BJU International
Volume 90 Issue 8 Page 769 - November 2002

Intersex
I.A. Hughes


Introduction

The term intersex conventionally refers to the appearance of the
external genitalia being at variance with normal development for
either sex and to present a problem of sex assignment. Generally the
problem is evident at birth, manifesting as ambiguous genitalia of
the newborn. Puberty is another stage when an intersex disorder may
arise by the masculinizing effects on the external genitalia of a
child hitherto raised as a female. How to establish a cause for
intersex and thereby formulate a management plan is probably one of
the most challenging clinical conditions for the practising
paediatric endocrinologist and appropriate surgical colleague.
Management in the longer term needs the skills of counsellors trained
and experienced in psychosexual issues affecting children and
adolescents. Genetic issues dominate the pathophysiology of intersex
and genetic counselling should be available to families needing
advice about recurrence risks, carrier detection and options for
prenatal diagnosis, and perhaps prenatal treatment. Clearly, the
management of intersex is complex and a multidisciplinary team
network of appropriate professionals is mandatory.

The approach to investigating and managing intersex requires an
understanding of normal prenatal sexual development. The complexity
of intersex management has been compounded by the use of confusing
terminology and lack of clarity in the use of definitions. For the
purpose of this review, sex determination refers to the formation of
the gonad (testis or ovary) and sex differentiation refers to the
formation of the genital phenotype (internal and external) consequent
upon hormonal effects. The composite of these two processes defines
sex development; further hormone-induced amplification of genital
phenotype occurs at puberty, followed by the acquisition of
reproductive potential. Sex outcome at birth is the result of a co-
ordinated and sequential series of developmental events controlled by
a network of temporally expressed genes and hormones. How an intersex
disorder may occur can be ascertained logically when normal sex
determination and differentiation is understood. When the
classification and causes of intersex are considered in this review,
the terminology will refer to the masculinized female, the under-
masculinized male and the true hermaphrodite as three principal
classes of disorders. It is recommended that the
terminology 'pseudohermaphrodite' with male or female prefixes
currently has no place in an enlightened era of intersex management
involving closer collaboration with the patient and family.
Geneticists, particularly those working on identifying sex-
determining genes, prefer the terms 'complete' and 'partial' sex
reversal, prefixed by the sex chromosomes (XY or XX).

Embryology of prenatal sex development

There is compelling evidence that internal and external prenatal sex
development is constitutively female. The studies by Jost [1] in
rabbit embryos showed female phenotypic development when the ovary or
testis was excised, whereas a male phenotype could be restored with
testis engrafting. The imprint of his seminal work on the
understanding mammalian sex development is summarized in Fig. 1. The
dual formation of internal genital ducts in both sexes is described
in standard texts [2-4]; they comprise the Wolffian and Müllerian
ducts. The former develop from the mesonephric tubules and
differentiate into the vas deferens, epididymis and seminal vesicles
under the influence of high concentrations of androgens
(testosterone) acting locally. The Müllerian ducts stabilize in
the absence of anti-Müllerian hormone (AMH, secreted by Sertoli
cells) and form the uterus, Fallopian tubes and upper part of the
vagina.
While not generally regarded as a problem of intersex, the persistent
Müllerian duct syndrome in otherwise normally developed males is
caused by mutation of either the AMH or the AMH receptor gene [5].
External genital development is also common to both sexes initially,
the genital tubercle only enlarging from 10 weeks of gestation
onwards in the male as a result of androgen action. The genital
swellings later develop either into the scrotum or labia majora,
depending on exposure to androgens. In the developing male, the
urethral folds fuse ventrally to form the urethra, with the
urogenital orifice finally sited at the tip of the glans penis.
Failure in this developmental pathway results in hypospadias, the
commonest congenital anomaly in males.

Formation of the gonad precedes the events of internal and external
genital development by several weeks. A thickening of the coelomic
mesoderm on the medial aspect of the urogenital ridge at around 5-6
weeks of gestation is the first indication of a gonad which, at this
stage, is histologically indifferent. The development is juxtaposed
with mesonephric cells of the primitive kidney. The close
interdependence between gonad and kidney development is illustrated
by the absence of gonads and kidneys in mice with targeted disruption
of the wt-1 gene which prevents thickening of the mesoderm in the
urogenital ridge [6]. The human Denys-Drash and Frasier syndromes
resulting from mutations in WT-1 are characterized by renal disorders
and varying degrees of gonadal dysgenesis [7,8].

The first histological evidence for testis development is the
appearance of seminiferous cords from the condensation of primary sex
cords together with Sertoli cells. This occurs at 7 weeks'
gestation; by contrast, the ovary is not defined for about another 4
weeks. Interstitial cells derived from the mesenchyme are present by
9 weeks and differentiate as the steroid-secreting Leydig cells.
Primordial germ cells migrate to the site of gonad formation from the
wall of the yolk sac, via the mesentery of the hindgut. The migratory
pathway is dependent on chemo-attractants and cell adhesion
molecules, a process still to be fully explained [9,10]. While gonad
determination will take place even if germ cells fail to migrate,
there may be some sex-dependence to the pattern of germ cell
migration.

Prenatal male sex development is completed by the migration of the
testis into the scrotum; this is a two-stage process of
transabdominal migration and inguinoscrotal descent [11]. The first
phase of descent, at least in the mouse, is controlled by an insulin-
like peptide relaxin produced by the testis; when the gene encoding
this protein is disrupted, bilateral cryptorchidism occurs [12].
However, studies of the human homologue of this gene in boys with
undescended testes have only rarely reported mutations [13,14].
Inguinoscrotal descent is androgen-dependent, as exemplified by
abnormal gonadal positions in hypogonadotrophic hypogonadism and
syndromes of androgen insensitivity [15,16]. Sex determination and
sex differentiation is dependent in the male on a coordinated
interplay of temporally related genetic and hormonal control events.
The embryological events are schematically summarized in Fig. 2; the
following section briefly describes the key genes and hormones
involved in prenatal male sex development.

Genetic and hormonal control of fetal sex development

Sex development is constitutively female so that the active
imposition of male development requires factors to determine the
testis, regress the Müllerian ducts and differentiate the internal
and external genitalia as male. There is a panoply of genes involved
in testis determination, many yet to be identified. Syndromes of sex
reversal and studies on mouse embryos have been critical in
identifying key genes (see [17-19] for detailed reviews). The SRY
gene (sex-related gene on the Y chromosome) is central to testis
formation, as exemplified by the presence of testes in XX males and
the male outcome after transgenic insertion of the sry gene into XX
mouse embryos. Pairing of X and Y chromosomes at their
pseudoautosomal regions occurs during paternal meiosis. As SRY is
located close to the pseudoautosomal boundary, the gene can become
translocated to the X chromosome if X-Y interchange of genetic
material extends beyond the pseudoautosomal boundary. SRY encodes a
protein which contains a central region of 80 amino acids and is
homologous with a large family of high mobility group (HMG) nuclear
proteins. The SRY protein functions as a transcription factor by
bending DNA, thereby promoting protein-protein interactions.
Mutations in SRY are associated with gonadal dysgenesis and complete
XY sex reversal (Swyer's syndrome). However, only 15-20% of such
patients have an SRY mutation, thereby indicating that other genes
must be involved in testis determination. One such gene is SOX9,
which also encodes a protein containing an HMG-related motif of amino
acids, and is a transcription factor. Mutations in SOX9 lead to a
syndrome of campomelic dysplasia (severe thoracic and limb skeletal
defects) with gonadal and genital abnormalities in most. SOX9 is
probably activated by SRY as both genes are expressed in fetal
Sertoli cells in a temporally related fashion. However, other genes
must be regulated by these two key transcription factors and remain
to be identified. Notably, SOX9 up-regulates AMH gene expression.

Have any ovarian-determining genes been identified? So far, no genes
whose products direct the development of the ovary have been
identified. However, genes which appear to operate in an 'anti-
testis' fashion are described. Duplication of the short arm of the X
chromosome can lead to complete XY sex reversal. A gene identified in
this region, DAX1, is a member of the nuclear hormone receptor
family. It is postulated that over-expression of DAX1 either inhibits
SRY directly or inhibits its action as an up-regulator of SOX9.
Another gene, WNT4, located on chromosome 1p34 appears to be an anti-
testis gene based on sex reversal in an XY female with duplication of
1p31-p35. Both DAX1 and WNT4 are expressed in the fetal testis and
ovary initially, but persist only in the ovary. A simplified summary
of the key factors involved in gonad determination is shown in Fig. 3.

Fetal development of the female phenotype does not require
oestrogens. In contrast, male sex differentiation requires the
production of high concentrations of androgens during a critical
period (Fig. 2). Furthermore, the principal androgens, testosterone
and DHT, mediate their developmental effects through binding to a
specific androgen receptor (AR) in target tissues. Androgens are
synthesized by the Leydig cells, initially autonomously, but then
dependent on placental hCG secretion. Later in gestation with
declining hCG concentrations, androgen synthesis is controlled by LH
secretion from the fetal pituitary gland. Growth of the phallus
occurs during this later stage of gestation, hence the occurrence of
micropenis typically observed in a male infant with congenital
hypopituitarism. Optimal androgen production and action sufficient to
differentiate the internal and external genitalia is dependent on an
intact membrane bound LH/hCG receptor on Leydig cells, a sequence of
enzyme steps to synthesise testosterone from cholesterol, the
conversion of testosterone to its more potent metabolite DHT, and
finally androgen-induced (testosterone and DHT) activation of the AR
transcription factor. Logic dictates that a defect in any one of
these components would result in an XY intersex phenotype.

Intersex disorders - a pragmatic classification

Table 1 shows a simple classification of intersex which is both
descriptive and includes broad aetiological categories. Congenital
adrenal hyperplasia (CAH) and the other rare causes of a masculinized
female are not strictly disorders of sex determination and sex
differentiation.

The external genitalia in affected males are generally normal at
birth, thereby exposing them to a life-threatening adrenal crisis. It
is for this reason that many countries now screen newborns for CAH
using filter-paper blood-spot 17OH-progesterone measurements [22]. No
screening is currently undertaken in the UK.


The question of when surgery should be used in an infant with CAH and
ambiguous genitalia, and which procedure to be adopted, is the
subject of ongoing and unresolved debates [26-29]. There is no
unanimity on whether surgery should be early (2-6 months) or later (1-
2 years).



Intersex with an XY karyotype

An infant with ambiguous genitalia in whom the karyotype is 46,XY is
a problem for both diagnosis and management. Typically, there is
micropenis with chordee, perineo-scrotal hypospadias and a bifid
scrotum. Gonads may or may not be palpable. It has been reported that
the presence of impalpable gonads and severe hypospadias makes an
intersex state more likely, and warrants a karyotype test [31,32].
Intersex is not a diagnosis; even when XY infants with the signs
previously ascribed to intersex are investigated in detail, an
underlying specific cause is established only in a few. In general,
failure of masculinization of the external genitalia can result from
either inadequate production of, or response to, androgens.

Defects in androgen biosynthesis

Testosterone is synthesized in Leydig cells under the trophic control
of hCG in early gestation and later, fetal pituitary LH secretion.
Both peptides bind to a seven-transmembrane LH receptor to activate
intracellular steroidogenesis. The starting substrate is cholesterol
which is converted via a series of enzymatic steps to testosterone.
This androgen is also further metabolized to the more potent androgen
DHT, via the 5-reductase type 2 enzyme. Key steps necessary for
optimal androgen production are shown in Fig. 5, together with the
genes which, when mutated, cause varying degrees of XY sex reversal.

Inactivating mutations of the LH receptor can give rise to a range of
phenotypes which includes complete sex reversal, ambiguous genitalia
or isolated hypospadias [33]. How a single mutation type can cause
such a range of sex reversal is not currently known. Histology of the
gonads shows Leydig cell hypoplasia; serum LH levels are elevated and
there is no testosterone response to stimulation with hCG.

An intriguing phenomenon of 'double sex reversal' is observed in the
androgen biosynthetic disorders, 17-hydroxysteroid dehydrogenase
deficiency and 5-reductase deficiency [34,35]. In both disorders, the
external genitalia at birth are either female or partially
masculinized, yet there is marked virilization at puberty with intact
testes. It is not clear why masculinization does not occur prenatally
in response to hCG/LH stimulation. However, each of these two enzymes
operate as isoenzymes and mutations in only one isoenzyme cause
androgen biosynthetic disorders (type 3 and type 2 in the case of 17-
hydroxysteroid dehydrogenase and 5-reductase, respectively. It is
postulated that substrate usage by alternative isoenzymes results in
sufficient androgen production at puberty to cause virilization.
These are considerations which need to be borne in mind in the
context of the sex of rearing if a diagnosis is established soon
after birth. Figure 5 illustrates that measuring plasma
concentrations of androstenedione, testosterone and DHT before and
after hCG stimulation is essential to the biochemical diagnosis of
these enzyme deficiencies. Chromatographic analysis of androgen
metabolites in urine is also helpful, particularly for 5-reductase
deficiency. Once the clinical and biochemical parameters for
diagnosis have been established, mutation analysis of the 17HSD3 or
the 5RD2 genes is available in certain specialized centres.

Defects in androgen action

The androgen insensitivity syndrome (AIS) is a paradigm of resistance
to the action of a hormone, in this case androgens. The complete form
(complete AIS) is manifest as complete XY sex reversal and is not a
problem of intersex. However, the partial form of the syndrome
(partial AIS) is the commonest cause of fetal male under-
masculinization [19]. Partial AIS is defined as a defect in androgen
action of variable degree in an XY male with normal testis
determination, testosterone biosynthesis and metabolism of DHT. The
phenotype can range widely to include ambiguous genitalia, simple
hypospadias and even normal genital development in a male with
infertility.

Androgen action in target tissues, including the genitalia, is
mediated by ligand activation of the nuclear AR. This transcription
factor up-regulates androgen-responsive genes to elicit the
biological actions typical of androgens, i.e. muscle and skeletal
development, linear growth, genital development and voice changes at
puberty, stimulation of skin integuments, erythopoiesis and in
adults, spermatogenesis. These pleiotropic effects are assumed to
occur as a result of androgen binding to a single AR. The AR gene is
located on Xq11-12; complete AIS is invariably the result of
mutations in this gene [36]. Only a minority of patients who fulfil
the criteria to define partial AIS have identifiable mutations in the
gene [37]. As the AR is a transcription factor it is possible that
the same phenotype can arise from abnormalities in androgen-
responsive genes. These genes remain to be identified.

Once a mutation has been identified it is possible to recreate the
abnormal receptor by site-directed mutagenesis to study function in
vitro. While such work is of considerable value to analyse structure-
function relationships, the correlation between genotype and
phenotype in partial AIS is extremely variable. Nevertheless, the
presence of an AR gene mutation does not necessarily imply no
response to high doses of androgens given in vivo to a patient with
partial AIS.

Intersex: chromosome abnormalities and gonadal dysgenesis

Ambiguous genitalia of the newborn may occur as a result of
chromosome abnormalities or from a partial form of XY gonadal
dysgenesis. The commonest chromosome abnormality is 45XO/46XY
mosaicism associated with mixed gonadal dysgenesis. The phenotype may
include signs consistent with features of Turner syndrome
(particularly short stature) as well as the abnormal genital
development. Sometimes there may be normal male development; this is
consistent with the observation that most instances of XO/XY
karyotypes established prenatally are found in normal phenotypic
males [38]. The paediatric endocrinologist and surgeon therefore are
consulted on a skewed population of XO/XY patients. One gonad, at
least, can produce adequate testosterone levels after hCG
stimulation. The concomitant streak gonad should be removed because
of the risk of malignancy. A 46XX/46XY karyotype can be associated
with a variable phenotype, depending on the nature of the gonads.
True hermaphroditism is a histological diagnosis based on the
presence of ovarian (including follicles) and testicular tissue in
the individual. Internal and external genital development can be
variable. The most frequent karyotype is 46,XX.

Syndromes and intersex

The number of syndromes which include a genital anomaly, usually
hypospadias, is legion. The genetic abnormality has been identified
in some, e.g. Smith-Lemli-Opitz, Robinow and Rubinstein-Taybi
syndromes. Rarely are mutations identified in these genes when
analysed in patients with isolated genital anomalies.

Hypospadias is not an intersex disorder but familial forms of
hypospadias may occur from mutations in known genes such as the AR
gene. There are families with non-X-linked forms of hypospadias,
including affected sibling pairs and father-son pairs. Further
studies are needed to identify possible autosomal gene loci for this
common congenital anomaly.

Intersex and the environment

In recent years it has become necessary to consider the role of
environmental factors in the causes of male reproductive tract
disorders [39]. There is evidence from examining wildlife and
epidemiological studies in man that chemicals in the environment may
act as endocrine disrupters during critical stages of genital
development. A testicular dysgenesis syndrome has been hypothesized
to occur in males to account for the fall in sperm count and quality,
coupled with an increase in testis cancer, hypospadias and
undescended testes. The causal agent(s) inducing dysgenesis of the
testis have oestrogen-like or anti-androgenic properties; there is
abundant experimental evidence in vitro and in animals to show that
numerous compounds to which the population is exposed have these
properties. The effects are not so severe as to elicit a state of
intersex; nevertheless there was a clear association between the
occurrence of hypospadias in a cohort of 7000 male births in the
South-west of England and the frequency of mothers who were
vegetarian during their pregnancy [40].

Intersex - the initial investigation

There are numerous investigations which may need to be undertaken,
subsequent to an initial screen for the infant with ambiguous
genitalia. There is no substitute for an initial adequate history and
physical examination. Family history and information about exposure
to potential teratogenic compounds are particularly relevant. The
examination must also consider the relevance of any extra-genital
anomalies.

The initial investigations which should be undertaken are:

Chromosomes: fluorescent in situ hybridisation on interphase spreads
with X,Y specific probes; full karyotype.
Hormones: serum 17OH-progesterone at 24-48 h; serum testosterone;
plasma renin; save serum, DNA, urine.
Biochemistry: serum electrolytes, plasma glucose.
Imaging: pelvic ultrasonography for uterus/cervix; renal
ultrasonography.

It is mandatory to undertake a karyotype in an apparently normal male
with bilateral impalpable testes; it is also the author's view that
the karyotype should be assessed in a male with hypospadias and
impalpable testes. Further investigations may be required, consequent
on the karyotype result and 17OH-progesterone level:

Chromosomes: high-resolution karyotype (mosaicism, e.g. XO/XY).
Fluorescent in situ hybridisation studies (mosaicism).
Possible karyotype in fibroblasts.
Hormones: serum gonadotrophins, serum AMH; hCG stimulation test (1500
U daily for 3 days); androstenedione, testosterone, DHT; urinary
steroid profile.
Imaging: pelvic ultrasonography; MRI (locate gonads); urogenital
sinogram (also urethroscopy).
Genital: skin biopsy (at the time of surgery); androgen binding
studies; DNA/mRNA for mutation analysis; gonadal biopsy (at the time
of surgery).


The most difficult is the XY intersex infant; the hCG stimulation
test is useful as a marker of the presence of testes and whether they
produce androgens normally. The information may also predict what is
likely to occur at puberty. Serum AMH measurement has been proposed
as a reliable marker of the presence of testes [41,42], but the test
is not yet universally available.

Conclusion

Intersex or ambiguous genitalia of the newborn is a rare disorder
which needs prompt investigation, logically based on a sound
knowledge of normal sex determination and differentiation. The
complexity of the problem requires a multidisciplinary team working
in specialist centres. Longer-term management was not the focus of
this review. Nevertheless, the personnel involved in decisions on the
sex of rearing must be aware of changes in practice which continue to
develop, particularly in the timing and nature of any surgical
intervention [43].

References

1 Jost A. Problems of fetal endocrinology: the gonadal and
hypophyseal hormones. Rec Progr Horm Res 1953; 8: 379-418
2 Langman J. Special embryology: urogenital system. In Medical
Embryology: Human Development Normal and Abnormal. 2nd Edn.
Baltimore: Williams & Wilkins, 1969
3 Hughes IA. Intersex. In Freeman NV, Burge DM, Griffiths DM,
Malone PSJ eds, Surgery of the Newborn. Edinburgh: Churchill
Livingstone, 1994: 781-91
4 Pinsky L, Erickson RP, Schimke RN. Genetic Disorders of Human
Sexual Development. Oxford: Oxford University Press, 1999
5 Rey R, Picard J-Y. Embryology and endocrinology of genital
development. Bailliere's Clin Endocrinol Metabolism 1998; 12: 17-33
6 Hastie ND. The genetics of Wilms' tumour - a case of disrupted
development. Ann Rev Genet 1994; 28: 523-8
7 Little M, Wells C. A clinical overview of WT1 gene mutations.
Human Mutat 1997; 9: 209-25
8 Koziell A, Charmandari E, Hindmarsh PC, Rees L, Scrambler P,
Brook CGD. Frasier syndrome, part of the Denys Drash continuum or
simply a WT1 gene associated disorder of intersex and nephropathy?
Clin Endocrinol 2000; 52: 519-24
9 Anderson O, Heasman J, Wylie C. Early events in the mammalian
germ line. Int Rev Cytol 2001; 203: 215-30
10 Molyneaux KA, Stallock J, Schaible K, Wylie C. Time-lapse
analysis of living mouse germ cell migration. Dev Biol 2001; 240: 488-
98
11 Hutson JM, Beasley SW. Descent of the Testis. London: Edward
Arnold, 1992
12 Zimmermann S, Stedig G, Emmen JM et al. Targetted disruption
of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol
1999; 13: 681-91
13 Tomboc M, Lee PA, Mitwally MF, Schneck FX, Bellinger M,
Witchel SF. Insulin-like3/relaxin-like factor (RLF) gene: no
relationship with cryptorchidism. Ped Res 2000; 47: 538-41
14 Lim HN, Raipert-de Meyts E, Skakkeback NE, Hawkins JR, Hughes
IA. Genetic analysis of the INSL3 gene in patients with maldescent of
the testis. Eur J Endocrinol 2001; 144: 129-37
15 Bernstein L, Pike MC, Depue HL, Ross RK, Moore JW, Henderson
BE. Maternal hormone levels in early gestation of cryptorchid males:
a case control study. Br J Cancer 1988; 58: 379-81
16 Lim HN, Hughes IA, Hawkins JR. Clinical and molecular evidence
for the role of androgens and WT1 in testis descent. Mol Cell
Endocrinol 2001; 185: 33-41
17 Koopman P. The genetics and biology of vertebrate sex
determination. Cell 2001; 105: 843-7
18 Clarkson MJ, Harley VR. Sex with two SOX on. SRY and SOX9 in
testis development. Trends Endocrinol Metab 2002; 13: 106-11
19 Ahmed SF, Hughes IA. The genetics of male
undermasculinisation. Clin Endocrinol 2002; 56: 1-18
20 Acerini CL, Hughes IA. 21-Hydroxylase deficiency defects and
their phenotype. In Hughes IA, Clark AJL eds, Adrenal Disease in
Childhood: Clinical and Molecular Aspects. Basel: Karger, 2000: 93-111
21 White PC, Speiser P. Congenital adrenal hyperplasia due to 21-
hydroxylase deficiency. Endocr Rev 2000; 21: 245-91
22 Honour JW, Torresani T. Evaluation of neonatal screening for
congenital adrenal hyperplasia. Horm Res 2001; 55: 206-11
23 Forest M. Prenatal diagnosis, treatment, and outcome in
infants with congenital adrenal hyperplasia. Curr Opinin Endocrinol
Diabet 1998; 4: 209-17
24 New MI, Carlson A, Obeid J et al. Prenatal diagnosis for
congenital adrenal hyperplasia in 532 pregnancies. J Clin Endocrinol
Metab 2001; 86: 5651-7
25 Lajic S, Wedell A, Bui T, Ritzen EM, Holst M. Long-term
somatic follow-up of prenatally treated children with congenital
adrenal hyperplasia. J Clin Endocrinol Metab 1998; 83: 3572-880
26 Farkas A, Chertin B, Hadas-Helpren I. 1-Stage feminising
genitoplasty. 8 years of experience with 49 cases. J Urol 2001; 165:
2341-6
27 Creighton S, Minto C. Managing intersex. Editorial. Br Med J
2001; 323: 1264-5
28 Melton L. New perspectives on the management of intersex.
Lancet 2001; 357: 2110
29 Hrabovsky Z, Hutson JM. Surgical treatment of intersex
abnormalities: a review. Surgery 2002; 131: 92-104
30 Grumbach MM, Auchus RJ. Estrogen: consequences and
implications of human mutations in synthesis and action. J Clin
Endocrinol Metab 1999; 84: 4677-94
31 Kaefer M, Diamond D, Hendren WH et al. The incidence of
intersexuality in children with cryptorchidism and hypospadias:
stratification based on gonadal palpability and meatal position. J
Urol 1999; 162: 1003-7
32 McAleer IM, Kaplan GW. Is routine karyotyping necessary in the
evaluation of hypospadias and cryptorchidism? J Urol 2001; 165: 2029-
32
33 Themmen APN, Huhtaniemi IT. Mutations of gonadotrophins and
gonadotropin receptors: elucidating the physiology and
pathophysiology of pituitary-gonadal function. Endocr Rev 2000; 21:
551-83
34 Moghrabi N, Hughes IA, Dunaif A, Andersson S. Deleterious
missense mutations and silent polymorphism in the human 17-
hydroxysteroid dehydrogenase 3 gene (HSD173). J Clin Endocrinol Metab
1998; 83: 2855-60
35 Russell DW, Wilson JD. Steroid 5 alpha-reductase: two
genes/two enzymes. Ann Rev Biochem 1994; 63: 25-61
36 Ahmed SF, Cheng A, Dovey LA et al. Phenotypic features,
androgen receptor binding and mutational analysis in 278 clinical
cases reported as androgen insensitivity syndrome. J Clin Endocrinol
Metab 2000; 85: 658-65
37 Morel Y, Rey R, Teinturier C et al. Aetiological diagnosis of
male sex ambiguity: a collaborative study. Eur J Pediatr 2002; 161:
49-59
38 Chang JH, Clark RD, Bachman H. The phenotype of 45,X/46,XY
mosaicism: an analysis of 92 prenatally diagnosed cases. Am J Hum
Genet 1990; 46: 156-67
39 Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular
dysgenesis syndrome: an increasingly common developmental disorder
with environmental aspects. Human Reprod 2001; 16: 972-8
40 Hughes IA, Northstone K, Golding J and the ALSPAC Study Team.
Reduced birth weight in males with hypospadias. an index of androgen
dysfunction? Arch Dis Child 2002; 87: F150-1
41 Lee MM, Donahoe PK, Silverman BL et al. Measurements of serum
Mullerian inhibiting substance in the evaluation of children with
nonpalpable gonads. N Engl J Med 1997; 336: 1480-6
42 Rey RA, Belville C, Nihoul-Fékété C et al. Evaluation
of gonadal function in 107 intersex patients by means of serum anti-
Müllerian hormone measurement. J Clin Endocrinol Metab 1999; 84:
627-31
43 Diamond M. Pediatric management of ambiguous genitalia and
traumatized genitalia. J Urol 1999; 162: 1021-8

Abbreviations
AMH anti-Müllerian hormone
HMG high mobility group
AR androgen receptor
CAH congenital adrenal hyperplasia
AIS androgen insensitivity syndrome.