In utero exposure to both high and low dose diethylstilbestrol disrupts mouse genital tubercle development

image of female-mice

The resulting hypospadias phenotypes in male and female mice prenatally exposed to DES

2018 Study Abstract

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development (DSDs).

Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra.

This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol (DES), at a high, clinically relevant dose, and a low, previously untested dose, administered via water.

  • The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency.
  • Females exhibited hypospadias and urethral-vaginal fistula.

These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Sources

  • In utero exposure to both high and low dose diethylstilbestrol disrupts mouse genital tubercle development, NCBI PubMed PMID: 29931162, 2018 Jun 21.
  • Featured image credit Steph Hillier.
DES DIETHYLSTILBESTROL RESOURCES

Mouse hypospadias: A critical examination and definition

The current study describes the types of adult penile malformations induced developmentally by DES (hypospadias) in cohorts of mice treated with DES over the age range of E12 to P20, but examined in adulthood when enduring malformations are present

2016 Study Abstract

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation.

The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12 to P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0 to P10 period. In “estrogen mutant mice” (NERKI, βERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process.

Sources

  • Full text (free access) : Mouse hypospadias: A critical examination and definition, NCBI PubMed PMC5052099, 2016 Apr 5.
  • Featured image credit Aaron Logan.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias can be transmitted to the DES-exposed third-generation

Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains

2014 Study Abstract

Potential trans-generational influence of diethylstilbestrol (DES) exposure emerged with reports of effects in grandchildren of DES-treated pregnant women and of reproductive tract tumors in offspring of mice exposed in utero to DES.

Accordingly, we examined the trans-generational influence of DES on development of external genitalia (ExG) and compared effects of in utero DES exposure in CD-1 and C57BL/6 mice injected with oil or DES every other day from gestational days 12 to 18. Mice were examined at birth, and on 5-120 days postnatal to evaluate ExG malformations. Of 23 adult (>60 days) prenatally DES-exposed males, features indicative of urethral meatal hypospadias (see text for definitions) ranged from 18% to 100% in prenatally DES-exposed CD-1 males and 31% to 100% in prenatally DES-exposed C57BL/6 males. Thus, the strains differed only slightly in the incidence of male urethral hypospadias. Ninety-one percent of DES-exposed CD-1 females and 100% of DES-exposed C57BL/6 females had urethral-vaginal fistula. All DES-exposed CD-1 and C57BL/6 females lacked an os clitoris. None of the prenatally oil-treated CD-1 and C57BL/6 male and female mice had ExG malformations. For the second-generation study, 10 adult CD-1 males and females, from oil- and DES-exposed groups, respectively, were paired with untreated CD-1 mice for 30 days, and their offspring evaluated for ExG malformations. None of the F1 DES-treated females were fertile. Nine of 10 prenatally DES-exposed CD-1 males sired offspring with untreated females, producing 55 male and 42 female pups. Of the F2 DES-lineage adult males, 20% had exposed urethral flaps, a criterion of urethral meatal hypospadias. Five of 42 (11.9%) F2 DES lineage females had urethral-vaginal fistula. In contrast, all F2 oil-lineage males and all oil-lineage females were normal.

Thus, prenatal DES exposure induces malformations of ExG in both sexes and strains of mice, and certain malformations are transmitted to the second-generation.

Sources

  • Full text (free access) : Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains, NCBI PubMed PMC4254634, 2014 Oct.
  • Featured image credit figure/F1.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias in sons of women exposed to Diethylstilbestrol in utero

Increased risk of hypospadias in the DES grandsons, 2005

Abstract

BACKGROUND
Diethylstilbestrol (DES) is a synthetic estrogen that was widely prescribed to pregnant women before 1971. DES increases the risk of breast cancer in women who took the drug and the risk of reproductive tract abnormalities in their offspring. Dutch investigators have reported a 20-fold increase in risk of hypospadias among sons of women who were exposed to DES in utero. We assessed this relation in data from an ongoing study of DES-exposed persons.

METHODS
Several U.S. cohorts of women with documented exposure in utero to DES have been followed by mailed questionnaires since the 1970s. Comparison subjects are unexposed women of the same ages. In 1997, participants were asked about congenital abnormalities in their children. We calculated prevalence odds ratios for the risk of hypospadias in sons of exposed mothers relative to sons of unexposed mothers using generalized estimating equations to adjust for multiple sons per mother and controlling for maternal age at the son’s birth.

RESULTS
We obtained data from 3916 exposed and 1746 unexposed women. These women reported a total of 13 liveborn sons with hypospadias (10 exposed, 3 unexposed). The prevalence odds ratio for risk of hypospadias among the exposed was 1.7 (95% confidence interval = 0.4-6.8).

CONCLUSIONS
Our findings do not support a greatly increased risk of hypospadias among the sons of women exposed to DES in utero, as has been previously reported.

Sources

  • Hypospadias in sons of women exposed to diethylstilbestrol in utero, NCBI PubMed PMID: 15951681, 2005 Jul.
  • Featured image credit Bridget Coila.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias in sons of women exposed to DES in utero

These 2005 findings confirm an increased risk of hypospadias in the sons of women exposed in utero to DES and the transgenerational effect of DES

Abstract

Transgenerational effects of diethylstilbestrol (DES) have been reported in animals and humans. Alerted by two case reports, Klip H et al conducted a cohort study of all sons of a Dutch cohort of 16 284 women with a diagnosis of fertility problems and obtained a 67% response rate; their findings suggest an increased risk of hypospadias in the sons of women exposed in utero to DES. The mothers of 205 boys reported DES exposure in utero, and four of these boys were reported to have hypospadias. This defect was reported in only 8 of the remaining 8729 sons (prevalence ratio 21.3 (95% confidence interval (CI) 6.5–70.1)).

In a retrospective study, we analysed 32 406 computerized obstetrical and pediatric files at Port-Royal Maternity Center, covering births from 1 January 1993 to 31 December 2002. We compared the prevalence rate of hypospadias among 17 633 boys of mothers with and without in utero DES exposure.

The mothers of 240 boys had reported DES exposure in utero. Three (1.23%) were reported to have hypospadias. Obstetric records of the remaining 17 393 children reported hypospadias for only 44(0.5%◦) (prevalence ratio 4.99 (95% CI 1.2–16.8, p = 0.02)). All cases of hypospadias were medically confirmed by the pediatric files.

Our findings confirm an increased risk of hypospadias in the sons of women exposed in utero to DES and the transgenerational effect of DES.

The lower prevalence ratio in our study (4.99) than in the Dutch cohort (21.3) is probably due to the difference in the underlying populations: the Dutch cohort was selected for the criteria of infertility and is thus necessarily different from our general population.

Klip’s data came from questionnaires addressed to women who had consulted for infertility and been previously enrolled in a cohort. The analysis was based on a 67% response rate. We note that the 95% CI of the relative risk (RR) in the Klip study (6.5–70.1) is compatible with the RR in the present study. On the basis of both the studies, the true RR may range between 6.5 (lower range of Dutch study) and 16.8 (upper range of our study) and, given the possible bias of the Dutch study, the true RR may well be lower than 16.8.

Our study examined a large continuous series of woman who gave birth at Port Royal. During the study period, women were systematically asked about their own in utero exposure to DES. All of the obstetrical and pediatric records of our population were analysed.

The prevalence of hypospadias in our control series was 2.5 per 1000, which corresponds to the prevalence in the general French population −2.89 per 1000 male newborns. The prevalence in the control series for Klip’s population was only 0.9 per 1000.

The importance of understanding the mechanism of hypospadias warrants additional studies. Van Tongeren et al. point out that, especially in urban areas, mothers’ occupations (such as hairdressing and working in beauty salons) can increase exposure to potential endocrinedisrupting chemicals Van Tongeren et al (2002). Our study does not enable us to determine whether the mothers of sons with hypospadias were exposed to higher levels of or at higher rates to such chemicals.

Sources

  • Hypospadias in sons of women exposed to diethylstilbestrol in utero, NCBI PubMed PMID: 15906411, 2005 May.
  • Featured image credit Piron Guillaume.
DES DIETHYLSTILBESTROL RESOURCES

DES-induced persistent second-generation developmental abnormalities of the external genitalia

Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains

2014 Study Abstract

Potential trans-generational influence of diethylstilbestrol (DES) exposure emerged with reports of effects in grandchildren of DES-treated pregnant women and of reproductive tract tumors in offspring of mice exposed in utero to DES.

Accordingly, we examined the trans-generational influence of DES on development of external genitalia (ExG) and compared effects of in utero DES exposure in CD-1 and C57BL/6 mice injected with oil or DES every other day from gestational days 12 to 18. Mice were examined at birth, and on 5-120 days postnatal to evaluate ExG malformations. Of 23 adult (>60 days) prenatally DES-exposed males, features indicative of urethral meatal hypospadias (see text for definitions) ranged from 18% to 100% in prenatally DES-exposed CD-1 males and 31% to 100% in prenatally DES-exposed C57BL/6 males. Thus, the strains differed only slightly in the incidence of male urethral hypospadias. Ninety-one percent of DES-exposed CD-1 females and 100% of DES-exposed C57BL/6 females had urethral-vaginal fistula. All DES-exposed CD-1 and C57BL/6 females lacked an os clitoris. None of the prenatally oil-treated CD-1 and C57BL/6 male and female mice had ExG malformations. For the second-generation study, 10 adult CD-1 males and females, from oil- and DES-exposed groups, respectively, were paired with untreated CD-1 mice for 30 days, and their offspring evaluated for ExG malformations. None of the F1 DES-treated females were fertile. Nine of 10 prenatally DES-exposed CD-1 males sired offspring with untreated females, producing 55 male and 42 female pups. Of the F2 DES-lineage adult males, 20% had exposed urethral flaps, a criterion of urethral meatal hypospadias. Five of 42 (11.9%) F2 DES lineage females had urethral-vaginal fistula. In contrast, all F2 oil-lineage males and all oil-lineage females were normal.

Thus, prenatal DES exposure induces malformations of ExG in both sexes and strains of mice, and certain malformations are transmitted to the second-generation.

Sources

  • Full study (free access) : Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains, Chemosphere, NCBI PubMed PMC4254634, 2005 Nov.
  • A scanning electron micrograph (A) and a photo (B) of the penis of an adult prenatally oil-treated (A) mouse and a prenatally DES-treated mouse (B) featured image credit PMC4254634/figure/F1.
DES DIETHYLSTILBESTROL RESOURCES

DES Grandsons Hypospadias ; True Transgenerational Effect ?

image of Grandson

Hypospadias in sons of women exposed to diethylstilbestrol: a true trans-generational effect?

2005 Study Abstract

In May 2005, Pons et al. reported on an increased risk of hypospadias in male children of women exposed to diethylstilbestrol (DES) in utero. The authors have retrospectively reviewed the electronic files from 17 633 deliveries of male neonates in a 10-year period. The mothers of 240 male neonates had reported in utero DES exposure, three of whom (1.23%) presented with hypospadias vs 44/17 393 (0.5%) in the remaining male neonates (from non-DES-exposed mothers). The authors conclude that there is an increased risk of hypospadias in the male children of women exposed in utero to DES due to the transgenerational effects of DES. Although these results apparently compare favourably with the initial Dutch cohort, we would like to address the authors with our concerns regarding the interpretation of these additional data.

In utero DES exposure has been associated not only with an increased risk of preterm labour but also with an increased risk of intra-uterine growth retardation (IUGR). In turn, an increased risk of cryptorchidism and hypospadias has been associated with decreased birth weight. One might expect a higher rate of IUGR in the subgroup of neonates of women exposed in utero to DES compared with the control group in the Parisian cohort, as previously observed in the Dutch one. This information, essential to the interpretation of the data, may avoid causing the patients undue concern about hypothetical transgenerational adverse effects of DES (i.e. genetic or epigenetic changes in either germ or somatic cells).

Sources

  • Hypospadias in sons of women exposed to ditheylstilbestrol: a true trans-generational effect?, Prenatal diagnosis, NCBI PubMed PMID: 16302166, 2005 Nov.
  • Featured image credit Johan Mouchet.
DES DIETHYLSTILBESTROL RESOURCES

Birth Defects in DES Grandsons

Birth defects in children of men exposed in utero to diethylstilbestrol (DES)

2018 Study Abstract

OBJECTIVE
Prenatal exposure to diethylstilbestrol (DES) is associated with adverse effects, including genital anomalies and cancers in men and women. Animal studies showed birth defects and tumors in the offspring of mice prenatally exposed to DES. In humans, birth defects, such as hypospadias were observed in children of prenatally exposed women. The aim of this research was to assess the birth defects in children of prenatally exposed men.

METHODS
In a retrospective study conceived by a patients’ association (Réseau DES France), the reports of men prenatally exposed to DES on adverse health effects in their children were compared with those of unexposed controls and general population.

RESULTS
An increased incidence of two genital anomalies,

  1. cryptorchidism (OR=5.72; 95% CI 1.51-21.71),
  2. and hypoplasia of the penis (OR=22.92; 95% CI 3.81-137.90),

was observed in the 209 sons of prenatally exposed men compared with controls, but hypospadias incidence was not increased in comparison with either the controls or the general population. No increase of genital anomalies was observed in daughters.

CONCLUSION
With caution due to the methods and to the small numbers of defects observed, this work suggests an increased incidence of two male genital tract defects in sons of men prenatally exposed to DES. This transgenerational effect, already observed in animals and in the offspring of women prenatally exposed to DES, could be the result of epigenetic changes transmitted to the subsequent generation through men.

Sources

  • Birth defects in children of men exposed in utero to diethylstilbestrol (DES), Therapie, NCBI PubMed PMID: 29609831, 2018 Mar 3.
  • Featured image credit Danielle MacInnes.
DES DIETHYLSTILBESTROL RESOURCES

Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”

image pf hypospadias in human fetal penises

Defining a DES “programming window”

2016 Study Abstract

Hypospadias, an abnormality affecting the penile urethra, is one of the most prevalent congenital malformations afflicting human males. The morphology of hypospadias is markedly different in humans versus mice reflecting substantial differences in penile development in humans and mice. Estrogens such as diethylstilbestrol (DES) elicit mouse penile malformations, but the types of penile abnormalities differ depending on whether DES treatment is prenatal or neonatal.

A thorough investigation of the effects of DES over a wide age range of treatment may

  • elucidate the morphogenetic mechanisms involved in generating abnormal penile morphology and hypospadias
  • and reveal those penile elements more (or less) sensitive on a temporal basis to developmental exposure to DES.

Such an approach may also explain why certain effects of DES elicited and expressed during development resolve to normality in adulthood.

To define the actual “window of susceptibility” to the adverse effects of DES, pregnant mice and their neonatal pups were injected subcutaneously with 200ng/gbw DES every other day

  • from embryonic day 12 to 18 (DES E12-E18),
  • postnatal day 0 to 10 (DES P0-P10),
  • embryonic day 12 to postnatal day 10 (DES E12 to P10),
  • postnatal day 5 to 15 (DES P5 to P15),
  • and postnatal day 10 to 20 (DES P10 to P20).

Aged-matched controls received sesame oil vehicle. After euthanasia at 10, 15, 20 and 60 days, penises were analyzed by gross morphology, histology and morphometry.

Penises of all 5 groups of DES-treated mice were reduced in size, which was confirmed by morphometric analysis of internal penile structures, and are presumably mediated via signaling through estrogen receptors alpha and/or beta (ERα and ERβ), which have been previously detected in all of the structures affected by DES.

The most profound effects were seen in the DES E12-P10, DES P0-P10, and DES P5-P15 groups, thus defining a DES “programming window”.

For all parameters, DES treatment from P10-P20 showed the most mild of effects.

Adverse effects of DES on the MUMP cartilage and erectile bodies observed shortly after the last DES injection reverted to normality in the DES P5-P15, but not in the E12-P10 and P0-P10 groups, in which MUMP cartilage and erectile body malformations persisted into adulthood, again emphasizing a “window of susceptibility” in the early neonatal period.

Sources

  • Full study (free access) : Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”, Differentiation, NCBI PubMed PMC4803596, 2016 Jan 20.
  • Scanning electron micrographs of human fetal penises at 7 and 10 weeks of gestation. In (A) note the prominent urethral groove. In (B) the edges of the urethral groove are fusing in the midline to form the urethra, but the distal urethral groove is still widely open. Featured image credit PMC4803596/figure/F3.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias in DES grandsons : a cohort study

image of grandsons

Hypospadias: a transgenerational effect of diethylstilbestrol ?

2002 Study Abstract

BACKGROUND
Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero.

METHODS
We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero.

FINDINGS
16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7).

INTERPRETATION
Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.

Sources

  • Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study, Lancet, NCBI PubMed PMID: 11943257, 2002 Mar 30.
  • Featured image Donna Borzyskowski.
DES DIETHYLSTILBESTROL RESOURCES