Human Reproduction Update, human experimental data systematic review, 2019
Overall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.
OBJECTIVE AND RATIONALE
The objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.
For identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. Abstracts were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.
A total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.
Direct experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility.
Development of the male reproductive system and its subsequent function is impacted by events that occur in utero. Perturbations in testicular development or function during fetal life may result in male reproductive disorders that present postnatally. This includes anatomical abnormalities identified at birth, such as cryptorchidism and hypospadias, or disorders presenting in adulthood, including testicular cancer or infertility. These associated disorders are collectively referred to as the testicular dysgenesis syndrome (TDS). The development of TDS has been shown in rats to be influenced by a reduction in androgen production or action during a key period of fetal life, known as the masculinization programming window (MPW). The increasing incidence of TDS disorders over recent decades, highlights the potential importance of environmental impacts in their etiology. Environmental factors that have been proposed to affect fetal testis development and predispose to TDS disorders include environmental chemicals (e.g. plasticizers and pesticides), pharmaceuticals (e.g. analgesics, metformin and diethylstilboestrol) and lifestyle factors (e.g. diet, alcohol and smoking).
Pharmaceuticals : Diethylstilboestrol
Diethylstilboestrol (DES) is a synthetic estrogen that was used clinically to prevent spontaneous miscarriage and pre-term labor from the 1940 s until the early 1970 s. DES was withdrawn from clinical use after the demonstration of a causal role in the development of vaginal carcinoma in girls born to exposed mothers. In addition to the effects on female offspring, an association with structural abnormalities of the male reproductive tract was also described including epididymal cysts, microphallus and testicular hypoplasia.
Animal studies involving in-vitro culture of rat and mouse fetal testis, have reported a reduction in testosterone production following exposure to DES, similar to the results of previous in-vitro studies involving fetal mice and in-vivo studies in rats.
For TDS disorders, which are linked to a reduction in androgen action during fetal life, there is conflicting evidence regarding their association with maternal DES exposure. Three studies have reviewed the literature relating to exogenous estrogen exposure and male reproductive disorders. Whilst early studies reported that hypospadias was significantly associated with DES exposure, it has subsequently been pointed out that this related to urethral abnormalities resulting from exposure to exogenous estrogens (including DES), which may have resulted from abnormalities in penile development rather than an effect on urethral formation as a result of reduced androgen exposure. The meta-analysis of all available evidence revealed a significant association between DES exposure and hypospadias; however, it was concluded that any effect of DES on hypospadias is likely to be small. For cryptorchidism, an increased risk in association with DES exposure is reported; however, this was dependent on the statistical model used and was indicative of heterogeneity. A subsequent cohort study has reported an association between in-utero exposure to DES and an increased risk of cryptorchidism, however, only for those in whom the initial exposure occurred prior to the 11th week of gestation with no significant association following exposure after 11 GWs. Studies have demonstrated no effect of prenatal DES exposure on sperm counts or fertility; however, this is in contrast to a previous study demonstrating an association between prenatal exposure to DES and semen parameters in adult men. Importantly, this study included analysis of men born to a large cohort of mothers who participated in an RCT involving DES exposure during pregnancy.
Experimental evidence from human studies
To date, only two studies have investigated the effect of DES exposure on the human fetal testis (Table VI). In-vitro organ culture of first trimester human fetal testis exposed to DES (10−5 to 10−6 M) for 3 days did not alter testosterone production. Interestingly, this study compared effects of DES exposure in rodent and human fetal testis demonstrating contrasting results between species using an identical experimental system.
In a separate study using the xenograft model, exposure to DES (100μg/kg, three times weekly) for 35 days resulted in no significant difference in testosterone production by second trimester (15–19 GW) testis tissue. Interestingly, host mouse seminal vesicles were significantly increased in weight, which was indicative of increased testosterone production from the xenografted tissue over the entire grafting period. The reason for this unexpected increase in testosterone is unclear.
Whilst rodent studies have indicated a profoundly negative effect of DES exposure on testosterone production by the fetal testis, experimental studies utilizing human fetal testis tissues have failed to identify similar effects, which may relate to the presence of ESR1 in rodent Leydig cells, and the absence of this estrogen receptor in human fetal testis. Epidemiological data suggests that any potential effect of DES exposure on male reproductive development is likely to be of small magnitude. Taken together the results suggest an important species difference in terms of DES effects on fetal testosterone production which may explain why this frequently results in the development of male reproductive disorders in rodents, whilst associations between DES and subsequent male reproductive disorders in humans are rather modest. Whilst DES is unlikely to be used in pregnant women in the future, the findings of this study offer some reassurance regarding the potential of low-level exposure to environmental estrogens to affect human male reproductive development, given their extremely low potency compared with DES and the high exposures that resulted from therapeutic use of DES.
- Full study (free access) : Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data, Human Reproduction Update, doi.org/10.1093/humupd/dmz004, 14 March 2019.
- Featured image dmz004.pdf.
DES DIETHYLSTILBESTROL RESOURCES
The history of DES, lessons to be learned
“Health care professionals have to know the (full) history of DES to prevent future disasters with drugs prescribed.”
Abstract – 2005
At this moment, little is known about the implications of DES exposure for the third generation. Research has been done in mice to investigate the possibility of negative effects of DES on the male offspring of in utero exposed mice. The fertility of these males appeared to be the same as the fertility of the reference group. However, an increase in lesions of the testis and in tumours of the reproductive tract was reported, thereby providing the suggestion that effects of DES exposure can be transmitted to the third generation in mice.
Female offspring of mice exposed to DES in utero was also examined. In a group of 40 third generation mice ten uterine adenocarcinomas and five ovarian cystadenocarcinomas were seen, while tumours were absent in the control group. The development of these forms of cancer seemed to be age-dependent.
To find out whether these results would also be seen in humans, a group of 28 third-generation daughters was observed. While 61.5% of their mothers demonstrated cervical or vaginal changes associated with DES exposure in utero, none of these daughters showed any abnormality in the lower genital tract. However, this study is too small to draw conclusions but it seems that the carryover of DES effects in the female third generation is not of great significance.
Because the effects of DES in the third generation might be caused by biologic mechanisms, a study focused on the age at menarche in DES granddaughters. Fifty-two girls of mothers exposed to DES in utero were compared with 71 girls of unexposed mothers. The age at menarche was unaffected by DES administration to the grandmothers of these girls.
Next to the consequences in DES granddaughters, DES effects on the third-generation sons were also examined. As a result of two cases of hypospadia in sons of women that were exposed to DES in utero, a cohort study was done to find out whether an association between hypospadia in third generation males and DES exists. Hypospadia is a defect of the urogenital system in which the urethral opening is located on the ventral surface of the penis or on the scrotum. Sons of women with fertility problems (N ¼ 8934) were included in this study; 205 boys were sons of women exposed to DES in utero and the mothers of the remaining 8729 were unexposed. Four cases of hypospadia were reported in sons of exposed women compared with eight in the sons of unexposed. The prevalence rate ratio for hypospadia following from these results is 21.3, thereby suggesting an association between the administration of DES and the risk of hypospadia in the male third generation.
These outcomes suggest that there are no effects of DES exposure in females of the third generation, but that an increased risk of hypospadias exists for male offspring of women exposed to DES in utero. However, these results are obtained from small studies, so more research has to be carried out to obtain more reliable information.
- The history of DES, lessons to be learned, Pharmacy world & science : PWS, PMID: 16096877, 2005 Jun.
- Image credit rawdonfox.
DES DIETHYLSTILBESTROL RESOURCES
Journal of lower genital tract disease, 2005
Diethylstilbestrol (DES), a nonsteroidal estrogen, was widely used in the United States from 1940 through 1971 to prevent pregnancy loss.
In the late 1960s, an association was made with an increased incidence of clear cell adenocarcinoma in young women exposed in utero to DES. Additional study of these women over the next 35 years has shown an increased risk of other health problems including intraepithelial neoplasia, ectopic pregnancy, first trimester spontaneous abortion and second trimester pregnancy loss.
The National Institutes of Health continues to fund studies to follow cohorts of DES-exposed mothers, daughters, sons and third generation children. The Centers for Disease Control have conducted a large DES Education Project and have established guidelines for management.
The following six cases studies illustrate common problems seen in DES exposed daughters and management of problems encountered.
- Six cases of women with diethylstilbestrol in utero demonstrating long-term manifestations and current evaluation guidelines, Journal of lower genital tract disease, PMID: 15870516, 2005 Jan.
- Image credit earlymenopause.
DES DIETHYLSTILBESTROL RESOURCES
The multigeneration effect of DES provides a model to test the mechanism of transmission of cancer risk from one generation to the next
1989 Study Abstract
Animals of several species exposed perinatally to diethylstilboestrol (DES) have been evaluated for anomalies and tumours.
In male offspring, anomalies of the testis and epididymis have been reported, but evidence for tumours has been very limited.
Many anomalies and tumours have been recorded in female offspring, and some of these duplicate the anomalies and tumours reported in DES-exposed women, whereas others either have not yet been discovered or else do not occur in the human species.
A variety of abnormal physiological responses has been identified in animals exposed perinatally to DES. There were altered levels of hormones and receptors; responses to postnatal injection of hormones were often modified; and an increased susceptibility to other carcinogens has been established.
Several mechanisms have been postulated to explain tumour production later in life after perinatal exposure to DES. Deficiencies in immune function indicate a mechanism of impaired immune surveillance. The presence of DES and its metabolites in the fetus and neonate raise the issue of somatic mutation. Evidence for sister chromatid exchange, cell transformation in tissue culture and other toxic effects on chromosomes support the somatic mutation hypothesis. A third hypothesis is involvement of abnormal differentiation of the hypothalamus. Structural, hormonal and behavioural changes support this idea.
Possible additional problems in humans after exposure to DES, on the basis of animal model studies, are increased tumour frequency with ageing and transmission of cancer risk to the third generation. The multigeneration effect of DES provides a model to test the mechanism of transmission of cancer risk from one generation to the next.
The outcome of such experiments could have considerable impact on the understanding of the association between DES and cancer specifically and transplacental cancer generally.
- Animal models of prenatal exposure to diethylstilboestrol, IARC scientific publications PMID: 2680952, 1989.
- 3D Animal Models featured image credit psionicgames.
DES DIETHYLSTILBESTROL RESOURCES
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.
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.
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).
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.
- Family History is Underestimated in Children with Isolated Hypospadias: A French Multicenter Report of 88 Families, The Journal of Urology, Researchgate, DOI: 10.1016/j.juro.2018.04.072, April 2018.
- Image credit the anand stuff.
DES DIETHYLSTILBESTROL RESOURCES
A Long and Crucial History, IntechOpen, 2018
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.
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.
- 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
Diethylstilbestrol (DES): also harms the third generation,
Prescrire international, 2016 Dec
Diethylstilbestrol(DES) is a synthet- ic nonsteroidal oestrogen and endo- crine disruptor that was used in the 1950s-1970s to prevent spontaneous abortion, despite its lack of proven efficacy.
Millions of women worldwide took DES during pregnancy. In France, between 1951 and 1981, about 160 000 children were exposed to DES during the first trimester of their intrauterine life, and in some cases almost throughout the entire pregnancy. They are referred to as “DES daughters” and “DES sons“. In 2010, in France, about 25 000 DES daughters were aged 33 to 40 years: pregnancies among these women are foreseeable until about 2020.
In utero exposure to DES can have harmful effects. In particular, DES daughters have an increased risk of cancer and structural abnormalities of the uterus that can adversely affect their pregnancies.
What are the consequences of taking DES during pregnancy for the third generation, i.e. the children of DES children? To answer this question, we reviewed the available data in mid- 2016 using the standard Prescrire methodology.
According to a retrospective study conducted in France by Réseau DES France, published in 2016, which included 4409 DES grandchildren (2228 girls and 2181 boys) and about 6000 controls, about one-quarter of DES grandchildren are born prematurely. Preterm delivery exposes neonates to serious neonatal complications, including
- neurosensory disorders,
- and increased neonatal mortality.
The more premature the baby, the greater the risk of complications. In the Réseau DES France study, cerebral palsy was more frequent in the DES grandchildren group: 59/10 000, versus 6/10 000 in the control group.
A study conducted in the United States in about 4500 DES daughters found that preterm delivery occurred at a frequency of about 26%, much higher than that reported in controls. Neonatal mortality was 8 times higher among DES grandchildren, and the risk of stillbirth was twice as high. Other smaller studies have also shown an increased risk of preterm birth.
A cohort study conducted in about 5000 DES grandchildren found that the risk of malformations of any type was higher than in the unexposed control group.
Epidemiological studies, conducted in several countries, found an increased frequency of hypospadias in DES grandsons. The relative risk was about 5 in the largest study. Other, less robust studies found no statistically significant difference.
Several studies in several countries have shown a twofold increase in the risk of oesophageal atresia or tracheo- oesophageal fistula in DES grandchildren.
The data on congenital heart defects or musculoskeletal malformations are limited and uninformative.
Epidemiological studies have not identified a significant increase in the risk of gynaecological anomalies or cancers in DES granddaughters.
Limited data are available on the risk of malformations in the children of DES sons. The data obtained in rodents exposed to DES (and other endocrine disruptors) make it entirely plausible that in utero exposure to DES, in humans too, provokes epigenetic effects that are passed on to future generations not directly exposed to DES.
In practice, these data should be discussed with DES daughters, their partners and healthcare teams so that appropriate monitoring, clinical management and follow-up can be arranged for both mother and baby. The harms of taking DES during pregnancy last for decades and affect future generations.
- Diethylstilbestrol (DES): also harms the third generation, Prescrire international, NCBI PubMed PMID: 30758926, 2016 Dec 25.
- Image credit Rod Long.
DES DIETHYLSTILBESTROL RESOURCES
Current perspective of diethylstilbestrol (DES) exposure in mothers and offspring
- Diethylstilbestrol (DES) is a synthetic, non-steroidal estrogen of the stilbestrol group acting as an endocrine disruptor.
- Adverse pregnancy outcomes, infertility, cancer, and early menopause have been identified in women exposed to DES, their offspring, and subsequent generations.
- DES is one of the major disasters in medicine and it is mandatory to tackle and promote programs of DES-related cancer prevention.
2017 Review Abstracts
Diethylstilbestrol (DES) was an orally active estrogen prescribed to the pregnant women to prevent miscarriages. DES is known as a ‘biological time bomb’ and long-term effects of DES have been recorded in the mothers exposed to DES and their offspring (DES-daughters and DES-sons). Adverse pregnancy outcomes, infertility, cancer, and early menopause have been discovered in women exposed to DES, and some events occur in their offspring and subsequent generations. An increased risk of breast cancer is not limited to the DES-exposed daughters.
We were told that it “could take over 50 years” to detect the effects of DES exposure in future generations, due to the length of time required for diseases to manifest. It is predicted that cross-generational responses to the exposure of DES are possible due to epigenetic changes in the DNA.
The studies on the cohort of grandchildren (grandsons and granddaughters) whose mothers were exposed to DES prenatally (i.e., grandchildren had no direct DES exposure) are limited as they have just reached the age when relevant health problems could be studied.
It seems that the DES 3rd generation has also an increased risk for cancer. The epigenetic effects of DES could be manifested in this generation. DES could affect daughters of the exposed mothers as their oocytes might be developing at the critical stage, but transgenerational effect of DES, i.e., children of sons or daughters of DES mothers, may have an epigenetic basis.
In opposition to developmental epigenetics transgenerational epigenetics implies an absence of resetting of epigenetic states between generations. In fact,the exposure to endocrine disruptor chemical compounds is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues as recently observed by Ho et al.
In a multi-center study, an increase of congenital disabilities in the sons and daughters of the 3rd generation was observed. Currently, there are no large human studies that show adverse events of DES for granddaughters. Kaufman et al. conducted a small cohort study on 28 DES granddaughters and found neither CCA nor abnormalities of the lower genital tract. On the other hand, granddaughters may have more menstrual problems and higher infertility rate compared to non-exposed granddaughters. Direct maternal DES exposure during pregnancy can cause not only an alteration to the reproductive capacity of the woman, but also this alteration may be passed on to next generation, a phenomenon that is called ‘DES granddaughter effect’ and DES granddaughters may also harbor a higher risk of ovarian cancer rather than breast cancer. In DES grandsons, the incidence of hypospadia is 20 times higher than unexposed grandsons, but the risk of developing such anomaly seems to be low.
There is an urgent need to find ways to stop the inheritance cycle of DES and prevent adverse effects of DES in the future generations. The present article reviews the health implications of DES exposure and screening exams currently recommended to DES daughters and their offspring.
- Current perspective of diethylstilbestrol (DES) exposure in mothers
and offspring, NCBI PubMed, 28461243, August 2017.
- Image credit gstatic.
DES DIETHYLSTILBESTROL RESOURCES
- Diethylstilbestrol DES studies listed by topics and date of publication.