Approach to ambiguous

genitalia- Is it a boy or a girl?

Dr Lim Poi Giok
Paediatric Endocrinologist
HKL
 Gonadal development

Brennan J; Capel B Nat Rev Genet. 2004 Jul;5(7):509-21.

Testes determination
Summary of the molecular events in sex differentiation indicating
the genes in which molecular defects cause 46,XY DSD in humans
Phenotypic differentiation of the female and male urogenital tracts
Phenotypic differentiation of the external genitalia in female and male embryos
 Evaluation of infant with ambiguous genitalia

Who to evaluate?
• Overt genital ambiguity
• Apparent female genitalia: enlarged clitoris, posterior labial
fusion, inguinal/labial mass
• Apparent male genitalia: bilateral undescended testes, mild
hypospadias with undescended testes, isolated perineal
hypospadias, micropenis
• Discordance between genitalia and antenatal karyotype
 Investigation and evaluation of DSD

• Family and antenatal history

• General examination with specific attention to
syndromic/dysmorphic features, hyperpigmentation
(particularly of scrotum and genitalia) , midline defects
• Genital anatomy
 Initial evaluation- History

• Prenatal exposure to androgens (eg, progesterones, danazol,testosterone)

or endocrine disrupters (phenytoin, aminoglutethimide).
• Maternal virilization in pregnancy (placental aromatase deficiency,
luteoma).
• Family history of females who are childless or have amenorrhea (androgen
insensitivity).
• Family history of unexplained infant deaths (congenital adrenal
hyperplasia).
• History of consanguinity (or homogeneous population) (recessive
disorders, eg, CAH, or disorders of androgen biosynthesis).
 Genital anatomy

• Phallic/penile size-stretched length

• Presence of chordee
• Urethral position or degree of hypospadias
• Clitoral size
• Labial/scrotal development and rugosity
• Position of gonads, size and consistency
• Virilization
• Anogenital ratio
 Genital anatomy

Stretched phallic length of 63 normal

Penile length premature and full-term male infants
• measured from the pubic ramus to
the tip of the penis (excluding any
excess foreskin)
• Penile width (diameter) is measured
at the midshaft
• Normal penile length is ≥2.5 cm, and
normal penile diameter is ≥0.9 cm.
• Small phallus (micropenis) -↓
testosterone exposure in the 2nd or
3rd trimester and deficiencies of
growth hormone or gonadotropin

Fetal phallic growth and penile standards for newborn male

infants. J Pediatr 1975; 86:395.
Standards for stretched penile length of premature
infants and older children and adults

Peter A. Lee et al Pediatrics 2006;118;e488-e500

 Clinical evaluation

Urethral opening
• A single opening at the base of
the phallus
a. incompletely fused penile urethra
(hypospadias)
b. virilized urogenital sinus (eg,
internal connection between the
vagina and urethra).
• findings must be confirmed either
by cystoscopy/vaginoscopy or
radiographically, because the
physical examination can be
misleading
 Clinical evaluation

Clitoral size Clitoromegaly in a 46,XX infant

• Clitoral width = shaft of the clitoris with 21-hydroxylase deficiency
between the thumb and forefinger to
exclude excess skin and subcutaneous
tissue.
• Normal clitoral width = 2 -6 mm.
• Mean clitoral length > 9 mm =abnormal
• More prominent in preterm infants -
clitoral size developed 27 weeks gestation
but less fat in the labia majora
• Clitoromegaly secondary to androgen
exposure in a 46,XX infant can be caused
by CAH, ovotesticular or testicular DSD,
maternal androgen exposure, or, rarely, by
tumor infiltration of the clitoris (eg,
neurofibromatosis)
 Clinical evaluation

Gonads -position , size and consistency

• The scrotum, labia majora, and inguinal area should be carefully palpated
• 46,XY child, bilateral nonpalpable testes may arise from anorchia or
persistent Müllerian duct syndrome
• 46,XX child, non palpable testes -CAH
• Gonads palpable below the inguinal ligament (eg, in the labioscrotal folds)
are usually testes
Female virilization standards for CAH graded by Prader based upon the degree of
virilization of the urogenital sinus and the external genitalia
Virilization (cross-sectional view)

Virilization (external view)

White, PC, Speiser, PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000; 21:245.
 Clinical evaluation

Posterior labial fusion in the 46,XX

Anogenital ratio
patient with true hermaphroditism -
• independent of gestational age clitoromegaly and gonads palpaple in
and body size the labioscrotal folds
• distance between the anus and
posterior fourchette divided by
the distance between the anus
and the base of the clitoris
• A ratio of >0.5 suggests
virilization with some posterior
labial fusion
Proposed classification of DSD-Consensus Statement on
Management of Intersex Disorders

Peter A. Lee et al Pediatrics 2006;118;e488-e500

 Diagnostic Evaluation

• Karyotype
• Serum electrolytes, 17OH- Progesterone (after 48 hours), cortisol, ACTH,
DHEA
• SRY gene by FISH (SRY probes)
• Urinalysis (exclude proteinuria)
• Pelvic ultrasound
• Adrenal: ACTH/ ACTH stimulation test, Plasma Renin activity (PRA),
aldosterone
• Testis: DHEAS, androstenedione, testosterone, DHT, Anti- Mullerian
Hormone (AMH), HCG test with LH, FSH and Testosterone and
dihydrotestosterone at baseline and 72 hours
• Imaging: MRI, Genitogram
• Surgical- laparoscopy, gonadal biopsy
Investigation flow plan for assessment of ambigious genitalia

A L Ogilvy-Stuart and C E Brain Arch. Dis. Child. 2004;89;401-407

 Classification of Disorders of Sex Development (DSD)
46XX DSD
Disorder of ovarian development
• Ovotesticular DSD
• Testicular DSD (SRY+, duplicate SOX9)
• Gonadal dysgenesis

Androgen excess
• CAH (CYP21, CYP11β1, 3βHSD3)
• Androgen secreting tumour- Maternal luteoma
• Placental aromatase deficiency
• Drugs administered during pregnancy eg.progesterone, danazol

Other structural
• Cloacal extrophy
• Syndromes associated with Mullerian development
 Intepretation of results
Genital ambiguity with 46XX- virilised female
1. Apparent male genitalia, absence of palpable gonads= CAH.
↑ 17 OHP →21α- hydroxylase
↑ 11 deoxycortisol →11β –hydroxylase
↑ 17- hydroxypregnenolone, DHEA →3β- hydroxysteroid dehydrogenase
• CAH confirmed, monitor BUSE= salt wasting
• Other test: ↑ urinary sodium, DHEA, androstenedione, testosterone, ACTH, renin
USS

1. Excessive androgen- gonadal in nature – usually ovotestis

• Commonest 46XX (70.6%), mosiac (46XX/46XY) (20.2%)

3. Transplacental transfer of androgen

 46XY DSD
Disorder of testicular development
• Complete gonadal dysgenesis (SRY-, SOX9-)
• Mutations in WT1, SF1
• Duplications of DAX1
• Ovotesticular DSD
• Partial gonadal dysgenesis
• Testicular regression

Disorder of androgen synthesis

• Testosterone biosynthesis (StAR, 3βHSD , 17αOHD/17-20, 17ßHSD
• 5αreductase deficiency
• Leydig cell hypoplasia (LH deficiency or LH receptor defect)
• Deficient synthesis or action of AMH
End organ unresponsiveness
• Complete or partial androgen insensitivity

Others
• Urogenital malformations
• Dysmorphic syndrome
• Exogenous maternal oestrogens
 Sex chromosome DSD

• Turner syndrome (45X and variants)

• Klinefelter (47XXY)
• Mixed Gonadal dysgenesis (45X/46XY)
 Genital ambiguity 46XY- undervirilised male

1. Testicular dysgenesis/malfunction
• 46XY, low basal and hCG stimulated testosterone and low testosterone precursor
• Suggest gonadal dysgenesis (Lap/ testicular biopsy) or lipiod CAH
• Associated gene defects- bony or renal anomalies
• Basal gonadotrophins ↑
• < AMH – Mullerian structure present(Hypoplastic)
• Mosaic karyotype 45X/46XY- variable phenotype
 46XY Genital ambiguity

Biosynthetic defect
1. Inactivating mutation of LH receptor (Leydig cell hypoplasia)
 46XY, low basal and peak testosterone on hCG testing
 ↑ gonadotrophins

• Variable phenotype- predominantly female external genitalia, no uterus and

fallopian tubes: epididymis and vas deferans may be present

2. 5 α reductase deficiency (defective conversion of testosterone to

dihydrotestosterone)
 46 XY with normal basal and peak testosterone on hCG testing and ↑ T :DHT
ratio (T: DHT >10 )
• DHT dependant virilisation of external genitalia deficient = small phallus and
perineal hypospadias. Wolffian structure normal
 46XY Genital ambiguity

3. 46XY, low basal and peak testosterone on hCG testing with ↑testosterone
precursor
• CAH ( 3β hydroxysteroid, 17α/17,20 lyase deficiency
• Androstenedione: Testosterone >20:1 suggest 17 βhydroxysteroid
 46XY Genital ambiguity

End organ unresponsiveness

• 46 XY , normal or increased basal and peak Testosterone on hCG
test, and normal T: DHT ratio = partial androgen insensitivity
• Variable phenotype
• Trial of im Testosterone on penile growth
• Mutational analysis
 Disorder of androgen receptor

• X- linked recessive
• Newborn infants with ambiguous
genitalia
• Girls with inguinal hernias or labial
masses
• Women with primary amenorrhea
• Adolescent girls who become virilized
and develop clitoromegaly
• Adolescent boys who fail to undergo
normal male puberty or who have
persistent gynecomastia
• Adult men with undervirilization or
infertility associated with
azoospermia or severe oligospermia
 5 α Reductase Deficiency

Testosterone and DHT in 5-alpha-

Clinical features reductase 2 deficiency
• Consanguinity 38%
• Positive family history 41%
• Pseudovagina 54%
• Urogenital sinus 34%
• Hypospadias 21%
• Testes in inguinal canal, labia, 100%
scrotum
• Spermatogenesis impaired 100%
• Gynaecomastia rare

• Degree of virilization at puberty is striking and

change of gender reported 45% of 42
 Case 1- 4 years 5 months old Malay child

• Referred from HUSM for further evaluation of ambiguous genitalia

• Antenatal history
– Mother 41 years old Para 1, non consanguineous, spontaneous
conception
– Elderly primigravida
– Oligohydramnions
– No history of medicine/ contraception or herbal ingestions
– No history of hoarseness of voice, acne, hirsutism

• Birth history
– EMLSCS @ HUSM for fetal distress and oligohydramnions @ 35
weeks gestational age birth weight 1.59 kg
• Neonatal and Past Medical History (admitted at NICU until day 25 of life)

– Ambiguous genitalia
• ‘clitoromegaly’
• 17OHP 20 nmol/l
• Testosterone level 5.7 nmol/l
• Cortisol 90 nmol/l

– Treated as Congenital Adrenal Hyperplasia

• Fludrocortisone and hydrocortisone
• Karyotyping (24 May 2005)
– 46 XY with SRY gene
• USG abdomen and pelvic 28 May 2005
– No obvious uterine like structure seen. Normal kidney and bladder
• Left inguinal hernia at 1 month old
– Bilateral herniotomy with left orchidopexy done at 2
months old
• Testicular like structure at left inguinal
• Biopsy done at right inguinal – epididymal tissue
• Follow up at clinic, by 2 years old
– No signs and symptoms of adrenal crisis
– Thriving, no frequent febrile episode
– Active at home, prefer boyish activity and toys
– Hirsutism
– Hydrocortisone and fludrocortisone tapered down and off
• USG KUB & pelvis ( 28 May 2005 ) @ 1/12
– No obvious uterine structure seen
– Normal bladder and kidneys
• Repeat USG abdomen (03/10/06) @14/12
– No testes seen in both inguinal, renal and labial region
• Repeat USG pelvic (05/10/09) @ 4 years 5 months
– Non visualized testes or ovaries. Uterus present.
• MRI abdomen (11/12/06) @
16/12
– Non visualization of both
testes. Bladder and kidneys
appear normal
Genitogram
 Investigation

18/10/06 10/10/07 03/03/08

Na+ 137 139 136
K+ 4.0 4.8 4.1
Urea 4.9 3.5 5.4
Creatinine 51
11/05/06 01/11/06 03/01/08 07/01/08 31/01/08 11/04/08 02/03/09
Calcium 2.48
Testosterone <0.1 <0.1 0.35 0.35 0.2
(nmol/L)

Cortisol 349 124 256

(nmol/L)

DHEA.SO4 <0.4 <0.4 <0.4

(umol/L)

17-OHP 0.41
(ng/ml)

Androstenedione <0.3
(ng/ml)

DHT (nmol/L) 2.0

• Family history

48 years old 42 years old

Driver HUSM executive

No family history of infertility

or early neonatal death
Physical Examination
• Genitalia
– Phallus like structure
• Width: 0.8cm length: 2.3cm
• Labioscrotal fold, ruggated
– Tubular like structure at left labioscrotal fold
• Funnel shaped urogenital structure with 2 separate
holes
• HCG stimulation test
LH FSH Testosterone Androstenedione
Day 1 <0.5 1.4 0.35
Day 4 2

• Short Synacthen test

0 minutes 30 min 60 min

Cortisol 313 782 856
(nmol/L)
17 OHP (nmol/L) 1.7 6.3 8.4
 Summary

• 4 years 5 months old Malay child, ex premature 35 weeks, phenotypically

female/ ambiguous with presence of Mullerian structures; genotypically
46 XY with + SRY gene

Plan?

Laparoscopy: Left side- vas entering left inguinal canal

Right side- presence of ovotestis. Removed
HPE: Right gonad: Dysgenetic gonad with gonadoblastomatous change

Management: Raised as female. Will need hormonal supplementation at

puberty
• Possible gene involved SF 1, WT 1, DAX 1, WNT 4, DMRT 1
 Case 2- 11 .5 year old

• Birth history: Term SVD BW 3kg

Not noted ambiguous genitalia at birth
Raised as a girl
• Behaviour- Likes to wear jeans as with the other 4 sisters. Plays with dolls,
kitchen utensils
• Noted at age 10years, erection at night
• No breast development
• Pubic hair and voice deepening at at 10yr, Acne 11yr
• FHx of infertility- Maternal uncle married for 20yr no children
Case 2
 Examination

• Wt 37.4kg(50th C), Ht 143.5cm (25-50th C)

• Muscular ,Acne, visible larynx
• Genitalia- Phallus 5cm, chordee, width 3.2cm
• Rt gonad 4ml, mildly rugated Rt labioscrotal/ scrotum
• Lt labioscrotal fold rugated, testes undescended
• Single urethral opening- penoscrotal hypospadias
• PH 4
 Examination

• Wt 37.4kg(50th C), Ht 143.5cm (25-50th C)

• Muscular ,Acne, visible larynx
• Genitalia- Phallus 5cm, chordee, width 3.2cm
• Rt gonad 4ml, mildly rugated Rt labia scrotal fold
• Lt labioscrotal fold rugated, testes undescended
• Single urethral opening- penoscrotal hypospadias
• PH 4
 Investigation

• Karyotype: 46 XY, SRY +ve

• Testosterone 11.2nmol/L
• LH 9.9U/L, FSH 44U/L
• Genitogram- MCUG- vagina , no uterus
• Ultrasound pelvis- Lt testes in inguinal area, No uterus
• 17OHP- 2.6nnol/L
• Cortisol- 223nmol/L
• DHEAS- 1.2µmol/L(0.95-11.7)

• 46XY DSD
• Dilemma- Child has female gender identity

• Plan?
 Case 3- 3year 10 mth

• Referred by surgeon for ambiguous genitalia

• Birth Hx: FTSVD B.W 3.2kg. Assigned male gender from birth
• Normal development
• Non- consanguineous parents- 2nd child.
 Examination

• Wt 15kg (50th C), Ht 102cm(50th C)

• Genitalia- Stretched penile length(SPL) 3cm

Penoscrotal Hypospadias
Testes Rt 2ml in scrotum, Lt 1-2ml firm-lower inguinal
 Investigation

• Karyotype: 46XX
• Ultrasound: Both testes in the scrotal sac, no uterus visualized
• Genitogram/MCU- normal bladder, no filling contrast into other
tract (no Mullerian structure), short urethra (female)
• MRI- no soft tissue structure between bladder and rectum to
denote uterus. Both testes present

• Plan?
• EUA and testicular biopsy- Lt testes small and abnormal
• HPE: Lt ovotestes (46XX ovotestes)
• HCG stim: Testosterone D1 <0.5, D4 4.7nmol/L
• Androstenedione <1nmol/L (1-12mth 0.2-2.4)
• Lt gonadectomy. Hypospadias repair (im Testo)and pubertal
induction
 Management of child with ambiguous genitalia

Aims of management
• Establish a diagnosis
• Exclude and/or anticipate/treat associated life-threatening
conditions
• Successful gender assignment

 Initial stabilization- medical and psychosocial

 Family coping- helping parents understand genital ambiguity
 Long term management

 After diagnosis, long term management…….

• Supervision of general health, growth
• Hormonal supplementation-penile growth, puberty and
maintenance
• Surgery- repair, gonadectomy

• Disclosure to patient- when, how, how much

• Consistent management team- getting to know patient over
the years
• Privacy concerns
 Psychosexual Development

• Gender identity: sense of self as male or female

• Gender role: sex typical behaviours eg. toy preferences,
physical aggression
• Sexual orientation: pattern of erotic responsiveness

Influenced by multiple factors:

• Genes involved in sexual development
• Gender differences in brain structure
• Prenatal androgen exposure
• Societal and cultural factors
• Family dynamics
 Current controversies

1. Markedly virilized 46XX CAH infants

• Sex of rearing- evidence supports female
• Clitoral reduction
• Early separation of vagina and urethra
2. Significant micropenis- male
3. Ovotesticular DSD (true hermaphrodites)
 Conclusions

• More to DSD than diagnosis

• More research need to elucidate, on a case to case basis, what clinical
practices are likely to yield the best quality of life in affected individuals