The role of DES in the etiology of cryptorchidism

Diethylstilbestrol impairs the morphology and function of mouse gubernaculum testis in culture

2012 Study Abstract

Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen widely used in estrogen therapy.

In animal models, exposure to DES disrupts the outgrowth of the gubernacula, leading to testis maldescent. However, it remains unclear whether the effects of DES on gubernaculum are direct or indirect, and the underlying mechanisms are largely obscure.

In this study, mouse gubernaculum testis cells were isolated and treated by DES, and cell morphology and function were examined. The results showed that DES changed the morphology and inhibited the proliferation of gubernacular cells. Furthermore, DES increased intracellular [Ca(2+)] and induced F-actin rearrangement and stress fiber formation in gubernaculum testis cells in a dose-dependent manner.

Taken together, these data suggest that DES impairs the morphology and inhibits the proliferation and contractility of gubernaculum testis cells. The experimental model we established and our observations based on this model help provide new insight into the role of DES in the etiology of cryptorchidism.


  • Diethylstilbestrol impairs the morphology and function of mouse gubernaculum testis in culture, Cell biology and toxicology, NCBI PubMed, PMID: 22945456, 2012 Dec.

Cryptorchidism and endocrine disrupting chemicals

In utero DES exposure increases the risk of testicular dysgenesis syndrome ; linked to cryptorchidism, hypospadias, poor semen quality and testicular cancer

2012 Study Abstract

Prospective clinical studies have suggested that the rate of congenital cryptorchidism has increased since the 1950s. It has been hypothesized that this may be related to environmental factors.

Testicular descent occurs in two phases controlled by Leydig cell-derived hormones insulin-like peptide 3 (INSL3) and testosterone. Disorders in fetal androgen production/action or suppression of Insl3 are mechanisms causing cryptorchidism in rodents.

In humans, prenatal exposure to potent estrogen diethylstilbestrol (DES) has been associated with increased risk of cryptorchidism.

In addition, epidemiological studies have suggested that exposure to pesticides may also be associated with cryptorchidism.

Some case-control studies analyzing environmental chemical levels in maternal breast milk samples have reported associations between cryptorchidism and chemical levels.

Furthermore, it has been suggested that exposure levels of some chemicals may be associated with infant reproductive hormone levels.


  • Cryptorchidism and endocrine disrupting chemicals, Molecular and cellular endocrinology, NCBI PubMed, PMID: 22127307, 2012 May.
  • Featured image credit Ivan Bandura.

DES Sons Urogenital Abnormalities, 2009

Urogenital abnormalities in men exposed to diethylstilbestrol in utero: a cohort study


Diethylstilbestrol (DES), a synthetic estrogen widely prescribed to pregnant women during the 1940s70s, has been shown to cause reproductive problems in the daughters. Studies of prenatally-exposed males have yielded conflicting results.

In data from a collaborative follow-up of three U.S. cohorts of DES-exposed sons, we examined the relation of prenatal DES exposure to occurrence of male urogenital abnormalities. Exposure status was determined through review of prenatal records. Mailed questionnaires (1994, 1997, 2001) asked about specified abnormalities of the urogenital tract. Risk ratios (RR) were estimated by Cox regression with constant time at risk and control for year of birth.

Prenatal DES exposure was not associated with varicocele, structural abnormalities of the penis, urethral stenosis, benign prostatic hypertrophy, or inflammation/infection of the prostate, urethra, or epididymus. However, RRs were 1.9 (95% confidence interval 1.13.4) for cryptorchidism, 2.5 (1.54.3) for epididymal cyst, and 2.4 (1.54.4) for testicular inflammation/infection. Stronger associations were observed for DES exposure that began before the 11th week of pregnancy: RRs were 2.9 (1.65.2) for cryptorchidism, 3.5 (2.06.0) for epididymal cyst, and 3.0 (1.75.4) for inflammation/infection of testes.

These results indicate that prenatal exposure to DES increases risk of male urogenital abnormalities and that the association is strongest for exposure that occurs early in gestation. The findings support the hypothesis that endocrine disrupting chemicals may be a cause of the increased prevalence of cryptorchidism that has been seen in recent years.


  • Full study (free access) : Urogenital abnormalities in men exposed to diethylstilbestrol in utero: a cohort study, Environmental health : a global access science source, NCBI PubMed, PMC2739506, 2009 Aug.
  • Featured image credit Kyle Loftus.

DES-induced abdominal cryptorchidism

The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats

2009 Study Abstract

To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum.

Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting.

Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P<0.01). DTA in group B, C, D and E were (23.7+/-1.7) U, (38.8+/-1.9) U, (49.3+/-1.8) U and (58.6+/-2.1) U that were significantly larger than that in group A [(8.5+/-1.3) U] (q=46.12, 88.53, 120.44 and 141.37, respectively, P<0.01). There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0.9570+/-0.1490, 0.6760+/-0.1380, 0.0170+/-0.0040 and 0.0013+/-0.0003, respectively; the expressions of INSL3 protein were 0.8360+/-0.1520, 0.5310+/-0.1070, 0.0140+/-0.0020 and 0.0011+/-0.0003, respectively, which were significantly larger than the expression of INSL3 mRNA (1.801+/-0.126) and INSL3 protein (1.612+/-0.134) in group A (qmRNA=40.4840, 52.4402, 83.1585 and 82.0582, respectively, and qprotein=38.6151, 52.2747, 77.2756 and 76.1983, respectively, P<0.01). The expression of HOXA10 mRNA in group A, B, C, D, and E were 0.945+/-0.125, 0.940+/-0.119, 0.656+/-0.115, 0.544+/-0.118 and 0.463+/-0.114, respectively. Compared with the expression of HOXA10 mRNA in group A, the expression of group B was not significantly different (q=0.2213, P>0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, P<0.01).

DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.


  • The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats, Chinese medical journal, NCBI PubMed, PMID: 19534997, 2009 May.
  • Featured image credit pexels.

DES-induced intra-abdominal cryptorchidism

Prenatal exposure to diaethylstilbestrol in the rat inhibits transabdominal testicular descent with involvement of the INSL3/LGR8 system and HOXA10

2009 Study Abstract

Prenatal exposure to diaethylstilbestrol (DES) has been found to lead to intra-abdominal cryptorchidism, but the mechanism is still not completely clear. This study investigated the roles of the INSL3/LGR8 system and HOXA10 in DES-induced intra-abdominal cryptorchidism (DIIAC). The effect of DES on steroidogenic factor-1 (SF-1), that has been reported to control transcription of insulin-like factor 3 (INSL3), was also investigated.

Fifty pregnant female SD rats at embryonic day 13.5 (E13.5) were randomly assigned to five groups that received a subcutaneous injections of dimethyl sulfoxide (control), 2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg of DES. Male offspring were sacrificed at E19.5, and fetal mortality and the degree of transabdominal testicular ascent (DTA) were determined under a stereomicroscope. The mRNA expression of INSL3 and SF-1 in the testis and leucine rich repeat-containing G protein-coupled receptors 8 (LGR8) and homeobox-A10 (HOXA10) in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting.

Higher fetal mortality and DTA were induced by DES in a dose-dependent manner (P < 0.01). Compared with the control group, the expression of INSL3 and SF-1 mRNA were down-regulated in a dose-dependent manner (P < 0.01), as was INSL3 protein; HOXA10 in the 2.5 mg/kg group and LGR8 mRNA in the 2.5 mg/kg and 5 mg/kg groups were not significantly different (P > 0.05); HOXA10 mRNA in groups C, D, and E decreased significantly and LGR8 mRNA levels in groups D and E increased significantly (P < 0.05, P < 0.01, respectively).

DES can inhibit transabdominal testicular descent in a dose-dependent manner via down-regulating the expression of INSL3, which is induced by down-regulating the expression of SF-1. HOXA10 may not be involved in DES induced intra-abdominal cryptorchidism at 2.5 mg/kg, but is involved at 5, 10 and 20 mg/kg. LGR8 may not be responsible for DES-induced transabdominal testicular maldescent.


  • Full study (free access) : Prenatal exposure to diaethylstilbestrol in the rat inhibits transabdominal testicular descent with involvement of the INSL3/LGR8 system and HOXA10, Chinese medical journal, NCBI PubMed, PMID: 19493424, 2009 Apr.
  • Featured image credit

Testicular Dysgenesis Syndrome and the Estrogen Hypothesis: A Quantitative Meta-Analysis

It is clear that hypospadias, cryptorchidism, and testicular cancer are all positively associated with prenatal exposure to DES

2008 Study Abstract

Male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting from the disruption of gonadal development during fetal life, the testicular dysgenesis syndrome (TDS). The hypothesis that in utero exposure to estrogenic agents could induce these disorders was first proposed in 1993. The only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago, and other systematic reviews of the association between estrogenic compounds, other than the potent pharmaceutical estrogen diethylstilbestrol (DES), and TDS end points have remained inconclusive.

We conducted a quantitative meta-analysis of the association between the end points related to TDS and prenatal exposure to estrogenic agents. Inclusion in this analysis was based on mechanistic criteria, and the plausibility of an estrogen receptor (ER)-α–mediated mode of action was specifically explored.

We included in this meta-analysis eight studies investigating the etiology of hypospadias and/or cryptorchidism that had not been identified in previous systematic reviews. Four additional studies of pharmaceutical estrogens yielded a statistically significant updated summary estimate for testicular cancer.

The doubling of the risk ratios for all three end points investigated after DES exposure is consistent with a shared etiology and the TDS hypothesis but does not constitute evidence of an estrogenic mode of action. Results of the subset analyses point to the existence of unidentified sources of heterogeneity between studies or within the study population.


  • Full study (free access) : Testicular Dysgenesis Syndrome and the Estrogen Hypothesis: A Quantitative Meta-Analysis, Environmental Health Perspectives, PMC2235228, 2008 Feb.

DES-mediated fetal testis dysgenesis and cryptorchidism

2007 Study Abstract

There are numerous reports indicating that estrogenic molecules affect testicular descent both in rodents and humans. In fact, before the identification of Insl3 and LGR8 mutant mice, the main model for intraabdominal cryptorchidism involved the exposure of pregnant rodents to exogenous estradiol (E2).

In humans, the offspring of pregnant women treated with diethylstilbestrol (DES), a nonsteroidal estrogenic substance used during the 1950s to 1970s to prevent miscarriages, exhibited an increased incidence of cryptorchidism and hypoplastic testes. In rodents, we and others have shown that administration of DES or E2 during the second half of gestation resulted in a drastic reduction of Insl3 gene expression and testosterone levels, even though the expression of Sf-1 was unaltered. Recently, it has been shown that exposure to mono-N-butyl phthalate, an endocrine disruptor with weak estrogenic activities, also impairs Insl3 gene expression and testicular descent in rat embryos.

Collectively, these reports indicate that xenoestrogen-dependent cryptorchidism is caused by a down-regulation of Insl3 gene expression. However, the molecular mechanisms mediating estrogen-dependent inhibition of fetal Leydig cell function and cryptorchidism remain an important open question. The purpose of this study was to investigate the toxicogenomic effects of DES or E2 exposure on testicular gene expression, and to assess the contribution of the estrogen receptors (ERs) α or β in mediating these effects with an emphasis on Insl3 gene expression and testicular descent. We found 63 and 175 genes that were respectively down- or up-regulated by estrogen exposure in fetal testes; for more than half of these, this was mediated by ERα. The expression of Leydig-specific genes such as Insl3, cytochrome P450 17α-hydroxylase/17,20-lyase (Cyp17a1), steroidogenic acute regulatory protein (Star), and renin 1 (Ren1) was profoundly decreased upon exposure to E2 or DES but not affected in mutant testes lacking ERα. These data suggest that estrogenic exposure affects fetal Leydig cell endocrine functions, more precisely the steroidogenic function, Insl3 expression and testicular descent, via an ERα-dependent mechanism.


  • Full study (free access) : Estrogen Receptor α Is a Major Contributor to Estrogen-Mediated Fetal Testis Dysgenesis and Cryptorchidism, Endocrinology, Oxford University Press,, 01 November 2007.

Male reproductive health and environmental xenoestrogens

Exposure to DES during pregnancy results in an increased risk for several male reproductive disorders

1996 Study Abstract

Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood.

Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.

Occurrence of Abnormalities in the Reproductive System of the Sons of Women Exposed to Diethylstilbestrol during Pregnancy Exposure

Diethylstilbestrol (DES) was prescribed to more than five million pregnant women from the late 1940s to the early 1970s to prevent abortions and pregnancy complications. Dieckmann and co-workers performed a double-blind placebo-controlled study on the therapeutic value of DES during pregnancy in the early 1950s. DES was given to 840 pregnant women and placebo to 806 controls. Compliance was verified by a dye indicator in the urine during the whole study. The women entered the study between weeks 7 and 20 of pregnancy (the majority during weeks 10-12) and received increasing doses of DES until pregnancy week 35 (5-150 mg/day). This study clearly indicated that the medication was not efficacious in the indications for which it was used. Instead, in the reanalysis of the material of Dieckmann et al., DES was associated with significant increases in abortions, neonatal deaths, and premature births. When Herbst and co-workers reported the high incidence of a very rare cancer, clear cell adenocarcinoma of the vagina, in pubertal girls exposed to DES in utero, the U.S. Food and Drug Administration (FDA) banned the use of DES during pregnancy. Medical authorities in Europe that had allowed DES use for pregnant women soon followed FDA regulations. In Europe, approximately 200,000 French, more than 150,000 Dutch, 63,000 Czechoslovakian, and 7000 British women were exposed to DES, whereas in the United States 4.8 million women were prescribed DES during pregnancy. In addition, DES was used as an anabolic agent in livestock, and the general population that used dairy products and meat may have been exposed to the hormone via this route to an unknown, and probably variable, extent. Some of the DES-exposed daughters and sons have been followed since the 1970s and a significant number of abnormalities in the structure and function of reproductive organs have been described.

Structural Anomalies

Structural anomalies of the reproductive organs that are significantly more frequent in DES-exposed male subjects than in controls include meatal stenosis (12.9 vs 1.8%); hypospadias (4.4 vs 0%); epididymal cysts (20.8 vs 4.9%); testicular abnormalities, including hypoplastic testis, cryptorchidism, and capsular induration (11.4 vs 2.9%); and microphallus (4 cases vs 0 cases) (172-174). The data of Bibbo and Gill and their co-workers are based on the follow-up studies of the offspring of mothers who took part in the double-blind study of the effects of DES on pregnancy in 1953, and therefore the studies can be considered prospective. There were 308 men exposed to DES and 307 men exposed to placebo included in the study; 31.5% of men exposed to DES had an abnormality of their reproductive tract, whereas only 7.8% of controls had an anomaly. In the recent follow-up study of these males, it was found that the men who were exposed to DES before week 11 of gestation had twice as high a frequency of genital anomalies than did those who were exposed only later. This finding indicates the importance of the timing of the exposure (time of organogenesis). In a small study comprising 17 DES-exposed men, 12 nonexposed volunteers, and 11 fertile control men, genital anomalies (varicocele, epididymal cysts, absent testes) were reported in 13 of the DES-exposed subjects, 4 of the volunteers and 4 of the fertile normal controls. Whitehead and Leiter reported genital abnormalities in 29 of 48 men exposed to DES. Hypertrophy and squamous metaplasia of the prostatic utricle was found more frequently in aborted male fetuses that had been exposed to DES than in nonexposed controls, suggesting that DES-exposed males may have an increased risk of prostatic hyperplasia and/or cancer when aging. The data connecting DES exposure to several structural abnormalities of the male reproductive tract are convincing and leave little space for speculation on confounding factors. However, no association was found between first-trimester exposure to sex hormones, other than DES, and external genital abnormalities in a recent metaanalysis of 14 studies. In a large cryptorchidism study, no association between the disorder and exposure to estrogens during the pregnancy could be found.

Semen Quality

Gill et al. studied semen samples from 88 men exposed to DES and 85 men exposed to placebo, who were offspring of the mothers from the 1953 study performed by Dieckmann and co-workers. Sperm concentration of men exposed to DES was significantly lower than in the controls (83 million/ml vs 123 million/ml, p < 0.02). There was no difference in semen volume, whereas the total sperm count, sperm motility grade, the total number of motile sperm, the percentage of sperm with normal morphology, and the quality score were all statistically lower in men exposed to DES. Azoospermia was found only in men exposed to DES, and 20.5% compared to 3.5% of men who received placebo had a sperm concentration in semen of less than 20 million/ml. The groups did not differ in their testosterone, FSH, or LH levels. In a later study on the same men (20 controls declined to participate), sperm concentrations still differed significantly, whereas other semen characteristics were similar between the groups. Similar results were obtained in another study in which the mean sperm concentration of men exposed to DES was 66.4 million/ml compared to 101.7 million/ml in normal volunteers (p < 0.05). In this study, the zona-free hamster egg penetration assay was also performed: sperm from 14 of 17 men exposed to DES failed to penetrate more than 14% of the eggs (which is the reference value for the normal fertility range), suggesting infertility, whereas only 2 of 12 unexposed volunteers and none of 11 fertile normal controls had an abnormal test result. In the study performed by Whitehead and Leiter, only 33% of the men exposed to DES had normal semen quality. However, Andonian and Kessler found no difference in semen quality between 24 men exposed to DES and 24 age-matched control men. Again, the large 1953 study population that has been followed prospectively appears the most valid for evaluation of semen quality. On the basis of that finding, DES exposure resulted in a significant decrease in semen quality.

Semen quality and fertility are not in direct correlation. In the latest follow-up study of the Dieckmann cohort, no difference in the fertility between men exposed to DES and their controls were found. This is compatible with the earlier findings that the majority of the men exposed to DES had sperm concentrations well above the limit at which fertility is supposed to be disturbed (20 million/ml), although the mean sperm concentrations of exposed men were lower than those of controls.


Exposure to DES during pregnancy results in an increased risk for several male reproductive disorders, such as cryptorchidism, urethral abnormalities, epididymal cysts, and testicular hypoplasia. In addition, the semen quality of DES sons is worse than that of controls. Incidence of testicular cancer is approximately doubled among DES sons compared to the general population, but whether this represents a true increase of the cancer risk is equivocal.


  • Full study (free access) : Male Reproductive Health and Environmental Xenoestrogens, Environmental health perspectives, NCBI PubMed, PMC1469672, 15 March 1996.

Endocrine disruptor compounds and their role in the developmental programming of the reproductive axis

DES is the best documented compound

2007 Study Abstract

Different perturbations during fetal and postnatal development unleash endocrine adaptations that permanently alter metabolism, increasing the susceptibility to develop later disease, process known as “developmental programming.”

Endocrine disruptor compounds (EDC) are widely spread in the environment and display estrogenic, anti-estrogenic or anti-androgenic activity; they are lipophilic and stored for long periods in the adipose tissue. Maternal exposure to EDC during pregnancy and lactation produces the exposure of the fetus and neonate through placenta and breast milk. Epidemiological and experimental studies have demonstrated reproductive alterations as a consequence of intrauterine and/or neonatal exposure to EDC.

Diethystilbestrol (DES) is the best documented compound, this synthetic estrogen was administered to pregnant women in the 1950s and 1960s to prevent miscarriage. It was implicated in urogenital abnormalities in children exposed in utero and was withdrawn from the market.

The “DES daughters” are women with high incidence of vaginal hypoplasia, spontaneous abortion, premature delivery, uterine malformation, menstrual abnormalities and low fertility.

The “DES sons” show testicular dysgenesis syndrome, which is characterized by hypospadias, cryptorchidism and low semen quality.

This entity is also associated wtih fetal exposure to anti-androgens as flutamide.

The effects on the reproductive axis depend on the stage of development and the window of exposure, as well as the dose and the compound.

The wide distribution of EDC into the environment affects both human health and ecosystems in general, the study of their mechanisms of action is extremely important currently.


  • Endocrine disruptor compounds and their role in the developmental programming of the reproductive axis, Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion, NCBI PubMed, PMID: 17569302, 2007 Jan-Feb.
  • Featured image Jamie Street.

Exposure to endocrine disrupting chemicals and children’s health

Problems in epidemiological studies

2006 Study Abstract

Most endocrine disrupting chemicals are characterized by their properties to induce marked phenotypic changes in offspring such as congenital anomalies and neurodevelopmental dysfunctions. Although an increase in the prevalence of hypospadias or cryptorchidism has been reported in various countries, improvement in diagnostic techniques and more attention to the features of the diseases have also been emphasized.

Although there have been a few reports that hypospadias or cryptorchidism had been associated with diethylstilbestrol (DES), pesticides and so on, the associations between these diseases and endocrine disrupting chemicals remain unclear.

Recently, the association between maternal metabolic polymorphism or paternal smoking during pregnancy and these diseases has been reported.

There are also variable clinical features in children’s neurobehavioral development, and thyroid and immune functions in relation to exposure to endocrine disrupting chemicals such as polychlorinated biphenyls (PCBs) and dioxins.

Only a few Dutch studies have suggested that perinatal exposure to background level of PCB/dioxin confers immunity to allergy development. Genetic susceptibility to environmental endocrine disrupting chemicals may be related to adverse pregnancy outcomes.

It is suggested that well-designed epidemiological studies such as prospective cohort studies should be performed to elucidate this association.


  • Exposure to endocrine disrupting chemicals and children’s health: problems in epidemiological studies, Japanese journal of hygiene, NCBI PubMed, PMID: 16506651, 2006 Jan.
  • Featured image Ban Yido.