**3. 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 [9] 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 [10] 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 [11], 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 [1]. 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. [12], 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 [13]. The most recent Nurses' Health Study was conducted by O'Reilly et al., 2010 [14], 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 [10] 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 [15, 16], anxiety [1, 11, 17], schizophrenia-like behavior [18, 19], anorexia, and bulimia nervosa [20]. All these observations were synthesized by Pillard et al. [16] and Giusti [21]. On 1987, Katz et al. [18] 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. [16] 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. [14].

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

**11**

**Figure 2.**

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

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" [13] in which we detailed somatic and psychiatric disorders, associated or not, in the exposed

We have conducted a more recent analysis (2016) [22, 23] based on 1182

with the general population shows a dramatic increase (**Table 1**).

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

We were also interested by the effects on the brain of synthetic progestins on in utero-exposed children of our cohort (**Figures 3** and **4**). Currently, there is no research on these effects of in utero exposure of children to progestins given alone during pregnancy. Our recent observations [24, 25] were collected from 1200 families of the HHORAGES cohort, that is, 1934 children using always the same detailed questionnaire [13]. 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 (**Figure 3**). 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

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

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

*Total number of psychological/psychiatric disorders among the 982 (1002 total with 20 stillborns) DES-exposed* 

*and post-DES unexposed children. First-born children (intrafamilial control) unexposed are not ill.*

*DOI: http://dx.doi.org/10.5772/intechopen.80969*

pregnancies from 529 mothers (**Figure 2**).

children of our cohort.

indication in 1970.

did not present any disorder.

#### *Evidence for Link Between Mental Disorders and in Utero Exposure to Synthetic Hormones… DOI: http://dx.doi.org/10.5772/intechopen.80969*

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" [13] 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) [22, 23] based on 1182 pregnancies from 529 mothers (**Figure 2**).

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 (**Table 1**).

We were also interested by the effects on the brain of synthetic progestins on in utero-exposed children of our cohort (**Figures 3** and **4**). Currently, there is no research on these effects of in utero exposure of children to progestins given alone during pregnancy. Our recent observations [24, 25] were collected from 1200 families of the HHORAGES cohort, that is, 1934 children using always the same detailed questionnaire [13]. 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 (**Figure 3**). 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

**Figure 2.**

*Total number of psychological/psychiatric disorders among the 982 (1002 total with 20 stillborns) DES-exposed and post-DES unexposed children. First-born children (intrafamilial control) unexposed are not ill.*

*Psychopathology - An International and Interdisciplinary Perspective*

**humans after in utero exposure to DES/EE**

humans [8].

be upset by estrogens or other endocrine disruptors (EDs). Later and unequivocally, Newbold demonstrated the validity of the rodent model transposed to

**3. Behavioral disorders, psychoses, and depression demonstrated in** 

to a placebo [1]. 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. [12], 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 [13]. The most recent Nurses' Health Study was conducted by O'Reilly et al., 2010 [14], 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 [10] 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 [15, 16], anxiety [1, 11, 17], schizophrenia-like behavior [18, 19], anorexia, and bulimia nervosa [20]. All these observations were synthesized by Pillard et al. [16] and Giusti [21]. On 1987, Katz et al. [18] 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. [16] 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

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

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 [9] 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 [10] 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 [11], 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

**10**

by O'Reilly et al. [14].


#### **Table 1.**

*Prevalences of the psychological and/or psychiatric disorders in in utero-exposed children to DES and/or EE and comparison with the general population [23].*

#### **Figure 3.**

*Total HHORAGES cohort of 1934 children in which 3 groups can be observed.*

#### **Figure 4.**

*Division of the 115 children of the 46 "progestins" families: first-born unexposed, in utero exposed, and post exposed, that is, born after a previous exposed pregnancy.*

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

**13**

the Krebs' team.

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

reconstruction (one grandchild), and sexual ambiguity (one) and for girls (two)

*Comparative diagrams representing psychiatric disorder cases presented, respectively, by girls (a, c) and boys (b, d) exposed either to synthetic estrogens diethylstilbestrol (DES) and ethinyl estradiol (EE) (c, d) or to* 

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

As shown in **Figure 5**, 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

*DOI: http://dx.doi.org/10.5772/intechopen.80969*

hormonal sterility (two).

*synthetic progestins (a, b).*

**4. An epigenetic mechanism**

estrogens.

**Figure 5.**

*Evidence for Link Between Mental Disorders and in Utero Exposure to Synthetic Hormones… DOI: http://dx.doi.org/10.5772/intechopen.80969*

#### **Figure 5.**

*Psychopathology - An International and Interdisciplinary Perspective*

First-born pre-DES

**Group 1 Group 2 Group 3**

n = 180 n = 740-20 n = 262

Behavioral disorders 0% n = 109 (15.1%) n = 1 (0.4%) 3% Schizophrenia 0% n = 165 (22.9%) n = 6 (2.3%) 1% Eating disorders 0% n = 81 (11.3%) n = 2 (0.8%) 1.6%

Bipolar disorders, anxiety 0% n = 248 (34.4%) n = 9 (3.4%) 6.3% Suicide attempts 0% n = 612 (85%) n = 30 (11.5%) 0.3% Death 0% n = 32 (4.4%) n = 1 (0.4%) 0.02%

*Prevalences of the psychological and/or psychiatric disorders in in utero-exposed children to DES and/or EE* 

DES-exposed Post-DES General

population

*Total HHORAGES cohort of 1934 children in which 3 groups can be observed.*

*exposed, that is, born after a previous exposed pregnancy.*

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

*Division of the 115 children of the 46 "progestins" families: first-born unexposed, in utero exposed, and post* 

**12**

**Figure 4.**

**Figure 3.**

Depression

*and comparison with the general population [23].*

**Table 1.**

*Comparative diagrams representing psychiatric disorder cases presented, respectively, by girls (a, c) and boys (b, d) exposed either to synthetic estrogens diethylstilbestrol (DES) and ethinyl estradiol (EE) (c, d) or to synthetic progestins (a, b).*

reconstruction (one grandchild), and sexual ambiguity (one) and for girls (two) hormonal sterility (two).

As shown in **Figure 5**, 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.

#### **4. 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 [10] 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 [26, 27] 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 [28], 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 [29, 30]. 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 [31]. The ADAM TS9 gene is implicated in the control of organ shape, especially in the development and function of the uterus and reproductive organs [32] which are often abnormal after in utero DES exposure as well as in the control of the CNS development [33] and in several kinds of cancers [34].

**15**

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

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 [24]. 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 [35]. Negative mood symptoms have been reported by Andreen et al. [36] 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 [37], 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 [38]. 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 [39]. 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 [40]. Although some progestins have been banned from the market, others are not: our data demonstrated that caution should

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) (**Figure 2** and **Table 1**) 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

*DOI: http://dx.doi.org/10.5772/intechopen.80969*

**5. Discussion and conclusion**

*Evidence for Link Between Mental Disorders and in Utero Exposure to Synthetic Hormones… DOI: http://dx.doi.org/10.5772/intechopen.80969*
