1. A P r a c t i c a l A p p roa c h
to Ambiguous
Genitalia i n t he
Newborn Period
Sarah M. Lambert, MDa, Eric J.N. Vilain, MD, PhDb,
Thomas F. Kolon, MDa,*
KEYWORDS
Ambiguous genitalia Congenital adrenal hyperplasia
Disorders of sex development Neonates
CHAPTER characterize DSD and subcategories included
male pseudohermaphrodite, female pseudoher-
The evaluation and management of a newborn maphrodite, and true hermaphrodite. These terms
with ambiguous genitalia must be undertaken used gender in the nomenclature and were often
with immediacy and great sensitivity. The pediatric considered controversial or disparaging. There-
urologist, endocrinologist, geneticist, and child fore a revised nomenclature was proposed that
psychiatrist or psychologist should work closely incorporated genetic etiology and descriptive
with the family in pursuing a dual goal: to establish terminology while removing gender references.4
the correct diagnosis of the abnormality and, with The main categories include sex chromosome
input from the parents, determine gender based DSD, 46,XX DSD, and 46,XY DSD. Some condi-
on the karyotype, endocrine function, and tions can be placed into more than one category.
anatomy of the child. In this section the authors Additionally, although the majority of infants with
outline a practical approach to the neonate born 46,XX DSD will be diagnosed with congenital
with a disorder of sex development (DSD). adrenal hyperplasia (CAH), only approximately
50% of children with 46, XY DSD will have a defin-
Nomenclature itive clinical diagnosis.5
Genital ambiguity in the neonate has been
described for centuries and evidence for disorders
Diagnosis
of sexual differentiation exists from many ancient
civilizations.1 The actual incidence of DSD is diffi- Chromosomal sex is established at fertilization
cult to accurately determine because of the and the undifferentiated gonads subsequently
heterogeneity of the clinical presentation and the develop into either testes or ovaries. A child’s
varied etiologies. Using birth registries, some phenotypic sex results from the differentiation of
authors have attempted to estimate the incidence internal ducts and external genitalia under the
of ambiguous genitalia at birth; The estimated inci- influence of hormones and transcription factors.
dence of clinically detectable ambiguous genitalia Any discordance among these processes results
at birth in Germany is 2.2 per 10,000 births.2 in ambiguous genitalia or DSD. Currently, the
Congenital adrenal hyperplasia is estimated to main categories of DSD are 46,XX DSD, 46,XY
occur in approximately 1 per 16,000 births.3 DSD, sex chromosome DSD, ovotesticular DSD,
Historically, the term intersex was used to and 46,XX testicular DSD.
urologic.theclinics.com
a
Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, 39th Street and Civic
Center Boulevard, Philadelphia, PA 19104, USA
b
David Geffen School of Medicine at UCLA, 695 Charles Young Drive South, Los Angeles, CA 90095, USA
* Corresponding author.
E-mail address: kolon@email.chop.edu
Urol Clin N Am 37 (2010) 195–205
doi:10.1016/j.ucl.2010.03.014
0094-0143/10/$ – see front matter ª 2010 Elsevier Inc. All rights reserved.

2. 196 Lambert et al
46,XX disorder of sex development CAH resulting in genital ambiguity in boys is dis-
Girls with 46,XX DSD, the most common DSD, are cussed in detail later in this article.
¨
46,XX with normal ovaries and Mullerian deriva- The most common cause of CAH is inactivation
tives. The sexual ambiguity is limited to masculin- of CYP21, which catalyzes the conversion 17-OH
ization of the external genitalia that occurs as progesterone to 11-deoxycortisol, a precursor of
a result of exposure to androgens in utero. cortisol, and the conversion of progesterone to de-
Congenital adrenal hyperplasia (CAH), which oxycorticosterone, a precursor of aldosterone. A
accounts for the majority of patients with 46,XX spectrum of phenotypes from mild to severe clito-
DSD, describes a group of autosomal recessive romegaly is possible. Classic 21a-hydroxylase
disorders that arises from a deficiency in one of deficiency is comprised of two forms of CAH:
five genes required for the synthesis of cortisol a severe, salt-wasting type with a defect in aldo-
(Fig. 1). These five genes and the enzymes they sterone biosynthesis and a simple, virilizing type
encode are include CYP21: 21-hydroxylase; with normal aldosterone synthesis. A mild, non-
CYP11: 11b-hydroxylase, 18-hydroxylase and classic form also exists that can be asymptomatic
18-oxidase; CYP 17: 17a-hydroxylase and 17,20 or associated with signs of postnatal androgen
lyase; 3b2HSD: 3b–hydroxysteroid dehydroge- excess.9 There are two CYP11 genes: CYP11B1
nase; and StAR: side chain cleavage enzyme. and CYP11B2. CYP11B1 converts 11-deoxycorti-
Although these biochemical defects are character- sol to cortisol.10,11 Alternatively, CYP11B2
ized by impaired cortisol secretion, only defi- converts deoxycorticosterone (DOC) to cortico-
ciencies in CYP21 and CYP11 are predominantly sterone, corticosterone to 18-hydroxycorticoster-
masculinizing disorders, and 3b2HSD to a lesser one, and 18-hydroxycorticosterone to
extent. Although the female fetus is masculinized aldosterone. Hypertension, which occurs in about
because of overproduction of adrenal androgens two thirds of patients, is presumptively a conse-
and precursors, the affected boys have no genital quence of excess DOC, with resultant salt and
abnormalities. In contrast, 3b2HSD, CYP17, and water retention. Excess androgen secretion in ute-
StAR deficiencies block cortisol synthesis and ro masculinizes the external genitalia of the female
gonadal steroid production. Thus, boys have fetus. After birth, untreated male and female
varying degrees of undermasculinization, whereas neonates progressively virilize and experience
girls generally have normal external genitalia.6–8 rapid somatic growth and skeletal maturation.
Cholesterol
Side chain cleavage
17 hydroxylase 17,20Lyase Dehydroepiandrosterone
Pregnenolone 17-OH Pregnenolone
3 HSD 3 HSD 3 HSD
17hydroxylase 17,20Lyase
Progesterone 17-OH Progesterone Androstenedione
21α hydroxylase 21α h ydroxylase 17β HSD
Deoxycorticosterone 11-Deoxycortisol
Testosterone
11β hydroxylase 11β hydroxylase 5α reductase
Corticosterone Cortisol Dihydrotestosterone
aldosterone synthetase
18-OH Corticosterone
aldosterone synthetase
Aldosterone
Fig. 1. The steroid biosynthetic pathway.

3. Practical Approach to Ambiguous Genitalia Newborn 197
3b2HSD catalyzes three reactions: pregneno- ovary on the contralateral side. Furthermore, the
lone to progesterone; 17-OH pregnenolone to external genitalia are ambiguous with hypospa-
17-OH progesterone; and dehydroepiandroster- dias, cryptorchidism, and incomplete fusion of
one (DHEA) into androstenedione.6,8 Complete the labioscrotal folds. The genital duct differentia-
deficiency of 3b2HSD impairs synthesis of adrenal tion in these patients generally follows that of the
aldosterone and cortisol, and gonadal testos- ipsilateral gonad on that side, such as a fallopian
terone and estradiol. These newborns have severe tube with an ovary and a vas deferens with
CAH and exhibit signs of mineralocorticoid and a testis.15
glucocorticoid deficiency in the first week of life.
Masculinization occurs as a result of DHEA 46,XY disorder of sex development
conversion to testosterone in fetal placenta and The 46,XY DSD is a heterogeneous disorder in
peripheral tissues manifesting as mild to moderate which testes are present but the internal ducts
clitoromegaly. system or the external genitalia are incompletely
CYP17 also catalyzes three reactions: pregneno- masculinized. The phenotype is variable and
lone to 17-OH pregnenolone, 17-OH pregnenolone ranges from completely female external genitalia
to dehydroepiandrosterone, and progesterone to to the mild male phenotype of isolated hypospa-
17-OH progesterone.8 Phenotypically, affected dias or cryptorchidism. The 46,XY DSD can be
girls have normal internal and external genitalia, classified into eight basic etiologic categories: (1)
but demonstrate immature sexual development Leydig cell failure, (2) testosterone biosynthesis
because of an inability of the ovaries to secrete defects, (3) androgen insensitivity syndrome, (4)
estrogens at puberty. In mild defects, aldosterone ¨
5a-Reductase deficiency, (5) persistent Mullerian
secretion may be normal and hypertension duct syndrome, (6) primary testicular failure or
absent.12 vanishing testes syndrome, (7) exogenous insults,
StAR deficiency, also called lipoid adrenal and (8) gonadal dysgenesis.
hyperplasia, is a rare form of CAH and represents Leydig cell failure The presence of testosterone,
the most severe genetic defect in steroidogenesis. produced by testicular Leydig cells, induces
StAR deficiency is associated with severe gluco-
male differentiation of the wolffian ducts and
corticoid and mineralocorticoid deficiencies external genitalia. The 46,XY DSD can result from
because of a failure to transport cholesterol from Leydig cell unresponsiveness to human chorionic
the outer to the inner mitochondrial membrane,
gonadotrophin hormone (hCG) and luteinizing
which blocks conversion of cholesterol to preg- hormone (LH). The phenotypes of these patients
nenolone.13 These children are at risk for neonatal vary from normal female to hypoplastic external
demise because of missed adrenal crisis. male genitalia.
Neonates suspected to have adrenal insufficiency
should be closely monitored for hypoglycemia, hy- Testosterone biosynthesis enzyme defects Desc-
ponatremia, and adrenal insufficiency. Affected ribed earlier in this article for 46,XX DSD, defects
girls demonstrate normal internal and external in four of the steps of the steroid biosynthetic
genitalia. Most of these women undergo sponta- pathway from cholesterol to testosterone may
neous puberty but are at risk for irregular menses, also produce genital ambiguity in the male.7,8
ovarian cysts, and premature menopause.14 These defects include the less common forms of
congenital adrenal hyperplasia: 3b2HSD defi-
Ovotesticular disorder of sex development ciency, CYP17 deficiency, StAR protein deficiency,
Ovotesticular DSD requires expression of ovarian and 17bHSD deficiency. Although DHEA conver-
and testicular tissue. The most common karyotype sion into testosterone results in virilization in
in the United States is 46,XX, although 46,XY, females, this same process insufficiently masculin-
mosaicism, or chimerism (46,XX/46,XY) can occur. izes affected boys. Thus, male infants exhibit
Although mosaicism may occur from chromo- ambiguous genitalia with variable degrees of hypo-
somal nondisjunction, chimerism may result from spadias, cryptorchidism, penoscrotal transposi-
a double fertilization (an X and a Y sperm) or tion, and a blind vaginal pouch. Boys with CYP17
from fusion of two fertilized eggs. This fairly deficiency display a developmental spectrum
uncommon condition can be further classified from the normal female phenotype to the ambig-
into three groups: lateral ovotesticular DSD has uous hypospadiac male phenotype.12,16 The
a testis on one side and an ovary on the contralat- magnitude of the decreased masculinization in
eral (usually left) side; bilateral ovotesticular DSD the male infant correlates with the severity of the
has an ovotestis on each side; and, most block in 17a-hydroxylation. Affected boys with
commonly, unilateral ovotesticular DSD has an StAR deficiency have severe testosterone defi-
ovotestis on one side and either a testis or an ciencies and exhibit female external genitalia with

4. 198 Lambert et al
a blind vaginal pouch.17,18 No surviving patients (MIS), is secreted by the Sertoli cells from the
with 46,XY have demonstrated testis function at time of fetal seminiferous tubule differentiation
puberty. The affected 46,XY boys with 17bHSD until puberty. MIS binds to a receptor in the
deficiency have external female genitalia, inguinal ¨
mesenchyme surrounding the Mullerian ducts
testes, internal male ducts, and a blind vaginal before 8-weeks gestation causing apoptosis and
pouch. At puberty, these patients demonstrate an regression of the Mullerian duct.25 Because the
¨
increase in their levels of gonadotropins, andro- ¨
diagnosis of persistent Mullerian duct syndrome
stenedione, estrone and testosterone. Delayed viri- (PMDS) is often made at the time of inguinal hernia
lization may ensue if some testosterone levels repair or orchiopexy, this syndrome is commonly
approach the normal range.19,20 referred to as hernia uteri inguinalis. PMDS can
occur from a failure of the testes to synthesize or
Androgen insensitivity syndrome The broad
secrete MIS because of an AMH gene mutation
spectrum of androgen insensitivity syndrome or from a defect in the response of the duct to
(AIS) ranges from patients with 46,XY with MIS because of an AMH2 receptor gene mutation.
complete AIS, or testicular feminization, to partial PMDS is inherited in a sex-linked autosomal
AIS. This syndrome is the result of mutations of recessive manner and AMH mutations are most
the steroid-binding domain of the androgen common in Mediterranean or Arab countries with
receptor resulting in receptors unable to bind high rates of consanguinity.26 Most of these
androgens or receptors that bind to androgens
familial mutations are homozygous and the
but exhibit qualitative abnormalities and do not
patients have low or undetectable levels of serum
function properly. This disorder affects 1 in
MIS. In contrast, AMH2 receptor mutations are
20,000 live male births with a maternal inheritance
often heterozygous and are more common in
pattern, because the androgen receptor gene is
France and Northern Europe. These patients
located on the long arm of the X chromosome.21
usually have high-normal or elevated MIS
The external genitalia of a child with complete concentrations.27
androgen insensitivity resembles normal female
genitalia although the karyotype is XY and testes Congenital anorchia Congenital anorchia or van-
are located internally. These children are raised ishing testes syndrome encompasses a spectrum
as girls. Most children are not diagnosed until of anomalies resulting from cessation of testicular
puberty during an evaluation for primary amenor- function.28 A loss of testes before 8-weeks gesta-
rhea. Occasionally, it is also discovered that at tion results in patients with 46,XY with female
the time of inguinal hernia repair, or more recently, external and internal genitalia and either no
when the prenatal karyotype does not match the gonads or streak gonads. A loss of testes at 8 to
external phenotype of the newborn child. 10 weeks in development leads to ambiguous
5a-reductase deficiency 5a-reductase deficiency genitalia and variable ductal development. A loss
was first described as pseudovaginal perineal of testis function after the critical male differentia-
scrotal hypospadias.22 In this autosomal recessive tion period, which is at 12- to 14-weeks gestation,
condition, patients have a defect in the conversion results in a normal male phenotype externally
of testosterone to dihydrotestosterone (DHT). along with anorchia internally. Sporadic and
These patients have a 46,XY karyotype and familial forms of anorchia exist. The familial cases,
ambiguous external genitalia but normally differ- including some reports of monozygotic twins,
entiated testes with male internal ducts. However, support the presence of an as yet unidentified
at puberty, significant virilization occurs as testos- mutant gene in some patients with the syndrome.
terone levels increase into the adult male range
while dihydrotestosterone remains disproportion- Exogenous source Exogenous insults to normal
ately low. There are three genetic isolates of this male development include maternal ingestion of
disorder that have been described: the Dominican progesterone or estrogen or various environmental
Republic, the New Guinea Samba Tribe, and in hazards. As early as 1942, Courrier and Jost29
Turkey. Many of these patients undergo a change demonstrated an antiandrogen effect on the
of their gender identity from female to male after male fetus induced by a synthetic progestagen,
puberty.23,24 Virilization can be secondary to and more recently, Silver and colleagues30
slightly increased plasma DHT levels and to the showed an increased incidence of hypospadias
chronic effect of adult T levels on the androgen in male offspring conceived by in vitro fertilization.
receptor. They hypothesized that the increased risk may be
secondary to maternal progesterone ingestion.
¨
Persistent Mullerian duct syndrome Antimullerian
¨ Sharpe and Skakkebaek31 have further postulated
¨
hormone (AMH), or Mullerian inhibitory substance that the increase in reproductive abnormalities in

5. Practical Approach to Ambiguous Genitalia Newborn 199
men is related to an increase in the in utero expo- a benign growth, is the most common tumor.38
sure to environmental estrogens. Because of the 20% to 25% age-related risk for
malignant transformation into a dysgerminoma,39
Gonadal dysgenesis surgical removal of the gonad is recommended.
Dysgenetic 46,XY DSD exhibits ambiguous devel- Patients with a 45,XX/46,XY karyotype and normal
opment of the internal genital ducts, the urogenital testis biopsy could retain the testis if it is de-
sinus and the external genitalia. Dysgenetic testes scended or can be placed in the scrotum. These
can result from mutations or deletions of any of the children would then need a close follow-up of the
genes involved in the testis determination testis by monthly self examinations for tumor
cascade, namely SRY, DAX, WT1, and SOX9. formation.
The SRY gene is a single exon gene located on
the short arm of the Y chromosome.32 SRY gene Sex chromosome disorder of sex development
mutations usually result in complete gonadal Sex chromosome anomalies comprise another
dysgenesis and XY sex reversal or Swyer category of DSD. Klinefelter syndrome (47,XXY)
syndrome. The DSS locus (dosage-sensitive sex usually becomes evident during adolescence as
reversal) has been mapped to the Xp21 region, patients develop gynecomastia, variable androgen
which contains the DAX1 gene. Duplication of deficiency, and small atrophic testes with hyaliniza-
the DSS locus has been associated with dysge- tion of the seminiferous tubules. These patients
netic 46,XY DSD. The DSS locus has been theo- demonstrate azoospermia and increasing gonado-
rized to contain a wolffian inhibitory factor, which tropin levels. Boys with 47,XXY may develop
acts as an inhibitory gene of the testis determina- through nondisjunction of the sex chromosomes
tion pathway.33 Swain and colleagues34 have during the first or second meiotic division in either
shown that DAX1 antagonizes SRY action in parent, or less commonly, through mitotic nondis-
mammalian sex determination. Male patients junction in the zygote at or after fertilization. These
with Denys-Drash syndrome have ambiguous abnormalities almost always occur in parents with
genitalia with streak or dysgenetic gonads, normal sex chromosomes. The 46,XY/47,XXY
progressive nephropathy, and Wilms tumor. Anal- mosaicism is the most common form of the Kline-
ysis of these patients revealed heterozygous felter variants. The mosaics, in general, manifest
mutations of the Wilms tumor suppressor gene a much milder phenotype than patients with classic
(WT1) at 11p13.35 The WAGR syndrome (Wilms Klinefelter. Testes differentiation and a lack of
tumor, aniridia, genitourinary abnormalities, ovarian development in these patients indicates
mental retardation) is also associated with WT1 that a single Y chromosome with SRY expression
alterations.29 The urogenital anomalies seen in is enough for testis organogenesis and male sex
the WAGR syndrome are usually less severe than differentiation in the presence of as many as four
in Denys-Drash syndrome. The SOX9 gene has X chromosomes in some patients with Klinefelter.
been associated with campomelic dysplasia, an These testes are not truly normal, however, since
often lethal skeletal malformation with dysgenetic they are usually small and azoospermic. Although
46,XY DSD.36 Affected 46,XY children have there are sporadic reports of paternity, most fertile
phenotypic variability from normal boys to normal Klinefelter individuals have sex chromosome
girls, depending on the function of the gonads. mosaicism.40–42 Pure gonadal dysgenesis (PGD)
Swyer syndrome represents an uncommon form describes a 46,XX or 46,XY child with streak
of pure gonadal dysgenesis. These children have gonads or more commonly, a child with Turner
female external genitalia and have a uterus and fal- syndrome (45,XO or 45,XO/46,XX).
lopian tubes, however, the karyotype is 46,XY with
a Y chromosome that usually does not work and 46,XX testicular disorder of sex development
two dysgenetic gonads in the abdomen.37 Categories of 46,XX testicular DSD include boys
Partial gonadal dysgenesis refers to disorders with classic XX with apparently normal pheno-
with partial testicular development, including types, boys with non-classic XX with some degree
mixed gonadal dysgenesis, dysgenetic male pseu- of sexual ambiguity, and XX ovotesticular DSD.43
dohermaphroditism, and some forms of testicular Eighty percent to ninety percent of boys with
or ovarian regression. Mixed or partial gonadal 46,XX result from an anomalous Y to X transloca-
dysgenesis (45,XX/46,XY or 46,XY) involves tion involving the SRY gene during meiosis. In
a streak gonad on one side and a testis, often dys- general, the greater the amount of Y-DNA present,
genetic, on the other side. Patients with a Y chro- the more virilized the phenotype. Although 8% to
mosome in the karyotype are at a higher risk than 20% of boys with XX have no detectable Y
the general population to develop a tumor in the sequences, including SRY, about 1 in 20,000
streak or dysgenetic gonad. Gonadoblastoma, phenotypic boys have a 46,XX karyotype. Most

6. 200 Lambert et al
of these patients have ambiguous genitalia, but 2 cm in a term male neonate.47–50 One should
reports of boys with classic XX without the SRY describe the position of the urethral meatus and
gene do exist.26,33,38,39,43,44 This phenomenon the amount of penile curvature and note the
again raises the possibility of mutation of a down- number of perineal orifices.
stream wolffian inhibitory factor when cases of Another critical finding on physical examination
normal virilization are seen without the presence is the presence of a uterus that is palpable by
of the SRY gene. Some patients with 46,XX testic- digital rectal examination as an anterior midline,
ular DSD have the SRY gene translocated from the Y cord-like structure. Of course, a thorough general
to the X chromosome. However, for most patients, physical examination must also be performed. A
the genes responsible are not yet identified.45,46 blood pressure should be taken to rule out hyper-
tension. The presence of hyperpigmentation
should also be documented. Dysmorphic features
History and Physical Examination
indicating syndromic manifestations (eg, short
Patients with bilaterally impalpable testes or broad neck, widely spaced nipples, or aniridia)
a unilaterally impalpable testis and hypospadias should also be noted.
should be regarded as having DSD until proven
otherwise, whether or not the genitalia appear Patient Evaluation
ambiguous. Patient history should include the
In the immediate newborn period, all patients
degree of prematurity, ingestion of exogenous
require a karyotype and laboratory evaluation by
maternal hormones used in assisted reproductive
serum electrolytes, 17-OH progesterone, testos-
techniques, and maternal use of oral contracep-
terone, luteinizing hormone, follicle stimulation
tives during pregnancy. A family history should
hormone, and urinalysis. The karyotype can be
be obtained to document any urologic abnormali-
determined from peripheral blood or skin fibro-
ties, neonatal deaths, precocious puberty, amen-
blasts from genital skin. It is important to remember
orrhea, infertility, or consanguinity. Any abnormal
that chromosomal studies from an amniocentesis
virilization or cushingoid appearance of the child’s
do not negate the need for a postnatal karyotype.
mother should also be noted. Abnormalities of the
Once the karyotype is determined, serum analysis
prenatal maternal ultrasound are also helpful, such
will assist in narrowing the differential diagnosis. If
as discordance of the fetal karyotype with the
the 17-OH progesterone level is elevated, a diag-
genitalia by sonogram.
nosis of CAH can be made. Determining the 11-de-
For differential diagnosis and treatment
oxycortisol and deoxycorticosterone levels will
purposes, the most important physical finding is
help differentiate between 21-hydroxylase and
the presence of one or two gonads. If no gonads
11b-hydroxylase deficiencies. If the levels are
are palpable, all DSD categories are possible. Of
elevated, then a diagnosis of 11b-hydroxylase defi-
these, 46,XX DSD is most commonly seen fol-
ciency can be made, whereas low levels confirm
lowed by 45,X/46,XY. A palpable gonad is highly
21-hydroxylase deficiency. If the 17-OH proges-
suggestive of a testis, or rarely, an ovotestis,
terone level is normal, a testosterone to DHT ratio
because ovaries and streak gonads do not
along with androgen precursors before and after
descend. If one gonad is palpable, 46,XX DSD is
hCG stimulation will help elucidate the 46,XY DSD
less likely, whereas 45,X/46,XY, ovotesticular
etiology. A testosterone to DHT ratio of greater
DSD, and 46,XY remain possibilities. If two gonads
than 20 is suggestive of a 5 alpha reductase defi-
are palpable, 46,XY, and rarely ovotesticular DSD,
ciency. A failure to respond to hCG in combination
are the most likely diagnoses (Fig. 2).
with elevated LH and FSH levels is consistent with
Patients should be examined in a warm room
anorchia. It is important to remember that for the
supine in the frog leg position with both legs free.
first 60 to 90 days of life, a normal gonadotropic
It is important to determine size, location, and
surge occurs with a resultant increase in the testos-
texture of both gonads, if palpable. The unde-
terone level and its precursors. During this specific
scended testis may be found in the inguinal canal,
time period, hCG stimulation for androgen evalua-
the superficial inguinal pouch, at the upper
tion can be postponed. Serum levels of AMH and
scrotum, or rarely in the femoral, perineal, or
inhibin B can also be measured in the immediate
contralateral scrotal regions. One should also
postnatal period to document the existence of
note the development and pigmentation of the la-
normal testicular tissue.
bioscrotal folds along with any other congenital
anomalies of other body systems. An abnormal
Genetic Tests
phallic size should be documented by width and
stretched length measurements. Micropenis is The initial genetic testing is the assessment of the
defined as a stretched penile length of less than chromosomes by karyotype. This testing can be

7. Practical Approach to Ambiguous Genitalia Newborn 201
Palpate Gonads
0 1 2
FPH DSD
46XX Partial Gonadal Dysgenesis
MGD 46XYMPH
DSD
GD
D Ovotesticular DSD
TH Ovotesticular DSD (rare)
TH (rare)
46XY DSD
PH 46XY DSD MPH
Ovotestesticular DSD
H
Electrolytes, 17OH-Progesterone, Testosterone, LH FSH
Karyotype
Pelvic Ultrasound
Genitogram (possible)
17OH Progesterone (2000ng/dl)
Testosterone NL 17OH Progesterone
uterus Testosterone
uterus seen +/- uterus, NL Testosterone
ovaries Laparoscopy, Gonad Biopsy 2 testes
46X X DSD HCG stimulation:
(CAH)
(CAH) pre/post:
46XX 46X X DSD
46XX Testosterone/DHT
(maternal virilizing
maternal virilizing synd DHEA/androstenedione
syndrome)
11 deoxycortisol
DOC testis streak M PH
46XY DSD
ovary 46XY
46XY
ovotestis (46XX)
(46XX)
45X/46XY)
(45X/46XY)
11 deoxycortisol 11 deoxycortisol
DOC DOC
Ovotesticular DSD
TH Gonadal 1. 5a reductase def iciency
46XX 46XX Dysgenesis 2. AIS
(46XX/XY) 3. Steroid deficiency-male CAH
46X X DSD 46X X DSD (46XY) 4. Dysgenetic testes
21 Hydroxylase 11 Hydroxylase (45X/46XY) 5. Primary testicular failure
PURE (S-S) PARTIAL (S-T)
deficiency deficiency Pur e
45X 45X/46XY 6. Leydig cell failure
45X/46XX (46XY) 7. PMDS
46XX
(45X/46XY)
Fig. 2. Anatomic and endocrine approach to DSD evaluation. NL, normal; S, streak gonad; T, testis.
done by regular, meta-phasic, karyotype, but if the If the DSD does not have an obvious genetic
question asked is limited to the nature of the sex cause, whether it is syndromic or isolated, a screen
chromosomes, an interphasic FISH (fluorescent for CNVs (micro-deletions or micro-duplications) is
in situ hybridization) should be performed with X- recommended. This screen is performed in clinical
and Y-specific probes, providing the number of laboratories using micro-array–based compara-
and the quality of sex chromosomes in less than tive genomic hybridization (CGH) methods.
48 hours. If a mosaic is detected or suspected, Although the techniques and the sensitivity of the
a large number of interphasic nuclei can be as- array vary, all methods detect CNVs that can
sessed by FISH (typically 200–300) to evaluate then be confirmed by FISH. For best sensitivity,
the percentage of each clone accurately (Fig. 3). oligonucleotide-based arrays are preferred, and
The follow-up genetic tests are diagnostic. They may detect CNVs as small as 10 to 50 kb. The
are of two kinds: one is the direct sequencing of use of direct molecular sequencing and array-
a specific gene and the other the evaluation of CGH is presented in the diagnostic algorithm
copy number variants (CNV) by micro-array anal- shown in Fig. 1. The interpretation of genetic
ysis. If there are enough phenotypic features to testing can be challenging. If the test detects point
orient the physician toward a specific diagnosis, mutations or CNVs known to cause a DSD, the
direct sequencing of the causal gene is preferred. genetic diagnosis is certain, and the testing of
This sequencing would typically happen in cases parents as potential carriers will then be performed
of complete androgen insensitivity, or congenital for the purpose of genetic counseling.
adrenal hyperplasia, for which the diagnostic indi- If the test detects unknown point mutations or
cators are strong. Several genes can be sequenced CNVs, the investigation of parental DNA becomes
on a clinical basis by molecular laboratories (eg, AR, essential. A de novo (not present in parents) CNV
CYP21, SRY, SOX9, SF1), and the list is growing. or point mutation in patients with a DSD is likely

8. 202 Lambert et al
Fig. 3. (A) Genetic evaluation of XY patient with masculinized genitalia. AMHR, Anti-Mullerian Hormone
Receptor; CYP11A1, Cytochome p450scc; CYP17, 17a-hydroxylase; HSD3B2, 3b-Hydroxysteroid dehydrogenase
2; HSD17B3, 17b-hydroxysteroid dehydrogenase3; Nml, normal; POR, P450 Oxidoreductase; US, Ultrasound. (B)
Genetic evaluation of XX patient with masculinized genitalia. (Data from Fleming A, Vilain E. The endless quest
for sex determination genes. Clin Genet 2005;67(1):15–25).

9. Practical Approach to Ambiguous Genitalia Newborn 203
to be causative. An inherited, unknown variant thoughtfully and carefully construed to the family.
(present in one of the parents) is more difficult to A team approach that includes family discussions
interpret and requires a specialized genetics should begin immediately. The importance of
consultation. In all cases, pre- and post-test a clinical psychologist or psychiatrist who is
genetic counseling is essential to ensure that the familiar with DSD cannot be underestimated. In
family understands the interpretation of the test addition to support from the medical team, many
and the risk for recurrence in future pregnancies. families find support groups useful.55
Although the preferred gender assignment is not
Radiographic Tests always clear, a thorough examination of endocrine
function, karyotype, and potential for fertility
Examination of the internal genital can be achieved
should guide the determination. Current DSD
using many modalities, including abdominal and
guidelines recommend that all individuals receive
pelvic ultrasound, MRI, fluoroscopy, endoscopy,
a gender assignment in infancy and family partici-
or laparoscopy. Noninvasive, quick, and inexpen-
pation in that decision-making process is essen-
sive, an ultrasound should be the first radiologic
tial.4 A single-institution survey of parents of girls
examination obtained. Although only 50% accu-
with CAH reported that 52% of respondents
rate in detecting intra-abdominal testes,51 ultra-
were completely satisfied with the information
sound can detect gonads in the inguinal region
they were given during the neonatal period,
¨
and can assess Mullerian structures. For example,
whereas 43% were only partially satisfied.56 This
¨
the presence of Mullerian structures visible on
study reinforces the need for open and direct
a pelvic ultrasound can often differentiate between
communication between the family and medical
pure gonadal dysgenesis and complete AIS in an
team as a core component of satisfactory patient
adolescent with primary amenorrhea.52 Although
care. This sentiment is confirmed by a survey of
more expensive, MRI scan can further delineate
fellows in the urology section of the American
the anatomy. Ectopic gonads, testes, and imma-
Academy of Pediatrics that documented that the
ture ovaries have an intermediate signal intensity
overwhelming majority of pediatric urologists
on T-1 weighted images and a high signal intensity
believe a team approach and parental involvement
with an intermediate signal intensity surrounding
are recommended and important in the care of
rim on T-2 weighted images.53 A genitogram
patients with DSD.57 The decision of whether or
should be performed to evaluate a urogenital
not to pursue surgical intervention should be
sinus, including the entry of the urethra in the
based upon anatomy, functional status, and
vagina and the presence of a cervical impres-
a consensus of opinion between the family and
sion.51 Infants with intra-abdominal or non-
medical team; this decision must be individualized
palpable testes in whom ovotesticular DSD;
for each patient with DSD.
45,X/46,XY DSD; or 46,XY DSD is considered will
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