Early Female Transgender Identity after Prenatal Exposure to Diethylstilbestrol: Report from a French National Diethylstilbestrol (DES) Cohort.

Diagnostic of transsexualism and gender incongruence are terms to describe individuals whose self-identity does not match their sex assignment at birth. A transgender woman is an individual assigned male at birth (AMAB) on the basis of the external or internal genitalia who identifies and lives as a woman. In recent decades, a significant increase in the number of transgender people has been reported. Although, its etiology is unknown, biological, anatomical, genetic, environmental and cultural factors have been suggested to contribute to gender variation. In XY animals, it has been shown that environmental endocrine disruptors, through their anti-androgenic activity, induce a female identity. In this work, we described four XY individuals who were exposed in utero to the xenoestrogen diethylstilbesterol (DES) and were part of the French HHORAGES cohort. They all reported a female transgender identity starting from childhood and adolescence. This high prevalence of male to female transgenderism (1.58%) in our cohort of 253 DES sons suggests that exposure to chemicals with xenoestrogen activity during fetal life may affect the male sex identity and behavior.

Effects of prenatal exposure to synthetic sex hormones on neurodevelopment: a biological mechanism.

Since the middle of the 20th century, synthetic sex hormones (estrogens and progestins) have been administered to millions of pregnant or not women worldwide, mainly to avoid miscarriage or for comfort, although their mode of action and their effects on the mother and fetus were ignored. Despite the alerts and the description of somatic and psychiatric disorders in children exposed in utero, synthetic estrogens were prohibited for pregnant women only in the 1970s and 1980s, but some progestins are still authorized. In this review, we summarize the psychiatric disorders described in children exposed in utero to such hormones, focusing particularly on schizophrenia, bipolar disorders, severe depression, eating disorders, suicide and suicide attempts. Moreover, only in 2017 the mechanism of action of these xenohormones has started to be deciphered. Some studies showed that in the fetus exposed in utero, they alter the DNA methylation profile (mainly hypermethylation), and consequently the expression of genes implicated in neurodevelopment and in regulating the sexual organ morphogenesis and also of the promoter of estrogen receptors, located in the amygdala. These deleterious effects may be transmitted also to the next generations, thus affecting the children directly exposed and also the following generations.

Birth Outcomes in DES Children and Grandchildren: A Multigenerational National Cohort Study on Informative Families.

OBJECTIVE: Diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen belonging to the family of endocrine disrupting chemicals (EDCs), can cross the placenta and may cause permanent adverse health effects in the exposed mothers, their children (exposed in utero), and also their grandchildren through germline contribution to the zygote. This study evaluated pregnancy duration and birthweight (BW) variations in the children and grandchildren born before, during, and after maternal DES treatment in the same informative families, to rule out genetic, endocrine, and environmental factors. DESIGN AND SETTING: Nationwide retrospective observational study on 529 families of DES-treated women registered at the HHORAGES-France Association. The inclusion criteria were: (i) women with at least three pregnancies and three viable children among whom the first was not exposed in utero to DES, followed by one or more children with fetal exposure to DES, and then by one or more children born after DES treatment; (ii) women with at least one pre-DES or post-DES grandchild and one DES grandchild; (iii) confirmed data on total DES dose. Women with severe pathologies or whose illness status, habitat, lifestyle habits, profession, treatment changed between pregnancies, and all mothers who reported pregnancy-related problems, were excluded. RESULTS: In all, 74 women met all criteria. The preterm birth (PTB) rate was 2.7% in pre-DES, 14.9% in DES, and 10.8% in post-DES children (Cochran-Armitage test for trend, p = 0.0095). The mean BW was higher in DES than pre-DES full-term neonates (≥37 weeks of gestation) (p = 0.007). In grandchildren, BW was not different, whereas the PTB and low BW rates were slightly increased in children of DES women. CONCLUSIONS: These data within the same informative families show the DES impact on BW and PTB in DES and post-DES children and grandchildren. In particular, mean BW was higher in DES than pre-DES full-term neonates. This result may be in opposition to previous data from American cohorts, which reported lower BW in DES children, but is consistent with animal study. Our retrospective observational study highlights a multigenerational and likely transgenerational effect of this EDC in humans.

Diethylstilbestrol and autism.

It is acknowledged that diethylstilbestrol (DES), a synthetic diphenol with powerful estrogenic properties, causes structural anomalies of the reproductive tract and increases the risk of cancer and genital malformations in children and grandchildren of mothers treated during pregnancy. Conversely, data on DES effects on neurodevelopment and psychiatric disorders in in-utero exposed children and their descendants are rare, especially concerning Autism Spectrum Disorders (ASD). Recent studies presented in this review strengthen the hypothesis that in-utero exposure to DES and also other synthetic estrogens and progestogens, which all are endocrine disruptors, contributes to the pathogenesis of psychiatric disorders, especially ASD. A large epidemiological study in the USA in 2010 reported severe depression in in-utero exposed children (n=1,612), and a French cohort study (n=1,002 in-utero DES exposed children) in 2016 found mainly bipolar disorders, schizophrenia, major depression, suicide attempts, and suicide. Few publications described ASD in in-utero exposed children, mainly a Danish cohort study and a large Chinese epidemiological study. Molecular studies on endocrine disruptors demonstrated the transgenerational induction of diseases and DES epigenetic impact (DNA methylation changes) at two genes implicated in neurodevelopment (ZFP57 and ADAM TS9). We recently described in an informative family, somatic and psychiatric disorders in four generations, particularly ASD in boys of the third and fourth generation. These data show that the principle of precaution must be retained for the protection of future generations: women (pregnant or not) should be extremely vigilant about synthetic hormones.

Prenatal Exposure to Diethylstilbestrol and Multigenerational Psychiatric Disorders: An Informative Family.

BACKGROUND: Psychiatric disorders in children exposed in utero to diethylstilbestrol (DES) are still debated. We report here the impact of DES prescribed to suppress lactation on the children born after such treatment and their progeny, focusing particularly on psychiatric disorders. CASE PRESENTATION: We report here an informative family in which one or more psychiatric problems (e.g., bipolarity, suicide attempts and suicide, eating disorders) were detected in all children of second-generation (DES-exposed children; n = 9), but for II-2 who died at the age of 26 years due to rupture of a congenital brain aneurysm, and were associated with non-psychiatric disorders (particularly, endometriosis and hypospadias). In the third generation, 10 out of 19 DES-exposed grandchildren had psychiatric disorders (autism spectrum disorder, bipolar disorder, dyspraxia and learning disabilities, mood and behavioral disorders, and eating disorders), often associated with comorbidities. In the fourth generation (7 DES-exposed great-grandchildren, aged between 0 and 18 years), one child had dyspraxia and autism spectrum disorder. The first daughter of the second generation (not exposed to DES) and her children and grandchildren did not have any psychiatric symptoms or comorbidities. CONCLUSIONS: To our knowledge, the high prevalence of psychiatric disorders of various severities in two, and likely three generations, including DES-free pregnancies and DES-exposed pregnancies from the same family, has never been reported. This work strengthens the hypothesis that in utero exposure to DES contributes to the pathogenesis of psychiatric disorders. It also highlights a multigenerational, and possibly transgenerational, effect of DES in neurodevelopment and psychiatric disorders.

Multigenerational endometriosis : consequence of fetal exposure to diethylstilbestrol ?

BACKGROUND: Endometriosis, which affects 10-15 % of women of reproductive age, is an estrogen-driven condition influenced by environmental and genetic factors. Exposition to estrogen-like endocrine-disrupting chemicals (EDCs) has been reported to contribute to the fetal origin of this disease. CASE PRESENTATION: We report here an informative family in which all prenatally DES-exposed daughters and subsequent granddaughters presented endometriosis, whereas the unexposed first daughter and her progeny presented no gynecological disorders. Moreover, the only post-pubertal great-granddaughter, who presents chronic dysmenorrhea that remains resistant to conventional therapy, is at risk of developing endometriosis. The mother (I-2) was prescribed DES (30 mg/day for 3 months) to inhibit lactation after each delivery. CONCLUSIONS: Although a direct causal link between the grandmother's treatment with DES and the development of endometriosis in possibly three exposed generations remains speculative, this report strengthens the suspicion that fetal exposition to DES contributes to the pathogenesis of adult diseases, such as endometriosis. It also highlights a multigenerational and likely transgenerational effect of EDCs.

Experimental Evidence of 2,3,7,8-Tetrachlordibenzo-p-Dioxin (TCDD) Transgenerational Effects on Reproductive Health.

Previous studies have demonstrated that endocrine disruptors (EDs) can promote the transgenerational inheritance of disease susceptibility. Among the many existing EDs, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) affects reproductive health, including in humans, following direct occupational exposure or environmental disasters, for instance the Agent Orange sprayed during the Vietnam War. Conversely, few studies have focused on TCDD multigenerational and transgenerational effects on human reproductive health, despite the high amount of evidence in animal models of such effects on male and female reproductive health that mimic human reproductive system disorders. Importantly, these studies show that paternal ancestral TCDD exposure substantially contributes to pregnancy outcome and fetal health, although pregnancy outcome is considered tightly related to the woman's health. In this work, we conducted a systematic review of the literature and a knowledge synthesis in order (i) to describe the findings obtained in rodent models concerning TCDD transgenerational effects on reproductive health and (ii) to discuss the epigenetic molecular alterations that might be involved in this process. As ancestral toxicant exposure cannot be changed in humans, identifying the crucial reproductive functions that are negatively affected by such exposure may help clinicians to preserve male and female fertility and to avoid adverse pregnancy outcomes.

Protistology and Cell Biology at the Marine Arago Laboratory of Banyuls-sur-Mer (1961-2000): Personal Recollections.

The history of protistology and the introduction of modern methods of unicell observations is described in a large maritime laboratory over a period of forty years by the initiator of this new team. The development of this team and the doctoral theses developed there are described as well as the major discoveries made. The Arago Laboratory, which was then in 1960 a field laboratory mainly devoted to the collection of biological material, becomes a research laboratory specializing in the study of the major fundamental problems which govern life: the organization and expression of the genome, mitotic processes and their nuclear and cytoplasmic components, cell cycle and its regulation as well as molecular phylogeny. The biological models chosen were essentially the dinoflagellate protists in their great variety: autotrophs, heterotrophs, myxotrophs and able of proliferating at sea, thus disrupting their cell cycle. Coupled with the techniques of biochemistry and molecular biology which it was in its infancy, the most advanced observation methods used electron and confocal microscopy often after use of ultra-cold cryopreparations, necessary to preserve the antigenic sites and allow the highlighting new proteins. The dinoflagellate model was then abandoned in favor of unicellular micro-eukaryotes allowing the development of environmental genomics.

Diethylstilbestrol exposure during pregnancy with primary clear cell carcinoma of the cervix in an 8-year-old granddaughter: a multigenerational effect of endocrine disruptors?

To date, vaginal/cervical clear cell adenocarcinoma (CCAC) has not been reported in the granddaughters of women treated with diethylstilbestrol (DES) during pregnancy. We present an 8-year-old girl with a history of severe vaginal bleeding who was diagnosed with cervical CCAC. She underwent fertility-sparing surgery and radiotherapy. No sign of recurrence was detected throughout a 10-year follow-up. Her grandmother had received DES therapy during pregnancy with the patient's mother. Although no direct causal link is demonstrated, this case raises for the first time, the hypothesis of multigenerational effects of DES in girls and strongly suggests the need to follow the granddaughters of DES-treated women.

Adolescent ovarian thecoma presenting as progressive hyperandrogenism: case report and review of the literature.

Hyperandrogenism is frequent and under investigated in adolescent girls. A 15-year-6-month-old French girl presented with oligomenorrhea and slowly progressing virilization 2 years post-menarche. Medical history revealed prenatal pesticide exposure through maternal professional activity and recurrent premature thelarche. Severe hirsutism, mild facial acne and clitoromegaly were noted. Serum androgens (testosterone: 94 ng/dL, 4-androstenedione: 8.23 ng/mL) were high and non-classic 21-hydroxylase deficiency was excluded. Pelvic ultrasonography showed a left ovarian mass, confirmed by computed tomography scan. Tumor markers were negative. Laparoscopic surgery was performed. The pathological diagnosis was benign luteinized thecoma. Postoperatively, the menstrual cycle and serum androgens became normal and hirsutism slowly improved. Hyperandrogenism 2 years after menarche should be systematically investigated, even if slowly progressive, since it may be a symptom of a rare virilizing ovarian tumor, like thecoma.

Neurodevelopmental disorders in children exposed in utero to synthetic progestins: analysis from the national cohort of the Hhorages Association.

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: Group 1 (n = 18): firstborn unexposed children, Group 2 (n = 62): children exposed in utero to synthetic progestins, 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.

The Arago Laboratory of Banyuls and some of its Academicians.

Since its founding in 1881 by Henri de Lacaze-Duthiers (1821-1901), the Arago Laboratory of Banyuls has been one of the three marine stations of the University Pierre and Marie Curie-Paris 6. It is located in Banyuls (Banyuls-sur-Mer) in Northern Catalonia. The center hosts researchers and students from all over the world. Some became famous, including four Nobel Prize winners: André Lwoff (1965), Pierre-Gilles de Gennes (1991), Albert Fert (2007) and Jules Hoffmann (2011). This article focuses on five scientists closely related to the center. The first three are Henri de Lacaze-Duthiers (1821-1901), the founder; Édouard Chatton (1883-1947), eminent director of the center; and André Lwoff (1902-1994), who before being known for his work in bacterial genetics and virology was an outstanding protozoologist under the direction of Chatton. Lynn Margulis (1938-2011), a great friend of the Arago Laboratory and personal friend of the author, is also remembered. Finally, there is a mention of Walter J. Gehring (1939-2014), professor at the University of Basel, Switzerland. [Int Microbiol 19(4): 183-190 (2016)].

Association between fetal DES-exposure and psychiatric disorders in adolescence/adulthood: evidence from a French cohort of 1002 prenatally exposed children.

In utero diethylstilbestrol (DES) exposure has been demonstrated to be associated with somatic abnormalities in adult men and women. Conversely, the data are contradictory regarding the association with psychological or psychiatric disorders during adolescence and adulthood. This work was designed to determine whether prenatal exposure to DES affects brain development and whether it is associated with psychiatric disorders in male and female adolescents and young adults. HHORAGES Association, a national patient support group, has assembled a cohort of 1280 women who took DES during pregnancy. We obtained questionnaire responses from 529 families, corresponding to 1182 children divided into three groups: Group 1 (n = 180): firstborn children without DES treatment, Group 2 (n = 740): exposed children, and Group 3 (n = 262): children born after a previous pregnancy treated by DES. No psychiatric disorders were reported in Group 1. In Group 2, the incidence of disorders was drastically elevated (83.8%), and in Group 3, the incidence was still elevated (6.1%) compared with the general population. This work demonstrates that prenatal exposure to DES is associated with a high risk of psychiatric disorders in adolescence and adulthood.

Chromosomes of Protists: The crucible of evolution.

As early as 1925, the great protozoologist Edouard Chatton classified microorganisms into two categories, the prokaryotic and the eukaryotic microbes, based on light microscopical observation of their nuclear organization. Now, by means of transmission electron microscopy, we know that prokaryotic microbes are characterized by the absence of nuclear envelope surrounding the bacterial chromosome, which is more or less condensed and whose chromatin is deprived of histone proteins but presents specific basic proteins. Eukaryotic microbes, the protists, have nuclei surrounded by a nuclear envelope and have chromosomes more or less condensed, with chromatin-containing histone proteins organized into nucleosomes. The extraordinary diversity of mitotic systems presented by the 36 phyla of protists (according to Margulis et al., Handbook of Protoctista, 1990) is in contrast to the relative homogeneity of their chromosome structure and chromatin components. Dinoflagellates are the exception to this pattern. The phylum is composed of around 2000 species, and characterized by unique features including their nucleus (dinokaryon), dinomitosis, chromosome organization and chromatin composition. Although their DNA synthesis is typically eukaryotic, dinoflagellates are the only eukaryotes in which the chromatin, organized into quasi-permanently condensed chromosomes, is in some species devoid of histones and nucleosomes. In these cases, their chromatin contains specific DNA-binding basic proteins. The permanent compaction of their chromosomes throughout the cell cycle raises the question of the modalities of their division and their transcription. Successful in vitro reconstitution of nucleosomes using dinoflagellate DNA and heterologous corn histones raises questions about dinoflagellate evolution and phylogeny. [Int Microbiol 18(4):209-216 (2015)].

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

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

Methods for studying the nuclei and chromosomes of dinoflagellates.

Dinoflagellates are unicellular eukaryotic organisms whose nuclear structure, chromosome architecture, chromatin organization, DNA composition, and mitosis show original features. It has been necessary to adapt techniques and to create innovative methods for growing cells, isolating nuclei, and studies of their chromosomes by transmission electron microscope (TEM). Among these are innovative squash and whole-mount preparations for light and TEM observations of chromosome architecture and the spatial organization of nucleofilaments. Particular attention was given to adapt high-pressure freezing (fast-freeze fixation) techniques for the best preservation of delicate antigenic sites, and good immunodetection. The study of DNA replication with or without incorporation of bromodeoxyuridine (BrdU) was also refined to use confocal laser scanning microscopy. In this chapter, we describe methods that we have invented and/or improved from existing techniques in order to better understand this fragile chromosome architecture and the mechanisms intervening during mitosis and the cell cycle. These methods allowed us to detect specific DNA-binding proteins and the distribution of B-and Z-DNA in chromosomes during the cell cycle and mitosis, and to focus on the indissoluble link between chromosome structure and function.

Molecular characterization of iron-containing superoxide dismutases in the heterotrophic dinoflagellate Crypthecodinium cohnii.

Superoxide dismutases (SODs) are a family of antioxidant enzymes that catalyse the degradation of toxic superoxide radicals in obligate and facultative aerobic organisms. Here, we report the presence of a multi-copy gene family encoding SODs in the heterotrophic dinoflagellate Crypthecodinium cohnii. All the genes identified (sod1 to sod17) have been cloned and sequenced, and shown to encode potentially functional dimeric iron-containing SOD isozymes. Our data revealed a considerable molecular heterogeneity of this enzyme in C. cohnii at both genomic and transcriptional levels. The C. cohnii SOD1, overexpressed in Escherichia coli, was active and its structure obtained by homology modeling using X-ray crystal structures of homologues exhibited the typical fold of dimeric FeSODs. Phylogenetic studies including 110 other dimeric FeSODs and closely related cambialistic dimeric SOD sequences showed that the C. cohnii SODs form a monophyletic group and have all been acquired by the same event of horizontal gene transfer. It also revealed a dichotomy within the C. cohnii SOD sequences that could be explained by an ancestral sod gene duplication followed by subsequent gene duplications within each of the two groups. Enzyme assays of SOD activity indicated the presence of two FeSOD activities in C. cohnii cell lysate whereas MnSOD and Cu/ZnSOD were not detected. These activities contrasted with the SOD repertoire previously characterized in photosynthetic dinoflagellates. To explain these differences, a hypothetical evolutionary scenario is proposed that suggests gains and losses of sod genes in dinoflagellates.

Edouard Chatton (1883-1947) and the dinoflagellate protists: concepts and models.

Edouard Chatton contributed to our knowledge of single-celled protoctists, especially ciliates and dinoflagellates, free-living and/or symbiotic, in relation to the marine invertebrate animals in which they reside. More than the description of many new families, genera and species, and of their life cycles, he anticipated several major concepts of cell biology, including the fundamental difference between prokaryote and eukaryote protists, long time before the advent of electron microscopy. These concepts included: the reproductive ability of the kinetosome-centriole system; the homology of the kinetosome with the mitotic centriole of animal cells; and the different kinds of mitotic systems. Chatton trained more than thirty student collaborators, among them Andre Lwoff, who won the 1965 Nobel Prize in Physiology or Medicine. Later, the great cell biologist Hans Ris and I completed Chatton's light microscopy descriptions on syndinian mitosis dinoflagellate. We had at our disposal sophisticated electron microscopes as well as biochemical and molecular techniques and thus succeeded in corroborating the correct interpretation by Chatton of chromosome structure and mitotic cytology.

Evolution of plant-like crystalline storage polysaccharide in the protozoan parasite Toxoplasma gondii argues for a red alga ancestry.

Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates. Toxoplasma gondii displays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of the Toxoplasma gondii, green alga Chlamydomonas reinhardtii, and dinoflagellate Crypthecodinium cohnii storage polysaccharides. We show Toxoplasma gondii amylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that the Toxoplasma gondii amylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red alga Cyanidioschyzon merolae and, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan-water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes.