DES immunotoxicity

The comparative immunotoxicity of five selected compounds following developmental or adult exposure, 2006

Study Abstract

It is well established that human diseases associated with abnormal immune function, including some common infectious diseases and asthma, are considerably more prevalent at younger ages. Although not established absolutely, it is generally believed that development constitutes a period of increased immune system susceptibility to xenobiotics, since adverse effects may occur at lower doses and/or immunomodulation may be more persistent, thus increasing the relative risk of xenobiotic exposure to the immunologically immature organism.

To address this issue, a brief overview of immune maturation in humans is provided to demonstrate that functional immaturity alone predisposes the young to infection. Age-dependent differences in the immunotoxic effects of five diverse compounds, diethylstilbestrol (DES), diazepam (DZP), lead (Pb), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and tributyltin oxide (TBTO), which have undergone adult and developmental immunotoxicity testing in rodents, are then reviewed, as are human data when available. For all five chemicals, the developing immune system was found to be at greater risk than that of the adult, either because lower doses produced immunotoxicity, adverse effects were more persistent, or both.

Diethylstilbestrol immunotoxicity


Between 5 and 10 million pregnant women were given diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen, between 1938 and 1971 to prevent premature delivery or pregnancy loss. Its use was terminated when a rare form of reproductive system cancer was found in female offspring of DES-exposed mothers. Male and female reproductive systems malformations have been reported in children of treated women, as has anecdotal evidence of immune system dysfunction. DES was also used to increase weight gain in livestock, although this use is no longer permitted in most countries.

Effects on the Immune System in Humans

Both female and male children of DES-exposed mothers report a higher incidence of autoimmune diseases and asthma (Baird et al., 1996). In general, these diseases are considered to be the result of inappropriate immune system responses, or possible loss of homeostatic control, instead of immune system suppression.

Effects on the Immune System in Rodents

In utero exposure
Luster et al. (1978b) reported that a single injection of 0.1 mg DES/kg body weight on gestational day (GD) 16 did not affect the antibody response to the T-cell-dependent antigen, sheep red blood cells (SRBC), when evaluated in 7-wk-old male and female offspring of Swiss-Webster mice. The T-independent IgM response of female offspring to bacterial lipopolysaccharide (LPS) was suppressed by DES, but was similar to control responses when females were
immunized for a second time. In marked contrast, the male offspring response to LPS immunization was enhanced after both first and second immunizations, an effect attributed to the stimulating effect of estrogen on the antibody response to LPS. Delayed-type hypersensitivity responses (DTH) were suppressed in female, but not in male, offspring, even though thymus weights and T-cell responses to polyclonal stimulation were suppressed in both genders (Luster et al., 1979). Further studies suggest that DES targets early precursors of T lymphocytes in the fetal liver, accounting for thymic atrophy and suppression of DTH (Holladay et al., 1993), but not for defects in T-independent responses to LPS of female offspring.

Neonatal exposure
Nonspecific T- and B-cell proliferation was reported to be suppressed in 6-wk-old female NMRI mice given 5 μg DES/d (roughly 2.2 mg DES/kg/d) over postnatal days (PND) 1–5 (Kalland et al., 1979); suppression was still evident at 17 mo of age (normal life span ~24 mo). It is noteworthy that neither estradiol nor corticosterone exposure over PND 1–5 produced long-term suppression, and that lymphocyte proliferation was comparable to control values at 6 wk of age in females exposed to DES over PND 6–10. Lower doses (approximately 4.4, 44, or 440 μg/kg/ d) had no effect on proliferative response. The 5-μg DES/d exposure regimen also decreased NK cell activity in 6- to 8-wk-old female inbred C57Bl/6 (75%↓) and BALB/c (53%↓) mice and in outbred NMRI (28%↓) mice (Kalland, 1980a). NMRI or AKR/J female mice, exposed to 5 μg DES/d over PND 1–5, were also more likely to develop tumors after low dose injection of a known carcinogen (Kalland & Forsberg, 1981). A subsequent paper (Kalland, 1984) reported that, on a per cell basis, NK cells from DES mice were as active as cells from the control group, but that exposure reduced the number of NK cell precursors in the bone marrow. In other words, NK cells from experimental animals were as efficient as those from controls, but a deficiency in NK cell precursors produced functional suppression of NK activity at the whole animal level. The same postnatal exposure regimen (Kalland, 1980a) reduced the T-lymphocyte-dependent antibody response to SRBC by ~60%, and the T-independent response to bacterial LPS by ~40% when examined in 16- to 18-wk-old NMRI mice. Suppression of the T-dependent response was reportedly due to a defect in T-helper cells. DTH responses were likewise suppressed in 6- and 9-mo-old NMRI females exposed to approximately 2.2 mg/kg/d over PND 1–5 (Kalland & Forsberg, 1978). Kalland (1980b) also reported a persistent (at least 6.5 mo postpartum) decrease in the proportion of T cells in the spleens of DES-exposed mice.

Adult exposure
Luster et al. (1980) reported suppression of the antibody response to SRBC or LPS, and the DTH to keyhole limpet hemocyanin (KLH), in adult female mice exposed to 2 or 8 mg DES/kg/d × 5 d. The DTH was decreased in mice dosed with DES after, but not before, sensitization with KLH, suggesting that the suppressive effects of DES on DTH were not persistent. Using the same exposure regimen, resistance to bacterial or parasite infection was decreased and tumor incidence in animals challenged with tumor cells was increased at ≥2 mg DES/kg/d (Dean et al., 1980). T-cell-mediated resistance to a nematode infection was suppressed by 5 d of exposure to 0.2 mg DES/kg/d if exposure began on the day of infection; if exposure commenced 5 d before or 3 or 8 d after infection, decreased resistance was only observed at the highest dose (8 mg/kg/d) (Luebke et al., 1984).

Mode(s) of Action

DES is a potent estrogen, and likely affects immune function via the estrogen receptor (ER). Evidence includes similar effects of known estrogens (17β-estradiol) on the immune system of adult and neonatal rodents, blockade of certain immunotoxic effects by pharmacologic antagonism of the ER (Luster et al., 1984), and antagonism of estrogen-mediated immune system effects in mice lacking ERα (Staples et al., 1999). DES appears to target precursor cells in the bone marrow (adults and neonates) and fetal liver (neonates), producing a long-lasting or perhaps permanent reduction in numbers of precursor cells. This defect explains a significant portion of long-lived immunosuppressive effects (e.g., Kalland’s 1984 paper on suppressed NK activity), although the effects of adult exposure also includes damage to the thymic epithelium (Luster et al., 1984). The underlying mechanism of long-term suppression following exposure of the developing immune system to DES is not known, but the default assumption is that a critical cell population is lost to developmental exposure; either this purported population is refractory to estrogen-mediated ablation in adults or repair and recovery mechanisms are present in adults that are lacking in the developing immune system.

Data Gaps

There has been no systematic evaluation of persistent DES-mediated immunosuppression in adult animals. Dose-response data are not available for many of the of the developmental exposure studies that revealed persistent effects.


In utero exposure to 0.1 mg DES/kg during the last trimester of pregnancy suppressed T-cell- and B-cell-mediated responses only in female offspring. The gender dependence of effects was remarkable in that T-independent responses in male offspring were enhanced, yet suppressed in females. Exposure during gestation produced effects that persisted into the equivalent of young adulthood. In neonates there appears to be a critical developmental window during PND 1–5, during which exposure to DES produces persistent immune system defects that last well into adulthood or persist for most of the normal life span of the mouse. These effects are among the most persistent reported for any chemical. In adults, immunosuppression occurs at doses similar to those that produce immunotoxicity in developing animals. However, the immune system-related endpoints that have been evaluated over time in exposed adult animals (bone marrow cellularity, thymus weights) recover relatively quickly (Forsberg, 1984). In adults, recovery may occur so quickly that suppression of cell function or resistance to infection may require ongoing exposure to maintain suppression.


Immunotoxicity has been reported at similar doses when exposure occurs during late gestation, early postpartum, or as adults. However, the distinguishing feature of developmental exposure to DES is the persistence of effects, some of which are still apparent in very old mice. In contrast, immune system-related endpoints that have been evaluated (bone marrow cellularity, thymus, weights) suggest that adults recover relatively quickly (Forsberg, 1984).


  • The comparative immunotoxicity of five selected compounds following developmental or adult exposure, Journal of toxicology and environmental health. Part B, Critical reviews, NCBI PubMed, PMID: 16393867, 2006 Jan-Feb.
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Prenatal DES exposure linked to Hashimotos thyroiditis

Drug exposure, pregnancy outcome and fetal and childhood development occurring in the offspring of mothers with systemic lupus erythematosus and other chronic autoimmune diseases

2006 Study Abstract

Most autoimmune diseases occur more commonly in females and many of these young women wish to become mothers. For pregnancy to proceed successfully immunomodulation and physiological changes preparing the reproductive system need to occur.

Pregnancy occurring in a chronically ill mother who requires medications in order to maintain her own health and who may have already incurred significant organ pathology gives rise to several problems and so four questions arise:

  1. What will be the effect of the pregnancy on the underlying disease?
  2. What will be the effect of the disease on the outcome of pregnancy?
  3. How to manage the disease, just prior to, throughout and immediately after the pregnancy?
  4. The long term fetal and childhood effects of maternal disease and its management.

This paper reviews the current literature pertaining to these questions in patients with systemic lupus erythematosus (SLE) and other chronic rheumatic and autoimmune diseases.


… “Other evidence suggests that the risk of autoimmune disease, particularly Hashimotos thyroiditis, is also increased in individuals exposed to DES in utero.” …


  • Drug exposure, pregnancy outcome and fetal and childhood development occurring in the offspring of mothers with systemic lupus erythematosus and other chronic autoimmune diseases, Lupus, NCBI PubMed, PMID: 17153855, 2006.
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DES exposure linked to laterality process disruption

Obstetrical complications and subsequent schizophrenia in adolescent and young adult offsprings: is there a relationship?, 2004


Schizophrenia is a psychiatric disease affecting around 1% of the population, the negative signs of which are correlated with inactivity of the prefrontal dorsolateral cortex, while an increased, more deeply localized, activity in the mesolimbic pathway may explain the positive signs. Several events occurring during pregnancy are likely to be involved in its genesis: hormonal supplementation by diethylstilbestrol, severe maternal denutrition, exposure to influenza virus, repeated psychological stress.

From multicentric studies and meta-analyses in the psychiatric literature, the risk of schizophrenia appears to be multiplied by two if pregnancy is complicated, mainly by diabetes, Rhesus incompatibility, bleeding, preeclampsia, premature rupture of membranes and preterm birth. When delivery is linked to an abnormal presentation or happens via a caesarean birth for acute foetal distress, the time when the first signs of psychosis appear seems to be earlier in adolescence or in early adulthood.

Cerebral imaging of schizophrenic patients shows ventriculomegaly and gray matter reduction, mainly in hippocampal volumes and in the dorsolateral prefrontal cortex. Similar alterations in the neuronal pathways have been experimentally reproduced in rats after repeated prenatal stress and perinatal hypoxia. A region on the distal portion of chromosome 1 has shown evidence for linkage to schizophrenia.

Exposure to diethylstilbestrol (DES) in the second trimester of pregnancy is likely to disrupt the laterality process, leading to an outstanding number of left-handed individuals, be they boys or girls. In the same way data from the literature suggest that people exposed to DES have a higher risk of presenting depressive signs. Nevertheless it may not be the only cause, since it is obvious that the gynecological troubles which required hormonal prescription may have affected the course of pregnancy. Katz et al. reported a higher risk of psychosis after the mother took DES about four cases: DES doses varied from 7 to 12.8 g, but two pregnant women out of four also received progestatives (total dose of 1.950 mg of medroxyprogesterone to 3.600 mg of progesterone).

Therefore, a two factor model seems to be able to explain the onset of schizophrenia in which obstetrical complications may interact with a genetic liability and in which the consequences of hypoxic events may lie on a continuum ranging from cerebral palsy in some children to subtle cognitive and behavioural disturbances in others.


  • Obstetrical complications and subsequent schizophrenia in adolescent and young adult offsprings: is there a relationship?, European journal of obstetrics, gynecology, and reproductive biology, NCBI PubMed, PMID: 15140504, 2004.
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Gestational exposure to DES linked to maternal antibodies concentrations reduction

Perinatal exposure to low doses of PCB 153 and PCB 126 affects maternal and neonatal immunity in goat kids, 2006 Jan


Pregnant does (10 goats/group) were dosed orally either with polychlorinated biphenyl (PCB) 153 (98 microg/kg body weight/d) or PCB 126 (ng/kg body weight/d) dissolved in corn oil or with corn oil only (control group) from gestation day (GD) 60 until delivery.

An additional group (n = 5) of pregnant does received the synthetic estrogen diethylstilbestrol (DES; 0.4 microg/kg body weight/d) by intramuscular injection using the same treatment schedule as for the PCB groups.

Blood samples for immune analysis were collected at wk 0, 1, 2, 4, 6, and 8 of age. The effects of perinatal PCB exposure on postnatal humoral immune responses were examined by assessing the levels of total immunoglobulin G (IgG) and immunoglobulins to specific microbes at wk 0, 1, 2, 4, 6, and 8 of age, and immune responses following immunization of kids at 2 wk of age.

PCB 153 exposure suppressed maternal and neonatal immunity, as demonstrated by reduced transfer of maternal IgG and specific antibodies to the environmental microbes Arcanobacterium pyogenes, Mannheimia haemolytica, and reovirus (REO-1). Furthermore, PCB 153 reduced the level of maternal antibodies to Mycobacterium avium paratuberculosis and equine influenza virus (EIV-1) in the newborn kids. The antibody response against EIV-1 was significantly higher in PCB 153-exposed kids 2 wk following immunization.

PCB 126 exposure reduced the levels of maternal antibodies to REO-1. In contrast, gestational exposure to PCB 126 increased the concentrations of maternal antibodies to tetanus toxoid. No differences from controls in plasma total IgG levels at birth or colostrum IgG concentrations were observed in the PCB 126-treated does. However, a significant reduction in IgG levels from GD 60 until delivery was found in this group.

Gestational exposure to DES reduced the concentrations of maternal antibodies against A. pyogenes, M. haemolytica, M. avium Paratuberculosis, and REO-1.

These results suggest that perinatal exposure to low doses of PCB 126 and PCB 153 affects the maternal immunity in kids. The difference in responses between PCB 126 and PCB 153 treatment groups may strengthen the hypothesis that PCBs mediate immunotoxic effects through both AhR-dependent and -independent mechanisms. The observation that the effects produced by PCB 153 resembled those produced by DES raises the question of whether this congener may modulate immunity by estrogenic mechanisms.


  • Perinatal exposure to low doses of PCB 153 and PCB 126 affects maternal and neonatal immunity in goat kids, Journal of toxicology and environmental health, NCBI PubMed, PMID: 16291567, 2006 Jan.
  • Featured image Terrie Schweitzer.

Prenatal DES treatment alters two hepatic enzymes

Maternal exposure to low doses of DES altered mRNA expression of hepatic microsomal enzymes in male rat offspring, 2012


Our previous studies demonstrated that prenatal diethylstilbestrol (DES) treatment disrupts steroidogenesis but induces high-level expression of androgen receptor (AR) mRNA to inhibit the disruption of spermatogenesis.

This study examined which prenatal DES treatment influenced hepatic microsomal enzymes, CYP3A1, CYP2B1/2, CYP2C11, UGT2B1 (UDP-glucuronosyltransferase 2B1), and IGF-1 (insulin-like growth factor-1), in male rat offspring.

DES treatment decreased the mRNA expression levels of CYP3A1 and CYP2B1/2, but did not alter the expression of CYP2C11.

At 6 weeks, DES treatment increasd the mRNA expression levels of UGT2B1 and IGF-1.

These results suggest that prenatal DES treatment alters two hepatic enzymes (CYP3A1 and CYP2B1/2) and IGF-1 mRNA expression levels to counteract the low level of testosterone, but this disrupted UGT2B1 mRNA expression reduces the testosterone level.


  • Maternal exposure to low doses of DES altered mRNA expression of hepatic microsomal enzymes in male rat offspring, The Journal of veterinary medical science, NCBI PubMed, PMID: 21959891, 2012 Feb.
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DES exposure causes decreased testis weight and morphological demasculinization of males

Short-term study investigating the estrogenic potency of diethylstilbesterol in the fathead minnow (Pimephales promelas)

2012 Study Abstract

Diethylstilbestrol (DES) is a synthetic estrogen that has been “banned” for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world.

Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish.

In this study, sexually mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10, or 100 ng of DES/L of water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive end points were measured in the fish at the end of a 4 d exposure and after a 4 d depuration/recovery period in clean water. Accumulation of DES was sex-dependent, with females exhibiting higher tissue residues than males after the 4 d exposure. The observed bioconcentration of DES in the fish was about 1 order of magnitude lower than that predicted on the basis of the octanol-water partition coefficient of the chemical, suggesting relatively efficient metabolic clearance by the fish. Exposure to 1, 10, or 100 ng of DES/L caused decreased testis weight and morphological demasculinization of males (regression of dorsal nuptial tubercles). Diethylstilbesterol induced plasma vitellogenin (VTG) in both sexes at water concentrations ≥10 ng/L; this response (especially in males) persisted through the end of the 4 d recovery period. Hepatic transcripts of VTG and estrogen receptor-α also were affected at DES concentrations ≥10 ng/L. Evaluation of transcript profiles in the liver of females using a 15K-gene fathead minnow microarray revealed a concentration-dependent change in gene expression, with mostly up-regulated transcripts after the exposure and substantial numbers of down-regulated gene products after depuration. Genes previously identified as vitellogenesis-related and regulated by 17β-estradiol were significantly enriched among those differentially expressed following exposure to DES.

Overall, our studies show that DES causes a range of responses in fish at water concentrations comparable to those reported in the environment and that in vivo potency of the estrogen is on par with that of the better-studied estrogenic contaminant 17α-ethinylestradiol.


  • Short-term study investigating the estrogenic potency of diethylstilbesterol in the fathead minnow (Pimephales promelas), Environmental science & technology, NCBI PubMed, PMID: 22708615, 2012 Jul.
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Neonatal DES exposure alters adult hepatic physiology

Hepatotoxicity induced by neonatal exposure to diethylstilbestrol is maintained throughout adulthood via the nuclear receptor SHP, 2014


Liver physiology is sensitive to estrogens, which suggests that the liver might be a target of estrogenic endocrine disrupters (EED). However, the long-term consequences of neonatal exposure to EED on liver physiology have rarely been studied. The nuclear receptor small heterodimer partner (SHP) mediates the deleterious effects of neonatal exposure to diethylstilbestrol (DES) on male fertility.

As SHP is involved in liver homeostasis, we aimed to determine whether neonatal estrogenic exposure also affected adult liver physiology through SHP. Male mouse pups were exposed to DES in the first 5 days of life.

DES exposure leads to alterations in the postnatal bile acid (BA) synthesis pathway. Neonatal DES-exposure affected adult liver BA metabolism and subsequently triglyceride (TG) homeostasis. The wild-type males neonatally exposed to DES exhibited increased liver weight and altered liver histology in the adult age. The use of deficient male mice revealed that SHP mediates the deleterious effects of DES treatment. These long-term effects of DES were associated with differently timed alterations in the expression of epigenetic factors.

However, the molecular mechanisms by which neonatal exposure persist to affect the adult liver physiology remain to be defined. In conclusion, we demonstrate that neonatal DES exposure alters adult hepatic physiology in an SHP-dependent manner.


  • Hepatotoxicity induced by neonatal exposure to diethylstilbestrol is maintained throughout adulthood via the nuclear receptor SHP, Expert opinion on therapeutic targets, NCBI PubMed, PMID: 6628266, 2014.
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Subchronic toxicology of diethystilbestrol in the mouse

image of female-mice

The liver, bone marrow, and thymus are major target organs for DES, 1983


This study evaluated the subchronic (14-day) toxicity of selected (0.2, 1.0, and 4.0 mg/kg) daily subcutaneous injections of diethylstilbestrol (DES) in female (C57B1/6 X C3H)F1 mice.

Parameters observed included body and organ weights, gross organ morphology, histopathology, clinical chemistry, and hepatic microsomal enzyme activities.

The liver, bone marrow, and thymus are major target organs for DES.

  • Liver enlargement, with associated histopathological changes consistent with mild hepatitis, centrolobular necrosis, and sinusoidal changes were observed. Supporting the histological changes were alterations in serum enzyme levels and microsomal enzyme activity.
  • Bone marrow changes included decreases in the number of cells as well as the number of colony forming units per gram stem cells.
  • Toxicity to the thymus was evidenced by decreased thymic weights and lymphocyte depletion. The hepatic and thymic effects were observed at the lowest (0.2 mg/kg) dose. Although all parameters were not assessed for recovery, those that were evaluated returned to control levels by thirty days after treatment.


  • Subchronic toxicology of diethystilbestrol in the mouse, Drug and chemical toxicology, NCBI PubMed, PMID: 6628266, 1983.

Increased DES in hepatitis E virus-infected pregnant women promotes viral replication

Pregnant women with high DES and/or immunosuppression will be vulnerable to HEV infection, study says, 2018


Hepatitis E virus (HEV) infection causes subclinical diseases, leading to high mortality (>25%) in pregnant women. HEV replication is aggressively escalated in pregnant women, especially in the third trimester of pregnancy. Oestrogen plays an important role in pregnancy. However, the pathogenesis of HEV in pregnant women or immunosuppressive pregnant women (such as HIV-infected or organ-transplanted pregnant women) remains unclear.

We investigated the role of oestradiol in HEV infection in a cell culture system. HEV-infected pregnant women had significantly higher oestradiol levels compared with uninfected individuals. HEV infection was significantly increased in cells treated with analogues of oestradiol, diethylstilbestrol (DES) or 17β-oestradiol in a dose-dependent way. However, tamoxifen, an antagonist oestrogen, inhibited HEV replication. HEV infection inhibits oestrogen receptor (ER-α) expression.

Immunofluorescence and co-immunoprecipitation assays indicated that ER-α interacted with the helicase of HEV ORF1 indirectly. More importantly, HEV infection was exacerbated in immunosuppressive cells treated with an inhibitor of PI3K-AKT-mTOR signal pathway (LY296004) and supplemented with pregnant women serum with high oestradiol simultaneously.

These results strongly suggest that pregnant women with high oestradiol and/or immunosuppression will be vulnerable to HEV infection.


  • Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication, Journal of viral hepatitis, NCBI PubMed, PMID: 29345855, 2018 Jun.
  • Featured image Samuel Zeller.

Antenatal exposure to DES: lessons learned…future concerns

DES-exposed offspring : certain complications have no time limit and continued follow-up is necessary, 2007


The short- and long-term effects of the widespread use of diethylstilbestrol (DES) over 3 decades have become a distant memory for many clinicians. Others are too young to remember the flurry of activity in the early 1970s on the part of many medical centers to identify the offspring of women who were prescribed DES during their pregnancies.

This medication was given in an attempt to prevent multiple pregnancy-related problems such as miscarriage, premature birth, and abnormal bleeding.

The recognition of the association of DES with an increased incidence of cervical and vaginal cancers in very young women led the Food and Drug Administration to ban its use during pregnancy in 1971.

Other pregnancy-related problems for the daughters and genitourinary tract changes in the sons did not become apparent until years later.

Ongoing follow-up of these offspring has raised concerns for their future as well as their mothers’ future. Clinicians need to be up-to-date with current knowledge regarding risks for cancer and other health-related issues.

Abstract (Third-Generation Effects)

Animal studies have shown tumor growth in older third-generation mice (human equivalent to age 70).

Multigenerational studies in humans are currently underway.

Several small studies of teenage third-generation females have not shown the same type of changes as in their mothers. Sons of DES daughters are at increased risk for hypospadias.


  • Antenatal exposure to DES: lessons learned…future concerns, Obstetrical and gynecological survey, NCBI PubMed PMID: 17634156, 2007 Aug.
  • Image credit wise owl tea ‏.