Pediatr Surg Int (2015) 31:519–527

DOI 10.1007/s00383-015-3686-z

REVIEW ARTICLE

Genetic and environmental factors in the aetiology of hypospadias

Mathew George • Francisco J. Schneuer •
Sarra E. Jamieson • Andrew J. A. Holland

Accepted: 17 February 2015 / Published online: 6 March 2015

Ó Springer-Verlag Berlin Heidelberg 2015

Abstract This article reviews the current evidence and DDE p p’ Dichloro diphenyl dichloroethane
knowledge of the aetiology of hypospadias. Hypospadias DDT p p’Dichloro diphenyl trichloroethane
remains a fascinating anomaly of the male phallus. It may DES Diethylstilbestrol
be an isolated occurrence or part of a syndrome or field DGKK Diacylglycerol kinase kappa
defect. The increasing use of assisted reproductive tech- DHCR Dihydrocholesterol reductase
niques and hormonal manipulation during pregnancy may EDC Endocrine disrupting chemicals
have been associated with an apparent rise in the incidence ESR1 Oestrogen receptor 1
of hypospadias. Genetic studies and gene analysis have ESR2 Oestrogen receptor 2
suggested some defects that could result in hypospadias. FGFR2 Fibroblast growth factor
New light has also been thrown on environmental factors FGF8 Fibroblast growth factor
that could modulate candidate genes, causing altered de- FGF10 Fibroblast growth factor
velopment of the male external genitalia. GWAS Genome-wide association study
GXE Gene environment interactions
Keywords Hypospadias
Aetiology
Genetics
HCB Hexachlorobenzene
Endocrine
Gene deletion
Gene expression HCG Human chorionic gonadotrophin
HOXA13 Homeobox A 13
Abbreviations ICSI Intracytoplasmic sperm injection
ART Assisted reproductive technology IUGR Intrauterine growth retardation
ATF3 Activating transcription factor IVF In vitro fertilisation
BMI Body mass index LBW Low birth weight
BMP7 Bone morphogenic protein MAMLD1 Mastermind-like domain containing 1
SF 1 Splicing factor 1
SGA Small for gestational age
M. George
A. J. A. Holland (&) SNP Single nucleotide polymorphisms
Douglas Cohen Department of Paediatric Surgery, The
SOX9 SRY box 9
Children’s Hospital at Westmead, Discipline of Paediatrics and
Child Health, Sydney Medical School, The University of SRD5A2 Steroid 5 alpha reductase
Sydney, Locked Bag 4001, Westmead, NSW 2145, Australia SRY gene Sex-determining region Y
e-mail: andrew.holland@health.nsw.gov.au WT1 Wilms tumour 1
F. J. Schneuer
Northern Clinical School, Kolling Institute of Medical Research,
The University of Sydney, Reserve Road, St Leonards,
NSW 2065, Australia Introduction
S. E. Jamieson
Telethon Kids Institute, University of Western Australia, Perth,
Hypospadias has been defined as a condition in which the
WA 6009, Australia urethral meatus may be proximal to its normal glanular

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occurrence anywhere along the penile shaft, scrotum or
perineum [1, 2]. Traditionally it has been classified ac-
cording to the position of the urethral meatus, with the
meatus located at the coronal sulcus or more distal in an-
terior or distal hypospadias, between the coronal sulcus and
mid-shaft of the penis in middle hypospadias and proximal
to the mid-shaft in posterior of proximal hypospadias [2].
It continues to be one of the most frequent problems in
paediatric surgery, with an incidence of one in 200–300
live male births [2–4]. A spectrum of associated anomalies,
including varying degrees of chordee, a hooded incomplete
prepuce and a deficient corpora spongiosum appear com-
monly associated with hypospadias [2]. Hypospadiology
remains a constantly evolving discipline with new research
into the putative genetic, endocrine and environmental
aetiological factors [5]. Surgical correction of the defect
will generally be required and this may be associated with
subsequent psychosocial and psychosexual developmental
difficulties [2, 6]. We conducted a review of published data
concerning the aetiopathogenesis of hypospadias as a guide
to assist clinicians and parents of boys with this anomaly.
Methodology
An electronic survey of Medline, Ovid, Search Medica,
Elsevier Clinical Key, Google Scholar and the Cochrane
library was performed on all manuscripts published be-
tween 1950 and 2014 regarding aetiology of hypospadias.
The following keywords were used: ‘‘hypospadias, aeti-
ology and genetics’’. The reference lists of the full articles
were also manually searched to identify additional eligible
studies. All data included in this review were published in
English, although no language restriction was imposed.
The latest search was performed in October 2014. The first
search yielded 117 articles.
Known embryology of hypospadias
The genital tubercle develops during the 4th week of ges-
tation as the precursor of the phallus. Endodermal cells
migrate from the cloaca along its ventral midline to form
the urethral plate and on either side proliferating mes-
enchyme form the urogenital folds [2, 7, 8]. From 9th to
12th week, androgenic stimulation causes phallic devel-
opment. Elongation of genital tubercle occurs and the
urogenital folds migrate to the midline and fuse enclosing
the urethral groove. This process moves proximal to distal
creating the urethra.
As the plate tubularises, mesoderm within the urethral
folds differentiates into the corpus spongiosum, which then
fuses distally to the glans. Mesoderm also forms the
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Pediatr Surg Int (2015) 31:519–527
corpora cavernosa. Development of the penis proceeds at
different rates along the ventral and dorsal surfaces re-
sulting in temporary ventral curvature. Similarly the dorsal
prepuce extends beyond the glans before the ventral aspect,
which follows closure of urethral groove. Midline fusion of
the ventral prepuce results in the frenulum.
Hypospadias appears to represent an arrest of penile
development, with the meatus located at some point be-
tween the perineum and the distal glans [2, 7]. The un-
tubularized residuum of tissue in the midline represents the
urethral plate, extending from the meatus to the glans.
Associated anomalies may include a split glans, splayed
corpus spongiosum, incomplete prepuce and ventral
chordee.
Aetiology of hypospadias
Hypospadias may be syndromic or non-syndromic. Nearly
200 syndromes have been associated with hypospadias,
with the more common ones listed in Table 1 [9]. In con-
trast, in non-syndromic hypospadias the aetiology has been
presumed to be multifactorial with interaction of genetic,
hormonal and environmental factors [1].
Genetic mutations involved in hypospadias
Initial genetic research on hypospadias focused on the
identification of putative causal mutations. Most of these
mutations were identified in patients with more severe
forms of posterior or proximal hypospadias (Table 2). This
has led to the view that posterior hypospadias might more
likely be due to a monogenic aetiology when compared to
anterior or distal hypospadias, which could be polygenic or
multifactorial [1].
Indifferent stage
WT1 and SF1 genes play major role in development of the
urogenital system; mutations in these genes may cause not
only hypospadias but also other defects. Kohler et al. [10]
in 2009 found Sf1 mutations causing severe penoscrotal
hypospadias and chordee. Wang et al. [11] in 2004 showed
WT1 mutation in a boy with penoscrotal hypospadias and
micropenis.
Early patterning
The normal male genital development appears to be due to
interaction between urethral plate epithelium (UPE) the
genital tubercle (GT), which has polarising activity, con-
trolling a network of epithelial–mesenchymal interactions
Pediatr Surg Int (2015) 31:519–527 521

Table 1 More commonly known syndromes associated with hypospadias

Syndrome Gene or region Chromosomal location Percentage Inheritance
involved with
hypospadias

Smith Opitz Lemli DHCR7 Chromosome 11q13 50 % Autosomal recessive

WAGR or Chromosome 11p deletion WT1 11p13 – Micro-deletion in 11p13 region
syndrome
Hand Foot Genital syndrome HOXA 13 Chromosome 7p 14–15 – Autosomal dominant
Opitz G/BBB syndrome Midline 1 gene Deletions in chromosome Nearly all X linked recessive/Autosomal
22q11 dominant
Wolf Hirschorn syndrome Deletions in 4p – Gene(s) deletion in chromosome
chromosome 4 4p

Table 2 Summary of genetic mutations potentially involved in the
aetiology of hypospadias
Stage of development Genes involved
Indifferent stage or WT1, SF1
early embryonic
Early patterning BMP4, BMP7, HOXA4, HOXB6,
HOXA13, FGF8, FGF10, FGFR2
Masculinisation SRY, SOX9, AR, FKBP4, HSD3B2,
HSD17B3, SRD5A2, SRD5A1
Other genes ESR1, ESR2, ATF3, MAMLD1, MID1,
INSL3, BNC2
which when disturbed may lead to hypospadias [2]. Studies
by Beleza-Meireles et al. [12], in their study of 354 non-
syndromic boys from Sweden with hypospadias, found that
mutations are rare in these genes but gene variants may
influence the risk of hypospadias.
Masculinisation
SRY gene on the Y chromosome remains crucial for mas-
culinisation of the indifferent gonad [1]. SRY expression
induces a cascade of gene interactions involving SRY-box
9 (SOX 9), resulting in differentiation of the primitive
gonad into the testis. Garcia and Miranda in 2002 [9] found
sex chromosomal abnormalities in 4 out of 100 patients
with hypospadias, but no mutations in the SRY gene were
identified in 90 Chinese patients in a later 2004 study [11].
The AR (Androgen Receptor) gene has been extensively
investigated [13–16]. AR appears to be expressed in de-
veloping penis and urethra. Rare mutations in the gene
encoding AR in patients with hypospadias have been noted
[11, 13, 14, 17]. Also polymorphisms in AR have been
found to increase the risk of hypospadias. For example, the
expansion of polyglutamine (CAG) repeat in the N termi-
nus of AR decreases AR transactivation function leading to
under masculinisation [18, 19].
The 5 alpha-reductase type 2 gene (8.6 % of isolated
hypospadias), encoded by SRD5A2 gene on chromosome
2, converts testosterone to more potent dihydrotestosterone
and appears to be required for normal development of male
external genitalia [14]. Single nucleotide polymorphisms
(SNPs) in this gene have been associated with hypospadias
in some studies but not all [17, 20].
Other candidate genes
In addition to steroidogenesis, androgen versus oestrogen
balance remains important for development of normal male
genitalia. Oestrogen receptors 1 and 2 (ESR 1, ESR 2) are
found in the normal developing genital tubercle. These two
isoforms function analogously to the AR. They contribute
to sex steroid levels and activity and interact with AR to
regulate gene expression. Several SNPs in these genes have
been identified in population studies of hypospadias pa-
tients [12].
MAMLD1—Mastermind-like domain containing 1
(MAMLD1) gene, previously known as Chromosome 6
open reading frame 6 (CXorf6), seems to be expressed in
foetal Sertoli and Leydig cells around the critical period for
sex development. It is co-expressed with steroidogenic
factor (SF-1), a regulator of the transcription of genes in-
volved in testicular differentiation [1, 21, 22]. Mutational
studies in hypospadias patients have revealed several point
mutations in MAMLD1 gene [21–23].
Activating transcription factor (ATF) 3 is an oestrogen
responsive gene showing strong upregulation in hypospa-
dias patients [24]. A study of Swedish subjects showed that
ATF3 SNPs were significantly associated in 20 boys with
hypospadias compared to controls [24]. Two other studies
have shown a greater frequency of mutations or polymor-
phisms in cases than controls [25, 26].
To better understand these associations two genome-
wide association studies (GWAS), using individual geno-
typing of a large series of cases and controls, have been

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performed. The first, by van der Zanden et al. [27] in 2010, Table 3 Clinical, behavioural, occupational and environmental fac-
suggested that variants of the diacylglycerol kinase kappa tors investigated for their association with hypospadias in more than
one study
(DGKK) gene were strongly associated with hypospadias
A second GWAS, by Geller et al. [28] in 2014 investigated Factors frequently investigated
1006 isolated hypospadias patients compared with 5486
Factors consistently associated Factors consistently not associated
controls. Using replication genotying, the authors identified with hypospadias with hypospadias
18 loci associated with hypospadias of genome-wide sig- LBW/SGA Gestational diabetes
nificance as well as four suggestively associated loci, set- Placental insufficiency Maternal alcohol consumption
ting the scene for future sequencing studies to identify Maternal Hypertension
causative variants. Pre-eclampsia
Maternal intrauterine DES
exposure
Environmental factors and endocrine disruption Factors associated with Factors not associated with
hypospadias in most studies hypospadias in most studies
Research on environmental factors as a putative cause of ICS OCPs during pregnancy
hypospadias has been extensive but conclusive evidence on Prolonged TTP IVF
causation remains elusive (Table 3). High maternal BMI Hormonal stimulation for pregnancy
Primiparity induction
Multiple pregnancy Loratadine
Exogenous exposure to progestins and oestrogens
Pre-existing maternal diabetes Folic acid
Maternal use of antiepileptics Paternal age
Kallen et al. in 1992 and Carmichael et al. in 2005 ob-
Maternal smoking
served a twofold increased risk for hypospadias in women
Maternal exposure to water
who took progestins for preventing pregnancy loss [29, 30]. disinfection products
Despite this finding, progestins used in oral contraceptives Factors showing inconsistent results
have not been associated with hypospadias [29, 31]. This Preterm
inconsistency seems likely to be due to differences in both Iron supplementation
the dosage and type of progestins used for pregnancy sal- Maternal age
vage compared to that used in oral contraceptives, with Maternal vegetarian diet
differing mechanisms of action. Maternal fish consumption
Diethylstilbestrol (DES) is a synthetic form of the fe- Maternal and paternal exposure to pesticides
male hormone oestrogen. It was prescribed to pregnant Maternal occupational exposure to endocrine disruptors, heavy metals,
women between 1940 and 1971 to prevent miscarriage, phthalates
premature labour, and related complications of pregnancy. Maternal serum levels of PCB’s
In 1971, researchers linked prenatal DES exposure to clear Seasonal trend
cell adenocarcinoma in a small group of women [32]. The Factors not frequently investigated
daughters of women who used DES while pregnant, called Factors that seem to be associated Factors that do not seem to be
with hypospadias associated with hypospadias
‘‘DES daughters’’, have about 40 times the risk of devel-
Paternal subfertility Weight gain during pregnancy
oping clear cell adenocarcinoma of the lower genital tract
Absence of nausea and vomiting Maternal corticosteroids and
than unexposed women, in addition to other adverse out- in early pregnancy antibiotics
comes on the reproductive tracts of both male and female Bleeding during pregnancy Most paternal and maternal
offspring [33]. Complications during labour occupational exposures
In 2002, Klip et al. [34] found a 21-fold increased risk of Maternal use of
hypospadias in the male offspring of DES daughters, which antihypertensive
suggested that DES had a trans-generational effect in hu- Factors showing inconsistent results
mans. Other studies, although with a lesser increased risk, Early age at menarche
have confirmed this finding [35–38]. Proposed explanations Maternal thyroid disease
include that DES may cause genetic or epigenetic changes Fever in first trimester
in primordial oocytes, which are then transmitted to next Progestin/progestogens for threatened abortion
generation, or changes in somatic cells of the DES Paternal heavy metal exposure
daughters resulting in hormonal imbalance in adult life Living in rural or urban areas
[34]. Whilst perhaps less likely, Brouwers et al. [37] have Modified and reproduced with permission from Hum Reprod Update,
suggested that the influence of DES on the reproductive 2012, 18:260–83

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structures in the daughters interferes with normal foetal
male genitalia development.
Assisted reproductive technology
Only certain forms of Assisted Reproductive Technology
(ART) appear to be associated with hypospadias. Intracy-
toplasmic sperm injection (ICSI) has been found to be as-
sociated with an increased risk of hypospadias [4, 39–44],
whereas studies on In Vitro Fertilisation (IVF) either did not
report an increased risk or were inconclusive [39, 43–46].
Increased risk was also seen with hormonal stimulation in
Carmichael et al.’s study in 2005 [29]. The finding that ICSI
rather than IVF is associated with hypospadias suggests the
fact that paternal rather than maternal fertility problems
play a greater role in hypospadias [37, 38].
ART may also cause genomic imprinting disorders due
to epigenetic regulation interference. A study by Vottero
et al. [16] in 2011 showed that alterations in methylation
pattern at the androgen receptor gene, leading to an ab-
normal expression of the gene in foreskin tissue, might
contribute to the development of hypospadias. The objec-
tive of the study was to assess whether defects of the AR
gene (specifically CAG repeats and methylation pattern)
and DNA methyl-transferase (DNMT) family were present
in hypospadias patients. The study showed that AR gene in
target tissues from patients with hypospadias was more
methylated than in control children, resulting in a de-
creased expression of the AR gene. This alteration of the
AR gene might be involved in the pathogenesis of hy-
pospadias [16].
Maternal endogenous oestrogen levels
Free oestradiol increases with increasing Body mass index
and certain diets and a higher BMI have been associated
with hypospadias in several studies [47–50] but not all
[38]. Perhaps confusingly, Rankin et al. [51] in 2010, in a
small study of 8 cases found an association with under-
weight mothers but not for overweight or obese women.
First pregnancy and multiple pregnancies have been
associated with hypospadias, with higher oestrogen levels
the suggested cause [52, 53]. Most studies have found that
women in their first pregnancy [15, 46, 54–63] or with a
twin or triplet [37, 38, 43, 46, 54–56, 61, 64, 65] were at
increased risk of having a son with hypospadias but several
studies have not replicated these findings for primiparity or
for multiple pregnancies [57, 62, 63, 66, 67].
Pregnancy related
Placental HCG is needed for commencement of the foetal
testicular steroid production, prior to the establishment of
523
the foetal pituitary gonadal axis. Placental insufficiency
may result in inadequate beta HCG stimulation of foetal
testicular steroidogenesis and may cause hypospadias. This
explains the association of intrauterine growth retardation
(IUGR) and hypospadias [3, 48, 55, 58, 63, 64, 68].
Placental insufficiency increases risk of hypospadias.
Stoll et al. in 1990 [69] found a direct relation between
hypospadias and low placental weight whilst Fujimoto
et al. in 2008 [70] demonstrated an increased occurrence of
placental infarction in extremely low birth weight infants
with hypospadias. The association in LBW infants with
hypospadias seems to be stronger for more posterior forms
of hypospadias, with more posterior hypospadias also as-
sociated with more severe IUGR [38, 57, 67, 71, 72].
Nausea in early pregnancy implies a good HCG surge
[73]. Vomiting and nausea in early pregnancy has been
associated with a decreased hypospadias risk [48, 56]. This
may be explained by the fact that nausea is due to HCG
surge and this implies the absence of placental insufficiency.
Studies have reported a two–threefold increased risk of
hypospadias in infants of mothers affected by pre-
eclampsia and hypertension, perhaps as a result of a
compromised placental circulation [74–76].
Maternal smoking
A study by Brouwers et al. [37] showed an increased risk of
hypospadias with maternal smoking. In contrast, a recent
systematic review by Hakonsen et al. [77] concluded that
there was no clear link between maternal smoking and
hypospadias, with previous links perhaps the result of the
association between smoking and several important co-
factors, including education, socioeconomic status and
other confounding factors. Interestingly, Pierek et al. in
2004 [78] also suggested an increased incidence of hy-
pospadias with paternal smoking, although the same
problem co-factors similarly applies.
Diet and pesticides
A 2009 meta-analysis of nine studies evaluating occupa-
tional exposure to pesticides and hypospadias reported a
small positive association with maternal exposure (Pooled
risk ratios (PRR) of 1.36, CI = 1.04–1.77), and paternal
exposure (PRR of 1.19, CI = 1.00–1.41) to pesticides [79].
Diet constitutes a significant exposure route to pesticides
and their residues. This appears more frequently reported in
conventional as opposed to ‘organic’ food products. Few
studies have examined diet as a potential source of expo-
sure to Endocrine disruptive chemicals (EDCs). Maternal
choice of an organic alternative to food items during
pregnancy was not associated with a decreased risk of
hypospadias in the offspring [80].
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One study reported an apparent association with a high
intake of maternal Vitamin E, whilst the epidemiological
study by Giordano et al. suggested associations with fish,
liver and fresh market fruits [50, 81]. Maternal vegetarian
diet and high intake of legumes (high levels of lignans, a
type of phytoestrogen) were associated with increased risk
of hypospadias [82]. This finding supports the potential
connection between EDC exposures and hypospadias. Soy
products containing genistein have been shown to induce
hypospadias in a murine model [83].
Maternal drug use
Some of the drugs evaluated include the anti-epileptic
sodium valproate, the anti-histamine loratadine, the opioid
anti-motility drug loperamide, corticosteroids, antipsy-
chotics, antifungals [84, 85]. Studies have reported an as-
sociation with valproate and hypospadias. This may be
attributed to the gonadotropin release hormone agonism and
the resulting anti-androgenic effect. Corticosteroids, which
also have anti- androgenic properties, and loratadine, which
has oestrogenic property, can induce hypospadias ex-
perimentally, although this has not been substantiated in
humans [86–91]. Loperamide [92] antiretroviral therapy
[93] antihypertensives [74] nystatin [94] and paroxetine [95]
have all been associated with increased risk of hypospadias
but most associations were only been reported once.
Conclusion
The aetiology of anterior or distal hypospadias seems likely
to be multifactorial, with interactions between genetic and
environmental factors. Single genetic mutations causing
hypospadias would more probably lead to severe posterior
or proximal hypospadias and associated with other
abnormalities.
Many candidate genes have been implicated in hy-
pospadias, but have generally only reported small numbers
of cases and control patients and with findings that have
often not been able to be replicated in subsequent studies,
perhaps as a result of differences in patient populations. As
a result, any suggestion of causality needs to be interpreted
with caution. More recent reports of the use of GWAS,
including replication genotyping, appear more promising in
terms of identification of causative genes.
Normal male genitalia development requires a delicate
balance between androgens and estrogens. ART and EDCs
have tended to dominate the environmental factors that
may be involved in the pathogenesis of hypospadias. It is
important for foetal medicine specialists as well as sur-
geons to emphasise the potential for genital malformations,
including hypospadias, in male foetuses conceived by
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Pediatr Surg Int (2015) 31:519–527
ART. The fact that hypospadias is associated with ICSI
[26–32] suggests that paternal fertility issues play a major
role. Research should focus on identifying risk factors for
hypospadias in ART and provide a perfect hormonal milieu
for normal male genital development.
Murine models have suggested that EDCs can cause
hypospadias [84]. This cannot be readily extrapolated to
humans as hormone levels are likely to be quite different
and other factors may play a role in addition to the dis-
turbed hormonal milieu. A consistent association with
LBW, maternal hypertension and pre-eclampsia suggests
that placental insufficiency and deficient beta HCG have a
role to play in hypospadias aetiology.
Other environmental factors, including the impact of
pesticides, have not shown a consistent association in most
studies. Future hypospadias research needs to focus more
on the interaction between genetic and environmental
factors as well as gene-to-gene interactions. Eliminating
potential genetic factors and hormonal imbalance will be
the preventive for hypospadias in ART male foetuses.
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