Child with ‘46, XX’ disorder of sex development: clues to diagnose aromatase deficiency

Abstract

A diagnosis of congenital adrenal hyperplasia (CAH) in a ‘46, XX’ newborn with ambiguous genitalia is like a ‘knee jerk reaction’ of the paediatrician because of its higher frequency and life-threatening consequences if remain undiagnosed and hence untreated. Aromatase deficiency (AD), a rare cause of ‘46, XX’ disorder of sex development, mimics virilising CAH in many aspects; thus, the disease is often overlooked. Diagnosis of AD in women is much easier around puberty due to the presence of primary amenorrhoea, undeveloped breasts, androgen excess and tall stature with eunuchoid proportions. Diagnosing AD with confidence immediately after birth or during early childhood is a challenging task without genetic analysis. In resource-restricted settings, AD remains a diagnosis of exclusion particularly in this age group and history of maternal virilisation, non-progressive genital ambiguity, elevated gonadotrophins (follicle-stimulating hormone >>luteinising hormone), mildly delayed bone age with/without enlarged polycystic ovaries serve as important clues to the underlying AD.

Background

Aromatase deficiency (AD) is a rare cause of ‘46, XX’ disorder of sex development (DSD). After the first description of this autosomal recessive disorder back in 1991, a total of about 40 such cases have been reported in the world literature until.^{1 2} However, diagnosis is likely to be overlooked in ‘46, XY’ individuals and incidence perhaps is higher than what is reported. Together with its coenzyme P450 oxidoreductase (POR) aromatase is responsible for conversion of C-19 steroids (androgens) to C-18 steroids (oestrogens) and is encoded by the gene CYP19A1 situated on chromosome 15q21.1. In aromatase-deficient fetus, androgens like dehydroepiandrosterone sulfate (DHEAS) and 16-hydroxy-DHEAS produced by the fetal adrenals and liver, respectively, are not converted to oestrogens. Conversion of these relatively weak androgens to more potent ones like androstenedione and testosterone within the fetoplacental unit and subsequent transplacental passage of these androgens results in ambiguous genitalia with variable degree of masculinisation in a ‘46, XX’ fetus. The phenotype depends on the level of residual activity of the enzyme; lower the aromatase activity, greater is the degree of androgenisation of the external genitalia at birth.^{3 4} These potent androgens of placental origin enter maternal circulation as well. Signs of maternal hyperandrogenaemia in the form of acne and hirsutism may appear at around the end of the first trimester and maternal virilisation like male pattern baldness and voice changes have also been reported. These features, however, characteristically resolve spontaneously in postpartum period except deepening of voice that often persists. Both maternal and fetal androgen excess gradually disappear after delivery and genital masculinisation typically is non-progressive. ‘46, XX’ children with AD are often misdiagnosed as virilising congenital adrenal hyperplasia (CAH) if evaluated immediately after birth due to high androgen levels. Definitive diagnosis is established by CYP19A1 mutation analysis; however, in a resource-restricted setting the diagnosis can also be achieved with confidence after ruling out other causes of ‘46, XX’ DSD by detailed history, relevant hormonal and appropriate radiological investigations.

Case presentation

Case 1

A 2-year 8-month-old child, reared as girl, was referred to us for evaluation of ambiguous genitalia in the form of non-progressive clitoromegaly that was noticed immediately after birth. A diagnosis of CAH due to 3β-hydroxysteroid dehydrogenase deficiency was made by her paediatrician due to equivocal adrenal steroid profile. She had been on hydrocortisone replacement for initial 1 year that was stopped subsequently for re-evaluation of the adrenal cortical functions. Born out of a non-consanguineous union, she had no history suggestive of adrenal crisis. However, her mother had developed severe form of cystic acne which appeared during the second trimester of gestation and resolved spontaneously postdelivery. The mother denied androgenic drug intake during the pregnancy and did not notice voice change, hirsutism or clitoromegaly. Physical examination revealed the following:

Weight: 12 kg (25th–50th percentile); height: 84.5 cm (5th–10th percentile); blood pressure (BP): 88/58 mm Hg (50th−90th percentile for age and height). She had non-palpable gonads with Prader stage 1 genitalia in the form of clitoromegaly (clitoral length 16 mm), no labioscrotal fusion and two separate openings (figures 1 and 2). Clitoral index was 36 mm² and anogenital ratio was 0.45. Systemic examination was normal. Abdominal ultrasound (USG) of mother during the pregnancy was reportedly normal.

Figure 1.

Genitalia in case 1.

Figure 2.

Enlarged clitoris in case 1. Palpation and USG ruled out underlying neurofibroma. USG, ultrasound.

Case 2

A 3-year-old child with ambiguous genitalia was referred to us with a working diagnosis of CAH. Genital ambiguity was noticed at birth and the child was reared as a girl. She was the first child, born out of a consanguineous marriage at term without any history suggestive of salt wasting crisis. There was no history of any offending drug intake by her mother or maternal virilisation during the gestation. The girl had been delivered at home by a quack and her parents were told that the genital ambiguity was a variant of normal female genitalia. Diagnosis of CAH was considered by a paediatrician immediately prior to her presentation with us. She had never been on corticosteroid.

Physical examination of the child revealed: height: 86.3 cm (3rd–5th percentile), weight: 11 kg (3rd–5th percentile), BP of 80/60 mm Hg (systolic blood pressure–50th percentile, diastolic blood pressure< 90th percentile for age). Local genital examination revealed a phallic length of 15 mm without palpable gonads. There was almost complete labioscrotal fusion with scrotal rugosities and a single perineal opening consistent with Prader stage 3 genitalia (figures 3 and 4). Anogenital ratio was 0.7. Systemic examination was essentially normal.

Figure 3.

Genitalia in case 2.

Figure 4.

Single perineal opening in case 2 suggesting Prader 3 genitalia.

Relevant investigations done in both these children have been summarised in table 1.

Table 1.

Summary of relevant investigations

Parameter	Case 1 (at 6 months of age)	Case 1 (at 28/12 years of age)	Case 2 (at 3 years of age)	Age and sex-specific reference range (identical in 28/12 and 3 years)
Karyotype	46, XX	46, XX	46,XX
Sodium	136	137	138	136–145 mEq/L
Potassium	5.4	4.7	5.0	3.5–5.5 mEq/L
08:00 Serum cortisol	10.3 (ref: 1.4–27 μg/dL)	11.2	7.27	5-21 μg/dL
08:00 Plasma ACTH (iced sample)		13.2	65.2	<46 pg/mL
Serum cortisol (1-hour postinjection tetracosactide)		25.3	31.32	>18 μg/dL is normal
08:00 17-OH progesterone (17-OH-P)	2.46 (ref.:≤1.47 ng/mL)	0.61	0.46	≤1.39 ng/mL
17-OH-P (1-hour postinjection tetracosactide)	4.2 (<10 ng/mL is normal)
Antimullerian hormone	0.13 ng/mL	0.05 ng/mL	<0.01 ng/mL	Not defined; usually very low to undetectable
DHEAS	15 (ref.: <74 μg/dL)	and <15	15	≤22 μg/dL
Total testosterone
08:00 Fasting	5.47 (ref.: 2–5 ng/dL)	<7	14.8	2–10 ng/dL
Day 5 of post-hCG stimulation		14.64	16.11	Rise by more than twice the baseline value is supportive of functioning testicular tissue
Day 20 of post-hCG stimulation		13	12.68
Androstenedione		0.34	0.48	≤77 ng/dL
Follicle-stimulating hormone		67.67 and 90.42	35.88	0.7–3.4 mIU /mL
Luteinising hormone		3.06	1.8	≤0.26 mIU/mL
Bone age		16 months (figure 5)	18 months (figure 6)
USG pelvis		Infantile uterus, gonads not visualised	Uterus—1.7×0.5×0.8 cm, right ovary—5×4 mm, left ovary—5×3 mm, no follicles seen
Genitogram			Low vaginal confluence
MRI pelvis		Hypoplastic uterus; Ovaries not visualised	Hypoplastic uterus; ovaries are not defined

ACTH, Adrenocorticotropic hormone; DHEAS, dehydroepiandrosterone sulfate; hCG, Human chorionic gonadotropin; USG, ultrasound.

Investigations

Figure 5.

Hand X-ray in case 1 (chronological age: 2^8/12 years): bone age is 16 months by Greulich and Pyle’s chart.

Figure 6.

Bone age (18 months) is less than chronological age (36 months) in case 2.

Fluorescence in situ hybridisation (FISH) for a sex-determining region of the Y chromosome (SRY) in peripheral blood leucocytes was negative in both of them.

Differential diagnosis

The differential diagnoses of ‘46, XX’ DSD with non-progressive virilisation after birth are AD either isolated or a part of POR deficiency, maternal androgenic drug exposure during the pregnancy and thecoma/luteoma of pregnancy. All these situations may also be associated with maternal virilisation. ‘46, XX’ ovotesticular DSD is also associated with non-progressive genital virilisation after birth till onset of puberty. However, maternal androgens are characteristically normal in this condition.

Treatment

Parents of both the patients decided to raise their children as girls. Patient 1 is being kept under regular follow-up with a plan for serial bone age assessment while patient 2, in view of her more masculinised genitalia (Prader stage 3) has been refereed for feminising genitoplasty.

Outcome and follow-up

As aromatase-deficient individuals are at future risk of developing insulin resistance, abnormal lipid profile and metabolic syndrome, the parents of both the children have been counselled for regular follow-up. Oestrogen replacement therapy has been shown to have varied responses on these parameters and preventing development of multicystic ovaries later on. However, there is no consensus on the use of oestrogen treatment during the infancy and early childhood at present. We decided against immediate low-dose oestrogen therapy due to the absence of ovarian cysts in either of these children. Both of them shall be followed up yearly with clinical assessment and pelvic USG. Puberty induction with oestrogen has been planned at around 10–12 years of chronological age.

Discussion

The default diagnosis in a child born with ambiguous genitalia, non-palpable gonads, elevated androgens and 46, XX karyotype is CAH due to 21-hydroxylase deficiency as this disorder is far more common than other aetiologies of DSD. Features of adrenocortical insufficiency (glucocorticoid with/without mineralocorticoid deficiency) is absent in aromatase defect. A history of maternal virilisation also points against CAH except POR deficiency or apparent combined 17α-hydroxylase, 21-hydroxylase and aromatase deficiencies. The mother of the first child did notice signs of androgen excess appearing at 24th week of gestation that resolved spontaneously after delivery. The mother of the second child, however, refuted any such history. Maternal virilisation is not universal in AD as placental aromatase activity as low as 1% of normal is sufficient to prevent maternal virilisation.⁵ It has also been hypothesised that a lower fetal adrenal androgen output or adequate placental oestrogen production, especially during the third trimester of gestation can prevent maternal virilisation in some patients.⁶ Interestingly, however, we noticed maternal androgen excess in the first child with minimal genital virilisation (Prader stage 1) and lack of such history in the mother of the second child with severe degree of masculinisation (Prader stage 3). This discordance remains unexplained by the above postulations. Patients with virilising form of CAH demonstrate an advanced bone age secondary to excess circulating gonadal steroids while aromatase-deficient individuals show a delayed skeletal maturation around puberty. Bone age is minimally delayed also during early childhood in AD suggesting a possible role of oestrogen in appearance of epiphyses in addition to its well established effect on epiphysial fusion. Both the children had bone ages that were less than the chronological ages (16–18 months) and the finding is consistent with the earlier studies.^{3 7} Non-progressive genital abnormalities with normal electrolytes, cortisol, ACTH and 17-OH progesterone (basal and stimulated) along with non-advanced bone age and presence of uterus prompted us to look for non-CAH aetiologies.⁸ Non-progressive genital virilisation is also observed in maternal androgenic drug intake or androgenic tumours during pregnancy which was ruled out with confidence in our cases. ‘46, XX’ ovotesticular DSD may have similar presentation at times. Prolonged hCG stimulation tests (1000 IU hCG given IM on day 1, 3, 4, 9, 12, 16 and 19 and serum samples for testosterone were sent at baseline and on day 5 and 20) were done to rule out any functional leydig cells. No demonstrable increase in testosterone was observed in either of these patients. Moreover, very low/undetectable serum antimullerian hormone and negative FISH for SRY were also consistent with absence of testicular tissue. Majority of peripubertal and postpubertal girls with AD develop large ovarian cysts due to chronic follicle-stimulating hormone (FSH) stimulation.⁷ However, literature has been inconsistent as far as ovarian morphology is concerned. Normal sized ovaries without cysts, hypoplastic ovaries and even streak ovaries have also been reported.^{2 9} An early block in follicular development at the antral stage leading to absent secondary follicles and atresia of primary follicles with increased collagen deposition may ultimately result in streak ovaries in some patients. A novel mutation in CYP19A1 (568insC) that generates a null mutation is associated with hypoplastic ovaries suggesting testosterone excess, absent oestrogen in fetal life and probably aromatase gene itself play important roles in normal ovarian development.¹⁰ Similar finding has also been observed in another homozygous mutation IVS7-2A>G (c.744-2A>G) involving intron 7 of the CYP19A1 gene.¹¹ The hypothalamo–pituitary–gonadal (HPG) axis shows prominent bursts of activity twice before the onset of puberty: once during fetal life and then during first postnatal months, a period known as ‘mini puberty’. Gonadotrophin levels are low at birth in both sexes, start rising after about 1 week once the placental hormones are cleared from circulation of newborn during initial postnatal days and reach a peak around 3 months. In boys, both luteinising hormone (LH) and FSH decrease to prepubertal level by 6–9 months. In girls though LH dynamic simulates that of the boys, FSH may remain elevated up to 3–4 years. It has been speculated that minimum amount of circulating gonadal steroids is sufficient to maintain an restraining effect on pituitary gonadotrophins in prepubertal period probably due to enhance sensitivity of the higher centres to inhibitory effect of oestrogen and/or testosterone. In girls with AD, FSH values are consistently elevated (often in menopausal range) not only during mini puberty but also around 3–4 years of age.^{7 8} Oestradiol and oestrone levels, however, remain remarkably low during the same period and LH levels may not be elevated in all.^{3 7 12} Unstimulated FSH levels were markedly high in both of our patients; LH values, though disproportionately low as compared with FSH, were not prepubertal as expected in this age group.

Girls with Prader 1 genitalia (as in case 1) may not require feminising genitoplasty at all as fat deposition in mons pubis and labia may mask the clitoromegaly later on. So they are followed up till adulthood when a decision on neurovascular preserving reduction clitoroplasty is taken. However, there has been much controversy about time of genital reconstructive surgery using total/partial urogenital mobilisation and perineal reconstruction with/without clitoroplasty in more masculinised genitalia (Prader 3–5). Girls with low vaginal confluence (as in case 2) are usually operated in infancy/preschool age.

Diagnosing AD before puberty without genetic analysis in resource-restricted setting is difficult. Serial hormonal levels of both infant and mother would give a strong clue and may be sufficient to make the diagnosis. Progressive decline in androgen levels in both mother and child from birth to early infancy are helpful indicators of AD. The low maternal oestradiol observed immediate post-partum gradually becomes normal.¹³ The diagnosis is even more challenging in prepubertal children postinfancy. The oestrogen and the androgen levels during that period may either be too close to the lower detection limit of the assay or undetectable. An elevated FSH in appropriate clinical and biochemical background is a cheap and effective tool to reach the diagnosis with confidence in this age group. However, it has also to be kept in mind that though serum FSH (with/without LH) is elevated between 3 and 4 years of age, FSH and LH may either become undetectable or barely detectable during late childhood (around 8 years of age) before pubertal reactivation of HPG axis sets in.^{8 14}

Learning points.

• Aromatase deficiency is often misdiagnosed as virilising congenital adrenal hyperplasia (CAH), the most common cause of ambiguous genitalia in a ‘46, XX’ child if evaluated immediately after birth. Features common to both these conditions are masculinised genitalia, non-palpable gonads, visualisation of mullerian structures with/without ovaries and high circulatory androgens. Normal serum electrolytes, cortisol (basal/stimulated), 17-OH progesterone (basal/stimulated) and ACTH performed in early neonatal period effectively rules out CAH.

• History of maternal androgenisation that had regressed spontaneously postpartum, lack of signs/symptoms of glucocorticoid and/or mineralocorticoid deficiency, non-progressive genital masculinisation of the girl child is important clue to possible aromatase deficiency. Androgenic drug intake by the mother during conception and pregnancy-specific virilising tumour simulate aromatase deficiency in many aspects.

• Diagnosis of aromatase deficiency in a child presenting after infancy is difficult as circulatory androgens decline and fall within prepubertal range. Elevated follicle-stimulating hormone with pubertal luteinising hormone level, slightly delayed bone age with/without enlarged polycystic ovaries clinch the diagnosis with confidence even in absence of genetic confirmation.