Approach
The approach to a neonate with atypical genitalia involves a multidisciplinary team (MDT) of paediatric subspecialists.[17] This usually includes an endocrinologist, a neonatologist, a urological surgeon, a clinical psychologist and a specialist nurse. The core MDT should have links with a wider team, including specialists from genetics, biochemistry, relevant adult medical specialties, such as gynaecology and endocrinology, social services, and if possible a clinical ethics forum.[18] Ongoing communication with the family is crucial, as well as communication with the primary care physician.[18][19] The following concepts should guide the diagnostic evaluation of neonates with atypical genitalia:[1][18][20]
Sex assignment. A sex is not assigned until the evaluation has been completed. The child is referred to as 'baby', not boy or girl. The family should be encouraged to delay naming the baby until the sex has been assigned. Sex assignment may be undertaken even if an underlying genetic or endocrine diagnosis is not apparent.[21]
Following the initial assessment and investigations the baby should be evaluated at a centre with multidisciplinary expertise in the evaluation of neonates with atypical genitalia. In some situations, care at a local hospital may be appropriate, with early consultation with a specialised MDT in a regional centre.
Ideally, discussions with the family are led by one professional from the team, usually the endocrinologist. It is useful to gain the parents' agreement to discuss the initial results of investigations collectively, rather than informing them when each result comes through, as parents may place undue emphasis on the result of a karyotype or testosterone level and not appreciate the extent to which these should be interpreted in the light of results of other investigations.[21]
The needs, background, culture, and expectations of the parents must be understood and respected. Parents should be reassured that children with a disorder of sex development (DSD) can live successful lives and function well in society. Decisions regarding sex assignment are undertaken by the DSD multidisciplinary team with the parents. A discharge management plan will be agreed. Informing the parents regarding expected sexual development is also important, as this helps anticipate issues early.[21]
History
A family history may identify family members with similar problems indicating an autosomal-recessive or X-linked inheritance. This includes specific enquiry into a history of consanguinity, genital surgery, infertility, maternal virilisation during pregnancy (e.g., voice change or hirsutism), virilisation of a female at puberty (5 alpha-reductase deficiency), or other children born with atypical genitalia. Unexplained neonatal death in a family member could indicate an undiagnosed DSD associated with adrenal insufficiency.
Most genetic causes of atypical genitalia are due to autosomal-recessive conditions, such as congenital adrenal hyperplasia (CAH), 5 alpha-reductase deficiency, and other defects in testosterone biosynthesis. X-linked recessive inheritance could suggest androgen insensitivity (the androgen receptor gene is on the X chromosome). The antenatal history should include questions regarding maternal exposures to androgens or medications and signs of virilisation in the mother during pregnancy.
Physical examination
Although different disorders may present with similar findings on physical examination, there are often aspects of the examination that are crucial and will help guide the initial investigations.
General examination: as some causes of DSD are associated with adrenal insufficiency, it is important to confirm that vital signs (blood pressure, capillary refill, and heart rate) and blood glucose are normal with arrangements for appropriate monitoring in place. The presence of dysmorphic features and/or other congenital anomalies may suggest a syndrome that includes atypical genitalia (e.g., Smith-Lemli-Opitz syndrome).
Gonads: the presence of one or two gonads rules out 46,XX DSD secondary to 21 hydroxylase deficiency. Rarely, 46,XX ovotesticular DSD may present with inguinal gonads. The presence of two gonads is more likely to be from a cause of 46,XY DSD. The presence of only one palpable gonad could also suggest a diagnosis of mixed gonadal dysgenesis.
Phallus length: for a term male infant, a normal stretched penile length ranges from 2.5 cm to 4.5 cm and for a term female infant a normal clitoral length ranges from 0.2 cm to 0.85 cm with variation between ethnic groups.[21]
Neonates with 46,XX DSD due to 21 hydroxylase deficiency may have hyperpigmented labioscrotal folds.
The external masculinisation score (EMS) can be a useful adjunct to the clinical examination as a guide for the local clinician.[22][23] The external genitalia score (EGS) is a gender neutral alternative, which has a high level of correlation to the original EMS scoring system.[18][24][Figure caption and citation for the preceding image starts]: External masculinisation scoreCreated by the BMJ Knowledge Centre based on Davies JH, Cheetham T. Recognition and assessment of atypical and ambiguous genitalia in the newborn. Arch Dis Child. 2017 April [epub ahead of print]. [Citation ends].
Urethral opening: hypospadias occurs when a urethral opening is not present at the tip of the phallus. Hypospadias associated with separation of scrotal sacs or undescended testis may suggest an underlying DSD. If the urethral opening is at the base of the phallus, it could be a urogenital sinus in a virilised female. This occurs when the urethral and vaginal openings are connected internally and exit at the perineum through a common opening.
Other signs to note include:[21]
Midline defects (e.g., cleft palate)
Excess pigmentation (e.g., areolar pigmentation)
Cardiac or skeletal abnormalities.
Further investigation for DSD should be considered in infants with isolated micropenis, isolated clitoromegaly, isolated perineal hypospadias or any familial hypospadias, isolated bilateral undescended testes and those with an EMS of less than 11 (or an EGS of less than 10.5). The presence of a metabolic disorder, dysmorphic features or relevant family history should lower the threshold for further investigation.[18]
Key clinical presentations
Bilateral impalpable gonads in an apparent male baby
A neonate with bilateral impalpable gonads with an external male phenotype may in fact have a 46,XX karyotype with clitoral enlargement from excess androgen production in utero. These babies should not be discharged until CAH secondary to 21 hydroxylase deficiency has been excluded.
Inguinal herniae in an apparent female baby
A neonate may still have an underlying 46,XY karyotype and this presentation may occur in, for example, complete androgen insensitivity, 17 beta-hydroxysteroid dehydrogenase deficiency, and 5 alpha-reductase deficiency.
Asymmetric genitalia
Sex chromosome mosaicism (e.g., 45,X/46,XY) may cause asymmetric genitalia. At laparoscopy, a streak gonad and hemiuterus are typically identified on the side contralateral to the descended or palpable gonad.
Micropenis
For a term male infant, a normal stretched penile length ranges from 2.5 cm to 4.5 cm.[21] A length of <2.5 cm is considered abnormal in a full-term male baby, especially in the presence of undescended testes or other abnormalities.[21] There is variation in normal penile size between ethnic groups.
In preterm infant males, the penis is shorter and the length should be plotted on a centile chart available for preterm male babies.[25]
Disorders of testosterone biosynthesis or testosterone action may lead to micropenis (e.g., partial androgen insensitivity and 5 alpha-reductase deficiency).
Clitoral enlargement
A normal clitoral length in a term female infant ranges from 0.2 cm to 0.85 cm, with variation between ethnic groups.[21] CAH secondary to 21 hydroxylase deficiency is the commonest cause of cliteromegaly.
Hypospadias
Severe hypospadias may be associated with atypical genitalia. Severe hypospadias may have an underlying genetic cause in 40% of cases including partial androgen insensitivity and 5 alpha-reductase deficiency.[22][26] The neonate with hypospadias but with impalpable gonads has CAH secondary to 21 hydroxylase deficiency until proven otherwise.
Approach to a patient with atypical genitalia
Following undertaking a detailed history and examination (which must include palpation for gonads), the next step is to organise specific investigations. At initial presentation, regular electrolytes, pre-feed glucose concentrations, and regular blood pressure monitoring should be undertaken. Urgent chromosome analysis (karyotype) should be requested, with imaging in the first 48 hours and hormone studies after 48 hours of age. In those with no palpable gonads, a 17-OHP and other hormone studies should be taken after 48 hours of age. An ultrasound is useful to define the presence of a uterus (a Müllerian structure), presence of adrenal glands, and morphology of gonads. A urine protein-creatinine ratio should also be requested.
Chromosomal analysis
On initial evaluation, a chromosomal analysis should be performed with at least 30 metaphases to assess for mosaicism. Results can usually be obtained within 72 hours. A formal karyotype can take many days to perform and a more rapid result may be obtained by requesting an urgent fluorescence in situ hybridisation (FISH) or quantitative polymerase chain reaction (PCR) of the sex-determining region Y (SRY) gene. Other genetic studies can be performed when a specific diagnosis is being considered.[8]
High-throughput sequencing (HTS) assays and whole genome or exome sequencing (WGES) are methods increasingly used to target multiple DSD-related genes in a single analysis. Genetic counselling should be provided to parents before testing.[18]
Imaging
A pelvic and abdominal ultrasound scan (USS) is performed to determine whether a uterus (a Müllerian structure) is present. In an infant, maternal oestrogens enhance the ability to view the uterus on ultrasound in the first few weeks of life. Ovaries and fallopian tubes are often not visible on ultrasound. A USS is also useful to detect renal anatomy, adrenal anatomy (as adrenal glands are relatively large in the neonatal period), gonad site, and morphology.[27]
Hormonal studies
These investigations are usually undertaken after 48 hours of age. Although certain clinical presentations of atypical genitalia make some diagnoses more likely, the clinician should be aware that a definitive diagnosis cannot be made from clinical examination alone as there are varied presentations in many DSD.
No palpable gonads
In the absence of palpable gonads, CAH secondary to 21 hydroxylase deficiency must be excluded as it is the most likely diagnosis.
17 hydroxyprogesterone (17-OHP) levels should be obtained and will be markedly elevated in 21 hydroxylase deficiency.
Electrolytes should be obtained and monitored closely, as salt wasting may take a few days to develop, typically in the second week of life in 21 hydroxylase deficiency. Renin and aldosterone measurement will help assess mineralocorticoid activity.
If CAH is suspected and 17-OHP levels are not markedly elevated, adrenal precursors should be further evaluated to look for rarer causes of CAH, including 11 deoxycortisol and 11 deoxycorticosterone to rule out 11 beta-hydroxylase deficiency. A urine steroid profile is helpful in this scenario.
If the infant requires immediate steroid hormone therapy, adequate urine and serum samples should be collected before treatment is started and saved for later analysis.[18]
Gonad(s) palpable
Testosterone and dihydrotestosterone (DHT): a high testosterone-to-DHT ratio suggests a 5 alpha-reductase deficiency. A low testosterone-to-androstenedione ratio suggests 17 beta-hydroxysteroid-dehydrogenase deficiency.
Luteinising hormone (LH) and follicle-stimulating hormone (FSH): these help evaluate the hypothalamic-pituitary-gonadal axis. Low levels in the first week of life may not necessarily indicate hypogonadotrophic hypogonadism; however, an assessment at 2 to 4 months may be more revealing when there is a physiological increase in LH and FSH levels.
Adrenocorticotropic hormone (ACTH) stimulation test: this test is used in selected cases to investigate abnormalities of glucocorticoid synthesis in some forms of DSD.
Human chorionic gonadotrophin (hCG) stimulation test: this assesses the ability of Leydig cells of the testes to respond to hCG (an LH receptor analogue) and produce testosterone. The ratio of testosterone to DHT after hCG stimulation is used to ascertain the presence of 5 alpha-reductase deficiency. The hCG stimulation test can also identify a block in the testosterone biosynthetic pathway such as 17 beta-hydroxysteroid-dehydrogenase deficiency, where the ratio of testosterone to androstenedione would be low.
Anti-mullerian hormone (AMH) (previously called Mullerian-inhibiting substance): this can be obtained to assess testicular (Sertoli's cell) function in a baby with a suspected 46,XY DSD and in chromosomal DSD. AMH levels are age- and sex-dependent.
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