Early Assessment of Ambiguous Genitalia

Home , Aromatase deficiency, Leydig cell hypoplasia

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Determination of sex (through duplication of Xp21) and Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from ...... Wnt4 (through duplication of 1p35), have been associated with impaired gonadal development and undervirilisa- Early assessment of ambiguous tion in a small number of karyotypic 46 XY males. genitalia Mutations or duplications in the various genes responsible for gonadal A L Ogilvy-Stuart, C E Brain differentiation and the subsequent development of the internal and exter- ...... nal genital phenotype genes may be responsible for gonadal dysgenesis and A multidisciplinary problem in some cases complete sex reversal (table 1). o discover that there is uncertainty the differentiation of the gonad. The Wnt4 is also expressed in the about the sex of one’s newborn differentiation of the gonad in turn Mu¨llerian ducts and in the absence of Tbaby is devastating and often determines the development of both anti-Mu¨llerian hormone (AMH) (also incomprehensible for most parents. It the internal genital tracts and the known as Mu¨llerian inhibiting sub- is paramount that clear explanations external genitalia and thus phenotypic stance) and testosterone, Mu¨llerian and investigations are commenced sex, which occurs later in development structures develop, while the Wolffian promptly, and that no attempt is made (from about 5–6 weeks of gestation). ducts involute.4 AMH promotes regres- to guess the sex of the baby. Extreme Both male and female genitalia differ- sion of Mu¨llerian structures and as the sensitivity is required, and ideally the entiate from the same structures along only source of AMH in the fetus is the baby should be managed in a tertiary the urogenital ridge. At about 4 weeks testes, the absence of a uterus in a baby centre by a multidisciplinary team after fertilisation, primordial germ cells with ambiguous genitalia is evidence including a paediatric endocrinologist migrate from the yolk sac wall to the that there has been functional testicular and a paediatric urologist. Early involve- urogenital ridge that develops from the tissue (Sertoli cell) present. Testosterone ment of a clinical psychologist with mesonephros. The urogenital ridge also produced from Leydig cells promotes experience in this field should be man- contains the cells that are the precursors differentiation of the Wolffian ducts and datory. Other professionals including for follicular or Sertoli cells and steroid hence the internal male genitalia (vas geneticists and gynaecologists may also producing theca and Leydig cells. The deferens, epididymis, and seminal vesi- become involved. There must be access ‘‘indifferent’’ gonads form on the geni- cles). to specialist laboratory facilities and tal ridges. Testosterone is converted to dihydro- experienced radiologists. The incidence The development of the fetal adrenals testosterone [DHT] by the enzyme 5a- of genital ambiguity that results in the and gonads occur in parallel, as before reductase. DHT masculinises the exter- child’s sex being uncertain is 1 per migration, the potential steroidogenic nal genitalia from about 6 weeks gesta- 4500,1 although some degree of male cells of both originate from the meso- tion, and the degree of masculinisation undervirilisation, or female virilisation nephros. There are many genes and is determined by the amount of fetal

may be present in as many as 2% of live transcription factors that are expressed androgen present (irrespective of http://adc.bmj.com/ births.2 in both tissues (for example, SF1 and source) and the ability of the tissues to Parents require reassurance that DAX1), and hence mutations in these respond to the androgens. either a male or female gender will genes may affect both adrenal and Defects in any part of this pathway definitely be assigned. However the gonadal development (fig 1). In addi- (including gene mutations and chromo- outcome of some of the investigations tion, WT1 is expressed in the kidney and somal abnormalities (for example, may take some weeks, and registration gonad, hence the association of Wilms’ 46XY/46XX, 45X/46XY), inappropriate of the child’s birth should be deferred tumour and gonadal dysgenesis in hormone levels, or end-organ unrespon- until gender has been assigned. This Denys-Drash syndrome, for example. siveness) may result in genital ambi- on September 26, 2021 by guest. Protected copyright. may require communication with the The undifferentiated gonad is capable guity, with undervirilisation of an XY Registrar of Births, and a skilled clinical of developing into either an ovary or a individual, virilisation of an XX indivi- psychologist will help the parents in testis. The theory that the ‘‘default’’ dual, or the very rare true hermaphro- deciding what to tell family and friends programme generates an ovary is prob- dite (an individual with both ovarian in the interim. It is also helpful (if ably not correct, although the exact role tissue with primary follicles and testi- appropriate) to reassure the parents that of ‘‘ovarian determining’’ genes in cular tissue with seminiferous tubules their child is otherwise healthy. humans is unclear at present. In con- which may be in separate gonads or While not all intersex conditions are trast, testicular development is an active ovotestes). apparent at birth (for example, complete process, requiring expression of the androgen insensitivity may only become primary testis determining gene SRY, CLINICAL ASSESSMENT OF apparent in a child with a testis within and other testis forming genes such as AMBIGUOUS GENITALIA an inguinal hernia, or at puberty with SOX9. Transcription factors such as SF1 History primary amenorrhoea and lack of and WT1 are also required for develop- The history should include details of the androgen hair), only those presenting ment of the undifferentiated gonad, as pregnancy, in particular the use of any with genital ambiguity at birth will be well as for the activation of the other considered in this article. male pathway genes required for testis Abbreviations: AMH, anti-Mu¨llerian hormone; An understanding of sex determina- development and the consequent devel- CAH, congenital adrenal hyperplasia; DHEA, tion and differentiation is essential to opment of male internal and external dehydroepiandrosterone; DHEAS, direct appropriate investigations and to genitalia.3 dehydroepiandrosterone sulphate; DHT, dihydrotestosterone; EUA, examination under establish a diagnosis. DAX1 and Wnt 4 are two genes that anaesthesia; hCG, human chorionic Genetic sex is determined from the may act to ‘‘antagonise’’ testis develop- gonadotrophin; StAR, steroidogenic acute moment of conception and determines ment. Over-expression of DAX1 regulatory protein

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drugs (table 2) that may cause virilisa- Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from tion of a female fetus and details of any previous neonatal deaths (which might point to an undiagnosed adrenal crisis). A history of maternal virilisation may suggest a maternal androgen secreting tumour or aromatase deficiency. A detailed family history should be taken, including whether the parents are con- sanguineous (which would increase the probability of an autosomal recessive condition) or if there is a history of genital ambiguity in other family members (for example, an X-linked recessive condition such as androgen insensitivity).

Examination The general physical examination should determine whether there are any dysmorphic features and the general health of the baby. A number of syndromes are associated with ambiguous genitalia, for example, Smith- Lemli-Opitz syndrome (characterised by hypocholesterolaemia and elevated 7-dehydrocholesterol levels, and resulting from mutations affecting 7-dehydrocholesterol reductase), Robinow syndrome, Denys-Drash syndrome, and Beckwith-Wiedemann syndrome. Mid- line defects may point towards a hypothalamic-pituitary cause for micropenis and cryptorchidism. Hypoglycaemia may indicate cortisol deficiency secondary to hypothalamic- pituitary or adrenocortical insufficiency. The state of hydration and blood pres- sure should be assessed as various forms Figure 1 Normal sexual differentiation. SRY, sex determining region on Y chromosome; TDF, of congenital adrenal hyperplasia (CAH) http://adc.bmj.com/ testis determining factor; AMH, anti-Mu¨llerian hormone; T, testosterone; DHT, dihydrotestosterone; WT1, Wilms’ tumour suppressor gene; SF1, steroidogenic factor 1; SOX9, SRY-like HMG-box; can be associated with differing degrees Wnt4, Wnt = a group of secreted signalling molecules that regulate cell to cell interactions during of salt loss, varying degrees of virilisa- embryogenesis; DAX1, DSS-AHC critical region on the X chromosome. tion in girls or undervirilisation in boys,

Table 1 Consequences of mutations/deletions and duplications/translocations of genes involved in gonadal differentiation

Chromosome Gonadal Sex reversal/genital Mu¨llerian location development Associated disorder ambiguity development on September 26, 2021 by guest. Protected copyright.

Gene mutation or deletion (loss of function) WT1 11p13 Dysgenesis (R and =) WAGR syndrome Genital ambiguity (=) Variable (=) Denys-Drash syndrome Sex reversal or genital Variable (=) ambiguity (=) Frasier syndrome Sex reversal (=) Yes (=) SF1 9q33 Dysgenesis (=) Adrenal failure Sex reversal (= Yes (=) SRY Yp11.3 =R ovary Sex reversal or genital = (variable) ambiguity (=) DAX1 Xp21 = Dysgenesis Adrenal failure and No No hypogonadotrophic hypogonadism/impaired spermatogenesis SOX9 17q24.3–25.1 = Dysgenesis or Campomelic dysplasia Sex reversal or genital Variable ovary/ovotestis ambiguity (=) AMH 19p 13.3–13.2 Normal Yes (=)

Gene duplication or translocation (gain of function) SRY Y fragment Yp11.3 RR testis Sex reversal or genital No translocation ambiguity (R) DAX1 duplication dupXp21 = Dysgenesis or Sex reversal or genital Variable ovary/ovotestis ambiguity (=) Wnt 4 duplication Dup 1p35 = Dysgenesis Genital ambiguity (=) Yes SOX9 duplication dup17q24.3–25.1 RR Testis Genital ambiguity (R)No

WAGR, Wilms’ tumour, aniridia, genitourinary anomalies, mental retardation; Denys-Drash (exonic mutations) WT, diffuse mesangial sclerosis; Frasier (intronic mutations) no WT, focal segmental glomerulosclerosis. Other abbreviations as for fig 1.

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in babies with CAH. The gestation of the Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from Table 2 Disorders of sex differentiation baby can cause confusion—in preterm Virilisation of XX females girls the clitoris and labia minora are Increased fetal adrenal androgen production relatively prominent and in boys, the Congenital adrenal hyperplasia (3b-hydroxysteroid dehydrogenase deficiency, 21-hydroxylase testes are usually undescended until deficiency, 11b-hydroxylase deficiency) Androgen secreting tumour about 34 weeks gestation. Placental aromatase deficiency Fetal gonadal androgen production INVESTIGATIONS True hermaphrodite with both testicular and ovarian tissue Transplacental passage of maternal androgens Initial investigations ascertain whether Drugs administered during pregnancy: e.g. progesterone, danazol the child is an undervirilised male or a Maternal androgen secreting tumour, luteoma of pregnancy virilised female and from these a differ- Other causes ential diagnosis can be established and Dysmorphic syndromes Prematurity—prominent clitoris subsequent investigations planned Bisexual gonads (fig 3). The principal aims of the initial Hermaphroditism. Usual genotype 46XX investigations are to determine the most appropriate sex of rearing and in a Undervirilisation of XY males genetic female, to exclude CAH and Testicular dysgenesis/malfunction Pure XY gonadal dysgenesis avert a salt losing crisis. Rarer forms of Mixed gonadal dysgenesis–45X/46XY. May be associated with gene mutations on SRY, SOX9, or CAH (steroidogenic acute regulatory WT1 genes protein (StAR) deficiency, 3b-hydroxys- Dysgenetic testis teroid dehydrogenase deficiency) may Testicular regression syndromes True agonadism, rudimentary testis syndrome also present with an adrenal crisis in Biosynthetic defect—decreased fetal androgen biosynthesis undervirilised males. Leydig cell hypoplasia (LH deficiency or LH receptor defect) Testosterone biosynthesis (non-virilising CAH): (StAR, 3b-HSD, 17a-OHD/17–20 lyase, Smith-Lemli- Opitz syndrome) Karyotype 5a-reductase deficiency As the differential diagnosis and a Deficient synthesis or action of AMH—persistent Mu¨llerian duct syndrome. May be due to mutations number of subsequent investigations in AMH or AMH receptor gene or SF1 gene will depend on the genetic sex, an End organ unresponsiveness Androgen receptor and post-receptor defects (complete and incomplete androgen insensitivity urgent karyotype should be sent. Rapid syndrome) FISH studies using probes specific for Others the X (DX1) and Y (SRY) chromosomes Urogenital malformations can provide useful information, Dysmorphic syndromes although a full karyotype is required Exogenous maternal oestrogens for confirmation and exclusion of mosaicism. The latter may be tissue specific and may not be apparent from blood, but only from skin or gonadal biopsies. In cases with a mosaic karyo- or hypertension (table 3). Although the detect the presence of a gonad. They type, for example, XO/XY or XX/XY, the http://adc.bmj.com/ cardiovascular collapse with salt loss may be situated high in the inguinal gonads are often different. This may and hyperkalaemia in congenital adre- canal so careful examination is required. involve one being a streak gonad and nal hyperplasia does not usually occur A unilateral palpable gonad may be a the other a testis or ovotestis, or two until between the 4th to 15th day of life testis, ovotestis, or rarely an ovary ovotestes with different ovarian and and so will not be apparent at birth in a within an inguinal hernia. Congenital testicular components. well neonate, it should be anticipated adrenal hyperplasia must be excluded in until CAH has been excluded. Jaundice a phenotypically male baby with bilat- Determination of internal anatomy (both conjugated and unconjugated) on September 26, 2021 by guest. Protected copyright. eral undescended testes. As with the external genitalia, the may be caused by concomitant thyroid The length of the phallus should be degree of virilisation of the internal hormone or cortisol deficiency. The determined. The normal term newborn genital tracts is defined by Prader stages urine should be checked for protein as penis is about 3 cm (stretched length I–V (fig 2). The anatomy of the vagina a screen for any associated renal anom- from pubic tubercle to tip of penis) with or a urogenital sinus and uterus may be aly (for example, Denys-Drash/Frasier micropenis less than 2.0–2.5 cm, determined by ultrasound, and if neces- syndromes). although this does vary slightly depend- sary, further delineation by EUA/cysto- 5 ing on ethnic origin. Chordee should be scopy or urogenital sinogram. Examination of external genitalia noted as this may decrease the apparent Ultrasound is also useful in excluding and Prader staging length of the phallus and the penis may associated renal anomalies, particularly Examination of the external genitalia be ‘‘buried’’ in some cases. The presence if Denys-Drash syndrome is suspected or determines whether gonads are palpable of hypospadias and the position of the proven. It may also be used to visualise and the degree of virilisation or Prader urethral meatus should be determined. the adrenal glands. Ultrasound may also stage. Prader stages I–V describe The urine may be coming from more locate inguinal gonads, although it is increasing virilisation from a phenotypic than one orifice. The degree of fusion not sensitive for intra-abdominal female with mild cliteromegaly (stage I) and rugosity of the labioscrotal folds gonads. Ultrasound sensitivity and to a phenotypic male with glandular should be noted and the presence or accuracy depend on probe resolution; hypospadias (stage V) (fig 2). absence of a separate vaginal opening an experienced ultrasonographer is If both gonads are palpable they are determined. required. Identification of the gonads likely to be testes (or ovotestes) which Hyperpigmentation, especially of the may require magnetic resonance ima- may be normal or dysgenetic. Flat finger genital skin and nipples occurs in the ging (MRI) or laparoscopy. These latter palpation from the internal ring milking presence of excessive ACTH and pro- investigations are also useful for deter- down into the labial scrotal folds may opiomelanocortin and may be apparent mining the anatomy of the internal

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period, but AMH is undetectable in Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from Table 3 Forms of CAH causing genital ambiguity in the newborn female serum until the onset of puberty. StAR protein AMH may prove to be a more sensitive Type of CAH deficiency 3b-HSD 17a-OHD 11b-OHD 21-OHD marker for the presence of testicular Genital === R R tissue than serum testosterone levels, ambiguity both before and after the neonatal Salt wasting Yes Yes Hypertension Hypertension Yes androgen surge, and, consequently, Enzyme P-450cc 3b-HSD P-450c17 P-450c11 P-450c21 may obviate the need for hCG stimula- Steroidsq None DHEA, DOC, DOC, 11- 17-OHP, A4 17-OH-preg corticosterone deoxycortisol tion in the evaluation of certain intersex 9 SteroidsQ All Aldosterone, Cortisol, Cortisol Cortisol, disorders. Similarly, basal inhibin B has testosterone, testosterone aldosterone been shown to predict the testosterone cortisol response to hCG in boys, and therefore Defective gene StAR HSD3B2 CYP17 CYP11B1 CYP21 may give reliable information about Chromosome 8p11.2 1p13.1 10q24.3 8q24.3 6p21.3 both the presence and function of the StAR protein, steroidogenic acute regulatory protein deficiency, otherwise known as lipoid hyperplasia/ testes. Furthermore, inhibin B levels cholesterol desmolase deficiency; 3b-HSD, 3b-hydroxysteroid dehydrogenase deficiency; 17a-OHD, have been shown to demonstrate spe- 17a-hydroxylase deficiency; 11b-OHD, 11b-hydroxylase deficiency; 21-OHD, 21-hydroxylase cific alterations in patients with genital deficiency; DOC, 11-deoxycorticosterone; DHEA, dehydroepiandrosterone; 17-OHP, 17- hydroxyprogesterone; 17-OH-preg, 17-hydroxypregnenolone; A4, androstenedione. ambiguity which may aid the differential diagnosis of male undervirilisation.10 However, these assays are not routinely available in the UK. genitalia, and gonadal and genital skin (testosterone secretion normally rises biopsies can be performed at the time of in the fourth week of life, peaking at Assessment of gonadotrophins laparoscopy. 1–3 months6–8) and proceeding with the Assessment of the gonadotrophins may investigations as promptly as possible. give useful information. Raised basal Testosterone response to human While some would advocate that this gonadotrophins are consistent with pri- chorionic gonadotrophin (hCG) test is deferred beyond 2 weeks of age, mary gonadal failure. The gonadotro- To determine whether functioning this may not be necessary. In the new- phin response to GnRH is difficult to Leydig cells are present (that is, capable born period an increase in testosterone, interpret in the prepubertal child unless of producing testosterone in response to reaching adult male levels, would nor- exaggerated, which is consistent with LH), an hCG stimulation test is under- mally be expected after hCG. gonadal failure but adds little to the taken. This is also used to delineate a The hCG test can be extended to a basal levels alone. Pituitary failure block in testosterone biosynthesis from three week test if the three day hCG would give a flat response but is not androstenedione (17b-hydroxysteroid test is inconclusive. The same dose of diagnostic, and hypothalamic abnorm- dehydrogenase deficiency) or conver- hCG is administered twice weekly for alities such as Kallmann’s syndrome are sion of testosterone to DHT (5a-reduc- three weeks and testosterone, DHT, not excluded by a ‘‘normal’’ response tase deficiency). There are a variety of and androstenedione samples taken The optimal time to assess this is within protocols for the hCG test, but essen- 24 hours after the last hCG injection. the first six months of life when there is tially it involves taking baseline samples The clinical response in terms of testi- a gonadal surge in both sexes (girls http://adc.bmj.com/ for testosterone and its precursors dehy- cular descent and change in the size of greater than boys), and the axis is hence droepiandrosterone (DHEA) (or DHEA the phallus and frequency of erections maximally responsive. sulphate (DHEAS)) and androstene- should be documented. Photographs pre- and post-hCG, may be helpful. dione and its metabolite (DHT). One to Assessment of adrenal function three intramuscular injections of high Urine steroid profile dose hCG (500–1500 IU) are given at Anti-Mu¨ llerian hormone (AMH) and Output of adrenal steroids will be low in 24 hour intervals and repeat androgen inhibin B levels adrenal insufficiency. In CAH specific on September 26, 2021 by guest. Protected copyright. samples are taken at 72 hours or While hCG stimulation tests the func- ratios of metabolites will be altered, 24 hours after the last injection. The tion of the Leydig cells, both AMH and depending on the level of the enzyme neonatal gonad is more active than at inhibin B are secreted by the Sertoli block. any time in childhood until puberty, cells. Inhibin B is detectable for the first reaching a peak at about a 6–8 weeks of six months, rising again at puberty. age. A balance needs to be reached AMH levels are high in human male Synacthen test A standard short Synacthen test is between performing the hCG test when serum postnatally for several years useful in the following situations: the gonad is normally most active before declining during the peripubertal N Suspected CAH where a peak 17- hydroxyprogesterone (17-OHP) of 100–200 nmol/l is suggestive of 21- hydroxylase deficiency. (Higher reference ranges for preterm infants.) N Suspected CAH to assess adrenal cortical reserve (measurement of cortisol levels).

The dose of Synacthen is dependent on local protocol (example 36 mg/kg or a standard dose of 62.5 mg intravenously Figure 2 Differential virilisation of the external genitalia using the staging system of Prader, from or intramuscularly). Cortisol and 17- normal female (left) to normal male (right). Sagittal (upper panel) and perineal (lower panel) views OHP levels are normally measured at 0 shown. and 30 minutes only as the 60 minute

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CAH are DHEA, androstenedione, tes- Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from tosterone, ACTH, and plasma renin activity, all of which may be increased. From day 3 of life, the ratio of urinary steroid metabolites will be altered depending on the site of the block, and is very helpful in the diagnosis of 21-OHD and the rarer forms of CAH. A skilled ultrasound examination will show normal internal genitalia, but an EUA may be required to confirm the presence of normal Mu¨llerian structures, and to show the level of entry of the vagina into the urogenital sinus, in the case of a single perineal opening. The excessive androgen may be gonadal in origin—usually from an ovotestis or testis in a true hermaphrodite. While the commonest karyotype of the true hermaphrodite is 46XX (70.6%), the next commonest karyotype is a chromo- somal mosaicism containing a Y chromosome (usually 46XX/46XY) (20.2%).11 It is important to check fibroblast and/or Figure 3 Investigation flow plan for assessment of ambiguous genitalia. gonadal genotype in these babies, which may contain a mosaic cell line. Transplacental transfer of androgen value does not usually contribute The androgens may be derived from the may rarely occur if the mother has an further. A normal basal value, or even fetal adrenal gland (CAH and placental androgen secreting tumour (she may be a normal stimulated response does not aromatase deficiency), fetal gonad (the virilised as a result) or from drugs given exclude the evolution of adrenal insuf- testis or ovotestis in true hermaphrodit- during pregnancy. Placental aromatase ficiency, and may need to be repeated ism), or rarely, exogenously via trans- deficiency, by inhibiting the conversion depending on clinical suspicion. A basal placental passage from the mother of androgens to oestrogens, may cause ACTH level may be helpful, but in most (adrenal or ovarian tumours). virilisation of a female fetus, and in laboratories the turnaround time is Absence of palpable gonads in addition, maternal virilisation occurs slower than for cortisol. association with otherwise apparently from placental transfer of the excessive male genitalia should always alert one fetal androgens.12

Skin and gonadal biopsies to the possibility of a virilised female. By Table 2 lists conditions that cause http://adc.bmj.com/ Genital skin biopsies (2–4 mm) per- far the commonest cause of this is virilisation of a female fetus. formed at the time of examination CAH. Of the enzyme defects that under anaesthesia (EUA) or genito- cause virilisation in female fetuses, 21- Genital ambiguity with a 46XY plasty are useful to establish cell lines hydroxylase deficiency is the most fre- karyotype indicates an for androgen receptor binding assays quent (accounting for 90–95%, UK undervirilised male and analysis of 5a-reductase activity. incidence 1/15–20 000). Diagnosis is This is a genetically XY male with two The cell line is also a source of genomic made on a raised serum 17-hydroxypro- testes, but whose genital tract fails to on September 26, 2021 by guest. Protected copyright. DNA and RNA and/or subsequent mole- gesterone level. This level may be differentiate normally. There are numer- cular and functional studies. Karyotype increased in the first 48 hours in nor- ous presentations of genital anomaly, analysis for the presence of mosaicism mal babies, and may be significantly from apparently normal female (Prader may be indicated. Gonadal biopsies are increased in sick and preterm babies in I) with a palpable gonad through to an essential when considering diagnostic the absence of CAH. The other enzyme apparently normal male with hypospa- categories such as dysgenesis and true defects that can cause virilisation of dias (Prader V). hermaphroditism. A detailed histo- female fetuses are 11b-hydroxylase The three main diagnostic categories pathological report is essential. As spe- deficiency (where the 11-deoxycortisol are: testicular dysgenesis/malfunction, a cial treatment of the samples may be levels will be increased) and less com- biosynthetic defect, and end-organ required, prior discussion with the monly 3b-hydroxysteroid dehydrogen- unresponsiveness (table 2). pathologist or genetics laboratory ase deficiency (diagnosed by raised If the gonads are palpable, they are should take place. 17-hydroxypregnenolone and dehydro- likely to be testes (or rarely ovotestes). epiandrosterone) (table 3). Investigations are directed at determin- INTERPRETATION OF RESULTS If CAH is confirmed, the electrolytes ing the anatomy of the internal genita- Genital ambiguity with a 46XX need to be watched closely as salt lia, and establishing whether the karyotype indicates a virilised wasting occurs in 70% of cases of 21- testicular tissue is capable of producing female hydroxylase deficiency, usually between androgens. Investigations that may help The female fetus with ovaries and days 4 and 15. Mineralocorticoid defi- with the former include pelvic ultra- normal internal genitalia has been ciency induces a rise in serum potas- sound, examination under anaesthetic exposed to excessive testosterone, and sium levels (usually the first sign of salt with cystoscopy and laparoscopy. hence dihydrotestosterone (by conver- wasting) and sodium levels fall. Urinary Genital skin biopsy specimens can be sion of testosterone by 5a-reductase) sodium levels will be inappropriately taken at the time of endoscopies. which virilises the external genitalia. high. Further confirmatory tests for Occasionally urogenital sinogram or

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MRI can be helpful. Laparoscopy/laparo- This condition is rare in the UK, but consistent with X linkage. The majority Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from tomy and gonadal biopsy may be recognised in the Dominican Republic, of XY infants with undervirilisation required. where individuals are often raised as remain unexplained. female and convert to male in puberty, when body habitus and psychosexual Testicular dysgenesis/malfunction DNA ANALYSIS orientation becomes male. Virilisation A 46 XY karyotype, with low basal and Many of the causes of genital ambiguity improves but is incomplete. hCG stimulated testosterone and low have a genetic basis, and in these cases A biochemical diagnosis is made by testosterone precursors suggests genetic counselling will be required. For either gonadal dysgenesis (which may showing a ratio of T:DHT .30 after example, androgen insensitivity syn- require laparoscopy and testicular puberty or following hCG stimulation drome is X-linked recessive, and CAH biopsy)orlipoidCAH(causedbyan before puberty, and the ratio of 5a:5b is autosomal recessive. In addition, abnormality in the steroidogenic acute metabolites in a urine steroid profile will identification and characterisation of a regulatory (StAR) protein) be increased after 6 months of age. The number of mutations of the genes In the latter condition total adrenal urinary 5a:5b metabolites can also be failure is confirmed on Synacthen test, used if the gonads have been removed. involved in sexual differentiation has electrolytes, and urinary steroids. The diagnosis is confirmed by screening resulted in DNA tests which can be Because of the other associated gene for mutations in the 5a-reductase type II used in prenatal diagnosis. Iden- defects (table 1), there may be other gene (5RD5A2). tification of carriers will facilitate anomalies such as bony dysplasias or genetic counselling. renal anomalies, which should be A 46XY karyotype, low basal and hCG looked for. stimulated testosterone levels with Congenital adrenal hyperplasia The poorly functioning testicular tis- increased testosterone precursors Several laboratories within the UK will sue is likely to give a subnormal indicates a testosterone biosynthetic undertake DNA analysis for this condi- testosterone response to hCG and basal defect tion. Once the diagnosis has been made, gonadotrophins will usually be Those forms of CAH that cause under- a DNA sample from the proband should increased, consistent with primary virilisation of male genitalia include be taken. If a gene mutation is identi- gonadal failure. In addition, the dysge- 17a-hydroxylase/17,20-lyase deficiency fied, the carrier status of the parents netic testes may secrete inadequate and 3b-hydroxysteroid dehydrogenase may be determined and the family amounts of anti-Mu¨llerian hormone, deficiency (table 3). should be counselled about the possibi- and Mu¨llerian structures may be present The conversion of androstenedione to lity of antenatal screening of subsequent (although often hypoplastic) in children testosterone occurs predominantly in pregnancies, and of steroid treatment of with gonadal dysgenesis and a 46XY the gonad and a post-hCG stimulated the mother in an attempt to reduce karyotype. ratio of androstenedione to testosterone virilisation of a subsequent affected A mosaic karyotype, for example 45X/ of .20:1 suggests 17b-hydroxysteroid female fetus. In the UK, antenatal 46XY suggests gonadal dysgenesis. dehydrogenase deficiency. A urine ster- diagnosis and treatment is offered as There is a very variable phenotype both oid profile is generally not helpful in this part of a national British Society for in terms of internal and external geni- diagnosis before puberty. Molecular Paediatric Endocrinology and Diabetes talia, which is not dependent on the analysis of the HSD III gene supported study monitoring efficacy, percentage of each karyotype as deter- (HSD17B3) is sought as confirmation short and long term side effects, and http://adc.bmj.com/ mined by lymphocyte analysis. Over of the diagnosis. outcome measures. 90% of individuals with prenatally diagnosed 45X/46XY karyotype have a nor- End-organ unresponsiveness mal male phenotype, suggesting most A 46XY karyotype with genital Androgen insensitivity individuals with this karyotype escape ambiguity, normal or increased basal DNA analysis is now being undertaken detection and that an ascertainment and peak testosterone on hCG test, in Cambridge as part of a molecular bias exists towards those with clinically and a normal T:DHT ratio points to genetics service in collaboration with 13 14 partial androgen insensitivity Professor Ieuan Hughes. Samples are evident abnormalities. on September 26, 2021 by guest. Protected copyright. There is a variable phenotype, and sex of only analysed following collection of Biosynthetic defect rearing depends on the degree of phallic clinical, biochemical, and histological development, and sometimes cultural A 46XY karyotype, low basal and peak data that are consistent with an andro- testosterone level on hCG testing, considerations. The child may benefit gen insensitive pathophysiology. often with increased gonadotrophins from a trial of topical DHT cream or intramuscular testosterone (for example suggests a diagnosis of an inactivating Suspected 5a-reductase deficiency using 12.5–25 mg, monthly for three mutation of the LH receptor (Leydig and 17b-hydroxysteroid months) on penile growth to help cell hypoplasia) dehydrogenase deficiency anticipate response in puberty. This condition is associated with a DNA samples in patients with suspected The diagnosis is suggested by showing variable phenotype from a completely 5a-reductase deficiency and 17b-hydro- an abnormality in androgen binding in phenotypic female to undervirilisation xysteroid dehydrogenase deficiency can 15 genital skin fibroblasts, or a mutation in of varying degrees. also be processed through the the androgen receptor gene. This Cambridge laboratory. A 46XY karyotype with normal or requires DNA and a genital skin biopsy, increased basal and peak testosterone taken at the time of EUA or genital on hCG test, and an increased T:DHT surgery. Despite clear evidence of a Unusual cases of sexual ambiguity ratio is seen in 5a-reductase phenotype consistent with partial Mutations of developmental genes such deficiency androgen PAIS and normal production as DAX1, SOX9, and WT1 may account DHT dependent virilisation of external and metabolism of androgens, only a for rarer cases of sexual ambiguity. genitalia is deficient, resulting in a small minority of patients are found to have Samples should be taken and DNA phallus and perineal hypospadias. an androgen receptor gene mutation. extracted and either stored or forwarded Wolffian structures are normal but The likelihood of finding a mutation is to the relevant laboratories, depending spermatogenesis is usually impaired.16 increased if there is a family history on clinical suspicion.

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ASSIGNMENT OF SEX OF CONCLUSIONS 3 Ahmed SF, Hughes IA. The genetics of male Arch Dis Child: first published as 10.1136/adc.2002.011312 on 21 April 2004. Downloaded from undermasulinization. Clin Endocrinol REARING Genital ambiguity resulting in uncer- 2002;56:1–18. A decision about the sex of rearing tainty of sex at birth is uncommon. The 4 Vainio S, Heikkila M, Kispert A, et al. Female should be made as soon as is practic- most frequent cause in a genetic female development in mammals is regulated by Wnt-4 signalling. Nature 1999;379:707–10. able, usually based on the internal and is CAH, which may be life threatening if 5 Cheng PK, Chanoine JP. Should the definition of external genital phenotype and the there is a risk of a salt losing crisis. micropenis vary according to ethnicity? Horm Res results of the various investigations. Reaching a diagnosis, particularly in 2001;55:278–81. 6 Winter JS, Hughes IA, Reyes FI, et al. Pituitary- Cultural aspects may also be important undervirilised males, is currently not gonadal relations in infancy: 2. Patterns of serum in cases of severe ambiguity. always possible, but many may have a gonadal steroid concentrations in man from birth Assignment of sex of rearing can be partial androgen insensitivity syndrome to two years of age. J Clin Endocrinol Metab 1976;42:679–86. extremely difficult, particularly as there yet to be defined in molecular terms or 7 Ng KL, Ahmed SF, Hughes IA. Pituitary-gonadal is a paucity of data on long term milder variants of testicular dysgenesis. axis in male undermasculinisation. Arch Dis Child outcome in this area. Prompt counselling and investigations 2000;82:54–8. 8 Davenport M, Brain C, Vandenberg C, et al. The In the case of a virilised female (with the backup of recognised bio- use of the hCG stimulation test in the endocrine (usually CAH), there is usually the chemical and genetic laboratories) is evaluation of cryptorchidism. Br J Urol potential for fertility and these babies essential. The decision of sex of rearing 1995;76:790–4. are usually raised as girls. This is much 9 Rey RA, Belville C, Nihoul-Fekete C, et al. and the timing of surgery need careful Evaluation of gonadal function in 107 intersex easier if the diagnosis of CAH is made consideration within a multidisciplinary patients by means of serum antimullerian early. environment with full informed consent hormone measurement. J Clin Endocrinol Metab Decisions about nature and timing of 1999;84:627–31. of the family. 10 Kubini K, Zachmann M, Albers N, et al. Basal any surgery are made with the family inhibin B and the testosterone response to acknowledging the considerable psycho- human chorionic gonadotropin correlate in logical impact of having a child with ACKNOWLEDGEMENTS prepubertal boys. J Clin Endocrinol Metab 2000;85:134–8. genital ambiguity. There is considerable We thank Professor Ieuan Hughes and Dr John Achermann for their helpful comments. 11 Krob G, Braun A, Kuhnle. True hermaphroditism: debate as to the optimal timing of any geographical distribution, clinical findings, Email address for Professor Hughes for genital surgery.17 In the presence of chromosomes and gonadal histology. Eur J Pediatr mutation analysis and discussion on the 1994;153:2–10. marked clitoromegaly, clitoral reduction androgen receptor, 5a-reductase deficiency, 12 Shozu M, Akasofu K, Harada T, et al. A new is usually undertaken in infancy. Lesser and 17b-hydroxysteroid dehydrogenase: cause of female pseudohermaphroditism: degrees of clitoral enlargement may be [email protected]. Email address for Dr placental aromatase deficiency. J Clin Endocrinol Metab 1991;72:560–6. left until puberty when the child can be Achermann for mutation analysis and dis- 13 Chang HJ, Clark RD, Bachman H. Chromosome involved with the decision making. The cussion on early gonadal genes or cases mosaicism in 6,000 amniocenteses. Am J Med timing of any vaginoplasty is dependent associated with adrenal failure: j.achermann Genet 1989;32:506–13. @ich.ac.uk. 14 Wilson MG, Lin MS, Fujimoto A, et al. The on the anatomy of the internal and phenotype of 45,X/46,XY mosaicism: an analysis external genitalia and influenced by Web address for the British Society for Paediatric Endocrinology and Diabetes: of 92 prenatally diagnosed cases. Am J Hum local practice. However, there has been Genet 1990;46:156–67. www.bsped.org.uk. a move away from early vaginoplasty in 15 Laue L, Wu S-M, Kudo M, et al. A nonsense Arch Dis Child 2004;89:401–407. mutation of the human luteinizing hormone all infants. It may be appropriate to receptor gene in Leydig cell hypoplasia. Hum Mol delay surgery until puberty when a doi: 10.1136/adc.2003.011312 Genet 1995;4:1429–33. 16 Hochberg Z, Chayen R, Reiss N, et al. Clinical, single stage reconstruction can be http://adc.bmj.com/ ...... biochemical, and genetic findings in a large undertaken. Recent outcome data on pedigree of male and female patients with 5 clitoral sensation18 and success of early Authors’ affiliations alpha-reductase 2 deficiency. J Clin Endocrinol vaginoplasty19 20 in adult women who A L Ogilvy-Stuart, Neonatal Unit, Rosie Metab 1996;81:2821–7. 17 Creighton S. Surgery for intersex. J R Soc Med underwent genital surgery in infancy Hospital, Addenbrooke’s NHS Trust, Cambridge CB2 2SW, UK 2001;94:218–20. and childhood has induced a more C E Brain, Dept of Paediatric Endocrinology, 18 Minto CL, Liao LM, Woodhouse CRJ, et al. The cautious surgical approach.21 22 effect of clitoral surgery on sexual outcomes in Great Ormond Street Hospital, Great Ormond individuals who have intersex conditions with The appropriate sex of rearing of a Street, London WC1N 3JH, UK ambiguous genitalia: a cross sectional study. very undervirilised male requires as Lancet 2003;361:1252–7. on September 26, 2021 by guest. Protected copyright. much information as possible, from the 19 Alizai NK, Thomas DF, Lilford RJ, et al. Correspondence to: Dr A L Ogilvy-Stuart, Feminizing genitoplasty for congenital adrenal investigations discussed, as well as Neonatal Unit, Rosie Hospital, Addenbrooke’s hyperplasia: what happens at puberty? J Urol thorough, multidisciplinary discussions NHS Trust, Cambridge CB2 2SW, UK; 1999;161:1588–91. involving the parents, urologist, endo- [email protected] 20 Creighton S, Minto C, Steele SJ. 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