Journal of Andrology, Vol. 29, No.
5, September/October 2008
Copyright E American Society of Andrology
Endocrine Disruptors, Genital
Development, and Hypospadias
MING-HSIEN WANG AND LAURENCE S. BASKIN
From the UCSF Childrens Hospital, Department of Urology, University of California, San Francisco, California.
ABSTRACT: Hypospadias is one of the most common congenital
anomalies in the United States, occurring in approximately 1 in 125
live male births. Embryological studies have demonstrated that,
depending on where the urethral development arrests, the meatal
opening can be anywhere along the shaft of the penis or, in more
severe forms, within the scrotum or in the perineum. Currently, the
only available treatment is surgery. If left uncorrected, especially in
its severe form, there is risk of infertility and psychological effects,
such as avoidance of intimate relationships. The cause of
hypospadias is largely unknown; however, current epidemiology
and laboratory studies have shed new light into the etiology of
T he term endocrine disruptor was initially coined in
1991 by Theo Colborn, one of the worlds leading
authorities on endocrine-disrupting chemicals in the
environment and a senior scientist with the World
Wildlife FundUS (Colborn et al, 1993). Endocrine
disruptors are exogenous substances that behave simi-
larly to biologic hormones. They interfere with the
physiologic functions of the endogenous hormones by
affecting the release, binding, or metabolism of the
endogenous hormones. An example is the nonsteroidal
estrogen diethylstilbestrol (DES). Before its ban in the
early 1970s, physicians prescribed DES to pregnant
women hoping to prevent spontaneous abortions and
promote fetal growth. It was later noted that daughters
of DES-treated women have an increased incidence of
vaginal and cervical cancers. DES is just one of many
well-known examples of endocrine disruptors in hu-
mans.
Multiple animal studies have linked endocrine dis-
ruptors to adverse biological effects, raising concerns
that low-level exposure might cause similar effects in
humans (Giesy et al, 1994; Guillette et al, 1994, 1995;
Supported by a grant from the National Institute of Health R01
DK058105.
Correspondence to: Laurence S. Baskin, MD, Chief, Pediatric
Urology, Professor Urology and Pediatrics, UCSF Childrens Hospi-
tal, University of California, San Francisco, 400 Parnassus Avenue
A640, San Francisco, CA 94143 (e-mail: lbaskin@urology.ucsf.edu).
Received for publication January 14, 2008; accepted for publication
March 25, 2008.
DOI: 10.2164/jandrol.108.004945
499
Review
hypospadias. With recent advancements in molecular biology and
microarray technology, it appears that hypospadias is potentially
related to disrupted gene expression. Specifically, some of the
environmental chemicals are acting as antiandrogens and interfere
directly with the action of testosterone-related gene expression. In
this paper, we briefly review the normal development of male
external genitalia and the prevalence and environmental risk factors
related to hypospadias. In addition, we discuss some of the recent
laboratory findings that contribute to our current understanding of this
disease.
J Androl 2008;29:499505
Fry 1995; Sumpter and Jobling, 1995; de Solla et al,
1998). Within the last decade, several epidemiologic
studies have suggested environmental factors as a
possible cause for the observed increased incidence of
abnormalities in male reproductive health. Some exam-
ples include the increased incidence of testicular cancer,
a decline in semen quality, an increase in the frequency
of undescended testes and hypospadias, and a growing
demand for assisted reproduction (Chilvers et al, 1984;
Matlai and Beral, 1985; Campbell et al, 1987; Carlsen et
al, 1992; Adami et al, 1994; Auger et al, 1995; Irvine et
al, 1996; Swan et al, 1997; Moller, 1998; Andersen et al,
2000). However, some of the recent studies both in
Greenland and Denmark showed a stabilization or even
decline in the incidence of hypospadias (Giwercman et
al, 2006, Cortes et al, 2008).
In 2001, Skakkebk and others published a paper
suggesting that these observations are in fact related and
indicate a common cause. His conclusions were based
on the number of male reproductive abnormalities that
have risen sharply over the past 50 years, concurrent
with the swift growth of the chemical industry and its
associated release of chemicals into the environment. He
coined the term testicular dysgenesis syndromes
(TDS), which encompasses cryptorchidism, in situ germ
cell carcinoma of the testis/overt testicular cancer,
reduced semen quality, and hypospadias. Additional
signs include presence of microliths in the testes, Sertoli
cellonly seminiferous tubules, or immature tubules with
undifferentiated Sertoli cells (Skakkebk et al, 2001,
Skakkebk, 2004).
500
Figure 1. In the male, the wolffian duct develops into the rete testis,
the efferent ducts, the epididymis, the vas deferens, and the seminal
vesicles. In the female, without the mullerian-inhibiting substance,
the mullerian ducts develop into the fallopian tubes, uterus, cervix,
and upper third of the vagina.
In the following review, we focus on the role of
environmental endocrine disruptors and penile malde-
velopment in the setting of hypospadias, with a brief
overview of the normal embryology of male external
genitalia.
Development of the Male External Urogenital System
Formation of the external male genitalia is a complex
process starting with genetic programming (ie, the
presence of the Y chromosome and its associated sex-
determining region Y [SRY] and its protein product,
testis-determining factor [TDF], which are necessary for
cell differentiation), hormonal signaling, enzyme activ-
ities, and tissue remodeling. At 3.5 weeks of gestation,
the wolffian system appears as 2 longitudinal ducts
connecting cranially to the mesonephros and caudally
draining into the urogenital sinus. Meanwhile, the
mullerian duct develops as an evagination in the
coelomic epithelium just lateral to the wolffian duct at
approximately the sixth week of gestation (Figure 1). In
Journal of Andrology N September October 2008
Figure 2. At 8 weeks of gestation, the external genitalia remain in the
indifferent stage. The urethral groove on the ventral surface of the
phallus is between the paired urethral folds (uf). The penile urethra
forms as a result of fusion of the medial edges of the endodermal
urethral folds. As development progresses, the ectodermal edges of
the urethral groove begin to fuse to form the median raphe. (A) By
1112 weeks, the coronal sulcus separates the glans from the shaft
of the penis. (B) By 16 weeks of gestation, the urethral folds have
completely fused in the midline on the ventrum of the penile shaft. (C,
D) Note the normal ventral penile curvature or chordee (vc) that
occurs during development and resolves by the 20th week. Note the
midline seam (ms).
addition, by the end of the fourth week of gestation,
both the hindgut and future urogenital system have
reached the cloacal membrane on the ventral surface of
the developing embryo. From this indifferent stage until
the end of the eighth week of gestation, the urorectal
septum continues to develop and divides the cloacal
membrane into anterior and posterior segments; with
the anterior aspect destined to be the urogenital
membrane and the posterior segment the future rectum.
Up to this point, the male and female genitalia are
essentially indistinguishable. With the surge in luteiniz-
ing hormone, coupled with the influence of testosterone
and 5[alpha]-dihydrotestosterone (DHT), masculiniza-
tion of the external genitalia occurs. One of the first
signs of masculinization is an increase in the distance
between the anus and the genital structures, followed by
elongation of the phallus, formation of the penile
urethra from the urethral groove, and the development
of the prepuce. By 1112 weeks, the coronal sulcus has
separated the glans from the shaft of the penis. At 1617
weeks of gestation, the urethral folds have completely
fused in the midline on the ventrum of the penile shaft
with medial fusion of the endodermal urethra and fusion
of the ectodermal edges (Figure 2; Hinman, 1993;
Moore and Persaud, 1998).
The glandular urethra, which consists of the squa-
mous epithelium, also completes its formation during
this period. The mechanism of the glandular urethral
formation remains controversial. Two theories have
been proposed: endodermal cellular differentiation,
wherein the glandular urethra formed by an extension
of urogenital sinus epithelium undergoes transdifferen-
tiation, and primary intrusion of the ectodermal tissue
Wang and Baskin N Endocrine Disruptors 501

Figure 3. Theories of human penile urethral development. The ectodermal ingrowth theory postulates that the glandular urethra is formed by
ingrowth of epidermis. More recent data support the formation of the entire urethra via endodermal differentiation alone.

from the skin of the glans penis (Figure 3). Anatomical urethra), the preputial tissues do not form ventrally
and immunohistochemical studies support the new (Figure 4).
hypothesis of endodermal differentiation, which shows
that the epithelium of the entire urethra is of urogenital Prevalence of Hypospadias
sinus origin. The entire male urethra, including the The cause of hypospadias is largely unknown, although
glandular urethra, is formed by dorsal growth of the epidemiologic studies have identified some risk factors.
urethral plate into the genital tubercle and ventral Rates are highest among whites and lowest among
growth and fusion of the urethral folds. Under proper Hispanics (Centers for Disease Control, 1988). Some
mesenchymal induction, the urothelium has the ability studies have found an association between hypospadias
to differentiate into a stratified squamous phenotype and maternal factors, such as primiparity and advanced
with characteristic keratin staining, thereby explaining age (Angerpointer, 1984). Clinical studies have pointed
the cell type of the glans penis (Yamada et al, 2003).
The future prepuce is formed at the same time as the
urethra and is dependent on normal urethral develop-
ment. At about the eighth week of gestation, low
preputial folds appear on both sides of the penile shaft,
which join dorsally to form a flat ridge at the proximal
edge of the corona. The ridge does not entirely encircle
the glans because it is blocked on the ventrum by the
incomplete developed glandular urethra. The process of
preputial formation continues until it covers all of the
Figure 4. Spectrum of hypospadias. (A) Anterior, where the meatus
glans, forming a midline seam. If the genital folds fail to is on the inferior surface of the glans penis. (B) Coronal, the meatus
fuse (ie, if there is a defect in the formation of the is in the balanopenile furrow. (C) Distal, on the distal third of the shaft.
502
to defects in testosterone metabolism or testosterone
receptors as a potential cause (Giwercman et al, 1993),
suggesting that exogenous hormones can potentially
interfere with endogenous hormonal functions and
influence the normal development of male genitalia.
The most recent hypothesis suggests that widespread use
of synthetic compounds, especially those with estrogenic
or antiandrogenic activities, is a contributing factor
(Giwercman et al, 1993; Sharpe and Skakkebk, 1993).
In 1999, the International Clearinghouse for Birth
Defects Monitoring Systems, a nongovernmental orga-
nization of the World Health Organization, reported an
increased rate of hypospadias in 7 European countries,
including Norway, Sweden, England and Wales, Hun-
gary, Denmark, Italy, and France in the 1960s, 1970s,
and 1980s (Paulozzi, 1999). Independently, the Centers
for Disease Control and Prevention (CDC) reported
their findings of a doubling of hypospadias from 1968 to
1993 in the United States (Paulozzi et al, 1997). The data
were based on collective analysis from 2 independent
surveillance systems. Specifically, data from the Metro-
politan Atlanta Congenital Defects Program (MoD/
MACDP/CBDMP, 2001), a population-based registry
that uses active case studies in 22 hospitals and clinics in
the Atlanta, Georgia, area, indicate that the rate of total
hypospadias almost doubled from 1968 to 1993 at an
annual rate of 2.9%. Concurrently, the Birth Defects
Monitoring Program, a program that gathered dis-
charge diagnoses of newborns across the country, also
reports an increase in hypospadias: from 20.2 per 10 000
live births in 1970 to 39.7 per 10 000 live births in 1993.
Overall, these longitudinal studies suggest an almost
doubling of hypospadias in the United States over a 14-
year period.
Environmental Factors
In the past, environmental effects were generally ruled out
as causes for hypospadias. However, in light of the
growing number of endocrine disruptors reported in
human tissue and the probability of shared exposure,
environmental contaminants are now being evaluated in
familial cluster studies. Reports of increase rates of
hypospadias have paralleled reports of other adverse
effects observed in male reproductive health, include
increased rates of testicular cancer, cryptorchidism, and
decreasing semen and sperm quality (Bergstrom et al, 1995;
Carlsen et al, 1995). Cheng et al (1996) found that 8% of
patients (n 5 252) with undescended testes also had other
urogenital anomalies, including hypospadias. A separate
study reports an increase in testicular cancer risk with
undescended testicles and hypospadias (Prener et al, 1996).
To understand the biology of antiandrogens, and
their roles as endocrine disruptors in the development of
Journal of Andrology N September October 2008
the external genitalia and urethra, we must first look at
testosterone. Under normal conditions, testosterone
dissociates from its carrier proteins in the plasma and
enters the cells via passive diffusion. Once intracellular,
testosterone binds to the androgen receptor (AR) and
forms a complex that can subsequently bind to the
androgen response element on DNA. With this binding,
multiple downstream androgen-responsive activities are
initiated, one of which is the development of male
external genitalia. Antiandrogens can directly interfere
with the proper functioning of testosterone in multiple
ways. Examples include inducing a conformational
change within AR, increasing AR degradation, or
blocking the release of heat shock proteins from AR.
A well-known example of an antiandrogen is DDT
(dichlorodiphenyltrichloroethane). DDT was widely
used to combat mosquitoes spreading malaria, typhus,
and other insectborne diseases. In 1962, Rachel Carson
published the book Silent Spring, questioning the
environmental effects of the indiscriminate use of
DDT and its effect on ecology and human health
(Carson, 1962). A study in the male rat fetus found that
in utero exposure to p,p9-DDE, a lipophilic metabolite
of DDT, leads to feminization of the genital urinary
tracts; reduced anogenital distance, hypospadias, and
cryptorchidism (Kelce et al, 1995). Perhaps the most
alarming aspect of the study is that the experimental
dose used was within the range of human exposure.
Normal urogenital differentiation also relies on the
interaction between testosterone and epidermal growth
factors (EGFs). In vivo experiments show that EGF
alone can induce partial virilization of the external
genitalia and that sexual differentiation is inhibited
when EGF is depleted (Gupta et al, 1996). Dioxin
(TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin), a compo-
nent of agent orange, a herbicide and defoliant used by
the US Armed Forces in the Vietnam War, has gained
popular attention because of its association with various
cancers. It has been shown in rats to induce c-Src kinase
activity and reduces EGF receptor binding during
testicular development (El-Sabeawy et al, 1998). How-
ever, there are no definitive studies that have demon-
strated an association between agent orange and human
cancers. Chlorinated pesticides, such as dioxin, as well
as furans and polychlorinated biphenyls are known
cytochrome P450 (CYP450) agonists that interfere with
the normal aromatization of androgens (Haake et al,
1987; Paolini et al, 1996; Sanderson et al, 1997).
Several synthetic chemicals have consistently been
shown to induce hypospadias in laboratory animals.
Vinclozolin, a commonly used fungicide, induces female-
like AGD (anal-genital distance), retained nipples,
reduced sperm count and fertility, and hypospadias in
100% of male offspring that were exposed to the chemical
Wang and Baskin N Endocrine Disruptors
in utero (Gray et al, 1999a; Kelce et al, 1994).
Procymidone, another antiandrogenic fungicide, induces
hypospadias in all male rat offspring when mothers are
exposed during gestation (Ostby et al, 1999). It has been
shown that procymidone exerts its effects by inhibiting
DHT-induced transcriptional activities (Gray et al,
1999b). Mice exposed to vinclozolin have less robust
and atrophic urethras when compare with controls (Kim
et al, 2004; Buckley et al, 2006). Phthalates, ubiquitous
chemicals found in plastics, have gained attention
because of their hormonal effects in laboratory animals.
Male rodents exposed to dibutyl phthalate and diethyl-
hexyl phthalate have reduced AGD, retained nipples,
epididymal agenesis, undescended testes, and hypospadias
(Gray, 1998).
As mentioned previously, DES provides an excellent
model for studying interrupted genital development
because of exogeneous hormones in humans. Longitu-
dinal studies have shown that DES daughters have a
2.5-fold increase in breast cancer after age 40, in
addition to an increased risk for vaginal and cervical
cancers (Herbst et al, 1971). Sons of DES-exposed
mothers are at an increased risk for feminization of the
male fetus; a study noted a 20-fold increase in the
development of hypospadias (Brouwers et al, 2006). A
separate study confirmed this risk: 10 of 225 DES-
exposed male offspring developed penile malformations,
including strictures/stenoses and hypospadias, vs 0 in
patients who were unexposed (Henderson et al, 1976).
Molecular Mechanism of Hypospadias and
Endocrine Disruptors
The exact molecular events that lead to normal urethral
development and hypospadias remain largely unknown;
however, recent studies have revealed possible involve-
ment of sonic hedgehog (SHH), fibroblast growth
factors 8 and 10 (FGF8 and FGF10), and homeobox
A13 and D13 (HOXA13 and HOXD13) genes in early
genital tubercle outgrowth and patterning (Haraguchi et
al, 2001; Perriton et al, 2002; Morgan et al, 2003). As
discussed earlier, antiandrogen might play a role in the
development of hypospadias. It has been shown that
pregnant female mice exposed to synthetic estrogen
compounds gave birth to offspring with hypospadias
(Kim et al, 2004; Willingham et al, 2006). Microarray
analysis of tissue samples from hypospadias have
identified additional genes that are both estrogen
responsive and up-regulated (Wang et al, 2007). One
such gene found in the tissues/foreskin of human
hypospadias, which also demonstrated significant up-
regulation in the presence of estrogen, is activating
transcription factor-3 (ATF-3). A recently published
study utilizing human foreskin fibroblast treated with
503
estrogen showed an increase in staining for ATF-3
within 2 hours of treatment (Liu et al, 2006). This was
further substantiated by an increase in protein expres-
sion, and ATF-3 promoter activity (Liu et al, 2007).
Similar results were also noted in the mouse genital
tubercle, where quantitative reverse transcriptase poly-
merase chain reaction showed that ATF-3 mRNA is up-
regulated in estrogen-exposed specimen compared with
controls. A study of human tissues from 28 children who
underwent repair of hypospadias vs 20 tissue samples
from children who underwent elective circumcision,
demonstrated a significant increase in immunohisto-
chemical staining for ATF-3 in the hypospadias
population (86% vs 13%), (Liu et al, 2005). Studies
have shown that ATF-3 protein suppresses cellular
growth, we can hypothesize that its up-regulation
induces an arrest in urethral development as a potential
cause of hypospadias. Further studies of ATF-3 and
other estrogen-related genes would perhaps provide a
link between exogenous hormones and the development
of hypospadias.
With the increasing incidence of hypospadias paral-
leling the rate of increase in environmental pollutants, it
is imperative that we consider endocrine disruptors as a
potential cause for this anomaly. Additional molecular
studies will help us elucidate the role of these exogenous
hormones and offer insights into other male develop-
mental disorders.
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