Submitted: May 22nd 2018 – Reviewed: August 17th 2018 – Published: November 5th 2018.
© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  • Full study free access) : “Evidence for Link Between Mental Disorders and in Utero Exposure to Synthetic Hormones: A Long and Crucial History, IntechOpen, DOI: 10.5772/intechopen.80969, November 5th 2018.
  • Image credit futurebrand.
DES DIETHYLSTILBESTROL RESOURCES

Menstrual history and fecundity of DES-exposed women (in utero)

The Journal of reproductive medicine, 1984

Abstract

Sequential examination and interview of 349 women exposed to diethylstilbestrol (DES) and 375 women unexposed to the drug verified that DES exposure has no effect on age at menarche and indicated no differences in the age at 1st coitus, pregnancy, and live birth.

Records of the women in the DES Adenosis project were studied to determine DES dosage and timing, but starting date and total amount prescribed could not always be determined accurately.

Estimates indicate that about 1-2% of pregnant patients in the late 1950s and 1960s were exposed to the drug. As many as 30% of mothers were unaware that DES was given to them during pregnancy. The women were asked about marital status, age at menarche, menstrual history, pelvic inflammatory disease (PID), operations, birth control, and pregnancy experience.

Analysis of variance indicated that there is an age-related increase in oligomenorrhea in DES-exposed women as compared to unexposed women that disappears as the patients reach their late 20s.

Of 133 exposed women, 59 (44%) had induced abortions, and of 162 unexposed women 93 (57%) had induced abortions. DES-exposed women used oral contraceptives (OCs) but not as frequently or extensively as their unexposed counterparts.

Age at first coitus was 19.33 +or-2.95 years for the exposed and 18.89 +or-2.62 years for the unexposed, showing no significant difference.

In a higher percentage of DES-exposed women the cause of the couple’s infertility was undetermined. 44 (12.6%) of the exposed and 42 (11.2%) of the unexposed women had dilatation and curettage (D and C) for some reason other than abortion.

There appears to be no substantial effect of in utero exposure to DES on women’s ability to conceive. Whether or not exposure to DES has specific physiological effects on reproductive function, exposed women may alter their sexual and reproductive behavior in anticipation of those effects as well as experiencing them.

Sources

DES DIETHYLSTILBESTROL RESOURCES

Reproductive performance among DES exposed daughters compared with that of their mothers

The Ohio State medical journal, 1983

Abstract

The study was undertaken to determine what the experience of 158 diethylstilbestrol (DES) exposed daughters in the Toledo area has been and whether the rate of pregnancy loss is related to a similar rate in their mothers, indicating a familial factor in the etiology.

45 patients could produce written, documented evidence through their mothers’ prenatal records of having been exposed to DES. In these, not only the drug, but also the dose, stage of gestation, and duration were known. In 37 of these (82.2%), definite, benign DES related changes were found in the vagina and cervix. In another 49 patients documented evidence was not available, but all demonstrated the same gross and/or colposcopic changes, thereby giving presumptive evidence of DES exposure. These 94 patients make up Group A.

In 64 patients, no documented evidence of DES exposure was obtainable, since their mothers’ prenatal records had been lost or destroyed. The mothers themselves recalled using the drug with varying degrees of knowledge about the specifics of dose or duration. None of these daughters had objective findings, by gross or colposcopic examination, suggestive of DES effect. In 20 of them there was some evidence consistent with exposure. These were designated “equivocal findings” and included with the Group B patients.

In the group of 94 women who satisfied the criteria of DES exposure by documented history and/or characteristic physical findings (Group A), there were 39 who were sexually active without contraception. The remaining either denied coitus or were regular contraceptive users. 10 patients attempted to conceive for more than 1 year without success, for a primary infertility rate of 25.6%. 29 achieved a total of 46 pregnancies; 22 were delivered at term, and 24 aborted spontaneously, for a pregnancy failure rate of 52.2%.

Of the 64 patients with histories of DES exposure but no documented evidence (Group B), there were 24 sexually active without contraceptive use. In these 24 there were 3 who complained of primary infertility, which was defined as regular coitus without contraception for a period of at least 1 year, for a primary infertility rate of 12.5%. The remaining 21 patients had a total of 41 pregnancies. 31 of these resulted in full term live infants. 2 were electively aborted, and 8 were aborted spontaneously.

Reproductive histories were available in 105 of the mothers of these daughters. The mothers of the women in both groups had poor reproductive histories. The reproductive performance of Group B daughters was better than that of their mothers and closely approximated that usually quoted for the general population.

The performance of group A mothers was significantly worse than that of their mothers, in whom the pregnancy failure rate already was high.

The data indicate that the DES exposed daughters have a higher reproductive failure rate than their mothers, while that of DES unexposed daughters is lower. The existence of a familial factor is not supported.

Sources

  • Reproductive performance among DES exposed daughters compared with that of their mothers, The Ohio State medical journal, NCBI PubMed, PMID: 6646562, 1983 Nov.
  • Image credit sagepub
DES DIETHYLSTILBESTROL RESOURCES

Estrogen Metabolism in Postmenopausal Women Exposed In Utero to Diethylstilbestrol

Increased postmenopausal breast cancer risk in DES Daughters

2018 Study Abstract

Background
Prenatal diethylstilbestrol (DES) exposure is associated with adverse reproductive outcomes and cancer of the breast and vagina/cervix in adult women. DES effects on estrogen metabolism have been hypothesized, but reproductive hormone concentrations and metabolic pathways have not been comprehensively described.

Methods
Blood samples were provided by 60 postmenopausal women (40 exposed and 20 unexposed) who were participants in the NCI Combined DES Cohort Study, had never used hormone supplements or been diagnosed with cancer, had responded to the most recent cohort study questionnaire, and lived within driving distance of Boston University Medical School (Boston, MA). Parent estrogens and their metabolites were measured by high-performance liquid chromatography-tandem mass spectrometry. Age-adjusted percent changes in geometric means and associated 95% confidence intervals (CIs) between the exposed and unexposed were calculated.

Results

  • Concentrations of total estrogens (15.3%; CI, -4.1-38.5) and parent estrogens (27.1%; CI, -8.2-76.1) were slightly higher in the DES-exposed than unexposed.
  • Ratios of path2:parent estrogens (-36.5%; CI, -53.0 to -14.3) and path2:path16 (-28.8%; CI, -47.3-3.7) were lower in the DES exposed.
  • These associations persisted with adjustment for total estrogen, years since menopause, body mass index, parity, and recent alcohol intake.

Conclusions
These preliminary data suggest that postmenopausal women who were prenatally DES exposed may have relatively less 2 than 16 pathway estrogen metabolism compared with unexposed women.

Impact
Lower 2 pathway metabolism has been associated with increased postmenopausal breast cancer risk and could potentially offer a partial explanation for the modest increased risk observed for prenatally DES-exposed women.

Sources

  • Estrogen Metabolism in Postmenopausal Women Exposed In Utero to Diethylstilbestrol, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, NCBI PubMed PMID: 30049842, 2018 Oct;27.
  • Featured image credit aacrjournals.
DES DIETHYLSTILBESTROL RESOURCES