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A Teenage Girl with Unexpected Pubertal Changes.


A 9-year-old girl presented with persistent labial fusion for pediatric surgical evaluation. On examination, she was noted to have bilateral undescended gonads. A pelvic ultrasound was performed, which confirmed the testicular appearance of gonads and no visible uterus or Mullerian structures. She was referred to pediatric endocrinology.

She was born at term and was the second child of the family. She had no other significant medical problems. Her family history was unremarkable apart from her sister, who had mild, transient labial fusion. Her parents were not related.

The initial investigations revealed a 46, XY karyotype. The serum hormone results are shown in Table 1. After stimulation with 5000 U human chorionic gonadotropin (hCG), [7] the testosterone concentration was 170 ng/dl (5.9 nmol/L), but the dihydrotestosterone (DHT) result was unavailable due to an analytical issue. A urine steroid profile (USP) was inconclusive with individual analyte results within reference intervals (RIs). At this stage, the provisional diagnosis of complete androgen insensitivity syndrome (CAIS) was made and the endocrinologists opted to monitor the patient.

At 13 years of age, she went into puberty with pubic hair development, which led to further laboratory investigations (Table 1). USP revealed an increased etiocholanolone of 1.31 mg/day (4.5 [micro]mol/day) [RI, <0.35 (<1.2)] and a low androsterone of 0.09 mg/day (0.3 [micro]mol/day) [RI, <0.44 (<1.5)], resulting in extremely low 5[alpha]- to 5[beta]-reduced steroid metabolites ratio of 0.06 (RI, 0.5-1.9). Pregnanediol, pregnanetriol, and cortisol metabolites concentrations were within reference ranges. The testosterone/DHT ratio was 12.6 (RI, <10) after hCG stimulation.


Disorders of sex development (DSD) comprise a range of congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. DSD most often present in the newborn as ambiguous genitalia or at the time of puberty. Depending on the underlying condition and its severity, DSD can present with various clinical manifestations. The most common cause of DSD is congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency; some forms of CAH could be life-threatening without prompt diagnosis and treatment (1,2).

Sexual differentiation in humans consists of 3 sequential processes. The first step is establishment of the chromosomal sex at the time of fertilization of the ovum--the heterogametic (XY) sex is male and the homogametic sex (XX) is female. The second phase is transference of the chromosomal sex into gonadal sex, which involves differentiation of the bipotential gonad into a testis or ovary. The testicular differentiation requires the SRY (sex determining region Y) [8] gene located on the Y chromosome and several other genes. During the third phase, phenotypic sex develops and the functional and behavioral characteristics of the 2 sexes are determined (1).

The Wolffian (mesonephric) and Mullerian (paramesonephric) ducts form in both sexes. Three hormones, namely the anti-Mullerian hormone, testosterone, and DHT, control the formation of male phenotype. Anti-Mullerian hormone, a glycoprotein produced by Sertoli cells of the fetal testis around the 7th week of gestation, causes the regression of Mullerian ducts and prevents the formation of female genitalia (uterus and fallopian tubes). Testosterone is synthesized by the Leydig cells of the testis. Testosterone promotes male development in 2 ways. First, it acts directly to convert the Wolffian ducts into the epididymis, vasa differentia, seminal vesicles, and ejaculatory ducts. Second, in the urogenital sinus and urogenital tubercle, it acts as a prohormone for DHT, the third hormone of male virilization. DHT is responsible for the development of the prostate, penis, and scrotum. Male external genital development is completed by approximately 17 weeks gestation. In females, the lack of testosterone leads to Wolffian duct regression and lack of anti-Mullerian hormone permits Mullerian duct maturation into oviduct, uterus, cervix, and upper vagina. The female external genitalia undergo canalization in a nonandrogen-dependent fashion but fail to fuse as occurs in normal male development; the separation of the vagina and urethra is completed by 12 weeks gestation. Excess androgen exposure before this separation can cause labial fusion and development of a phallic urethra or urogenital sinus. Later exposure causes only clitoral enlargement and masculinization of labial folds (1,3).

The common causes of DSD can be classified as sex chromosome variations (sex chromosome DSD), disorders of ovary development and androgen excess (46, XX DSD), and disorders of testis development and androgenization (46, XY DSD) (1, 2). The sex chromosome DSD includes 45, X (Turner syndrome and variants), 47, XXY (Klinefelter syndrome and variants), 45, X/46, XY (mixed gonadal dysgenesis), and individuals with mosaic 46, XX/46, XY cell lines (1). The differential diagnosis of 46, XX DSD includes CAH (e.g., 21-hydroxylase deficiency and 11-[beta]-hydroxylase deficiency), gestational hyperandrogenism, testicular DSD, ovotesticular DSD, and translocation of the SRY gene. CAH is the most common diagnosis in virilized XX infants. Depending on the site of the steroid biosynthetic defect, patients may have characteristic biochemical abnormalities. The 46, XY DSD can be categorized as disorders of testis development (e.g., complete or partial gonadal dysgenesis), disorders of androgen synthesis [e.g., 3[beta]-hydroxysteroid dehydrogenase 2, 17[alpha]-hydroxylase/17,20-lyase, 17[beta]-hydroxysteroid dehydrogenase, or 5[alpha]-reductase type 2 deficiency (5ARD)], disorders of androgen action (e.g., androgen insensitivity syndrome due to mutations in the androgen receptor gene), and other conditions affecting sex development.

The diagnosis and management of patients with DSD require an experienced multidisciplinary team. Clinical presentation, imaging, biochemical investigations, and molecular genetic testing are usually helpful to differentiate various DSD (1, 2). Diagnostic algorithms do exist but, with the spectrum of findings and diagnoses, no single evaluation protocol can be used in all circumstances.

The biochemical investigations include the measurement of relevant hormones in serum (e.g., 17-hydroxy progesterone, testosterone, DHT, androstenedione, luteinizing hormone, and follicle stimulating hormone) and the measurement of various metabolites of the steroid pathway (Fig. 1) in urine using gas chromatography-mass spectrometry. USP is a highly specialized test with complex patterns of results that need to be interpreted by an expert in the field. In USP, both quantitative data of individual metabolites and diagnostic ratios ofprecursor metabolites to product metabolites are important (4). At present, USP is available in only a few reference centers and this limits the use of USP in routine DSD evaluation. hCG stimulation testing is used to assess a child with bilateral cryptorchidism for evidence of functioning Leydig cells. Testosterone, DHT, and androstenedione can be measured before and after hCG stimulation. This test is also useful in a few other 46, XY DSD (2). The ratios of testosterone to DHT and of androstenedione to testosterone may become exaggerated in the hCG stimulation test and therefore are useful in the diagnosis of 5ARD and 17[beta]-hydroxysteroid dehydrogenase type III, respectively.

Establishing a specific molecular diagnosis is useful in the management and in offering accurate genetic counselling for the family. In patients for whom a steroidogenic defect has been identified biochemically, targeted single-gene analysis will confirm the diagnosis. Next-generation sequencing, which allows the sequencing of multiple DSD genes in one analysis, and whole-genome and -exome sequencing to target DSD genes are becoming available in clinical practice (2). However, molecular diagnosis is limited by cost and accessibility at present.


The 5[alpha]-reductase enzymes, which convert testosterone to DHT, have broad substrate specificity for steroids containing a [[DELTA].sup.4], 3-keto configuration, including progesterone and 17[alpha]-hydroxyprogesterone. Two genes that encode different 5[alpha]-reductases have been isolated: isoenzyme 1 [encoded by the SRD5A1 (steroid 5 a-reductase 1) gene] and isoenzyme 2 [encoded by the SRD5A2 (steroid 5 [alpha]-reductase 2) gene]. SRD5A1 is expressed in embryonic tissues at low levels, at birth in nongenital skin and liver, and during adulthood in brain, liver, and nongenital skin. SRD5A2 is expressed in fetal genital tissues and in prostate, epididymis, seminal vesicle, genital skin, and liver during adulthood (3).


An autosomal recessive male DSD, termed pseudovaginal perineoscrotal hypospadias, was described in 1961 by Nowakowski and Lenz. These patients typically present as females at birth but have bilateral testis and male urogenital tracts in which the ejaculatory ducts end in a blind-ending vagina (5). In 1974, the syndrome of 5ARD was first described clinically and biochemically by Imperato-McGinley et al. and Walsh et al., in studies of 24 subjects from the Dominican Republic and in 2 siblings from Dallas, TX (6, 7).

5ARD results from loss-of-function mutations in the SRD5A2 gene, which vary from single point mutations to entire deletions of the gene. Affected fetuses possess fully functioning Leydig and Sertoli cells, but they cannot convert testosterone to DHT and thus present with variable degrees of undermasculinized external genitalia. The spectrum of female phenotype can vary from normal female to female with clitoromegaly to men with hypospadias, bifid scrotum, or micropenis to occasional phenotypically normal male. This reflects the extreme variability in the loss of enzymatic function caused by different underlying mutations (3). Other common features are prostate hypoplasia and absence of Mullerian structures. The diagnosis is usually made at birth, in infancy, or at the time of expected puberty when there is virilization and amenorrhoea in patients who have been raised as females but are genetically males (3). The phenotypic features can overlap with disorders of testosterone formation and with partial androgen insensitivity syndrome (PAIS). Gynaecomastia is only rarely seen in 5ARD, in contrast to PAIS (3).

The main biochemical features of 5ARD are normal-to-high male concentrations of testosterone, low or low-normal DHT, and increased testosterone/DHT ratio at baseline or after hCG stimulation test (1-3). Measurement of DHT is challenging because it is present in very low concentrations compared with testosterone. The separation of testosterone from DHT is essential to provide an accurate measurement of DHT. Based on the diversity of methods, the RI for testosterone/DHT ratio ranges from 10 to 30 (2, 3, 8, 9). The characteristic USP pattern in 5ARD is reduced ratios of 5[alpha]- to 5[beta]-reduced metabolites of C19 and C21 steroids (3, 8).

The diagnosis of 5ARD can be confirmed by mutational analysis of the SRD5A2 gene. To date, around 90 different mutations have been described in the SRD5A2 gene; the majority are missense mutations. Homozygous defects are more frequent than compound heterozygote states (3).

Management of patients with 5ARD is challenging and should have a multidisciplinary approach including the family physician, endocrinologists, clinical geneticists, and psychologists. Management and further follow-up will vary based on the chosen gender identity (3, 8).


The main differential diagnoses of this XY female with normal male testosterone concentration at puberty were CAIS, PAIS, or 5ARD. The USP results of increased etiocholanolone, low androsterone, and extremely low 5[alpha]- to 5[beta]-reduced steroid metabolites ratio were consistent with 5ARD. The increased testosterone/DHT ratio was also a feature of 5ARD. The diagnosis was confirmed by genetic analysis. There were no mutations in the androgen receptor gene. SRD5A2 gene testing revealed homozygous c.377A>G (p.G1n126Arg) mutation, which


1. How would you explain the differential diagnosis of an undervirilized genetic male?

2. What tests could be used to investigate disorders of sex development?

3. What is the most probable diagnosis in this patient?


* DSD are important but challenging conditions for which good clinical and laboratory coordination is essential.

* Clinical features of various DSD are overlapping. Therefore, a good understanding of human development and steroidogenesis is essential for the accurate and early diagnosis.

* The 5a-reductase enzymes convert testosterone to DHT. Two genes that encode different 5a-reductases have been isolated: isoenzyme 1 (encoded by the SRD5A1 gene) and isoenzyme 2 (encoded by the SRD5A2 gene).

* 5ARD results from loss-of-function mutations in the SRD5A2 gene. Affected fetuses possess fully functioning Leydig and Sertoli cells but cannot convert testosterone to DHT and present with variable degrees of undermasculinized external genitalia, normal-to-high male concentration of testosterone, low or low-normal DHT, and increased testosterone/DHT ratio at baseline or after hCG stimulation test.

* Accurate measurement of urinary steroid concentrations and the ratios of different metabolites can help diagnose CAH and a number of DSD. Dynamic function testing and next generation sequencing can also have an important role in the diagnosis of DSD. has been previously described in 1992 in patients with 5ARD (10).

Author Contributions: All authors confirmed they have contributed to the intellectual content ofthispaper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation ofdata; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts ofinterest.


(1.) Houk CP, Levitsky LL. Evaluation of the infant with atypical genitalia (disorder of sex development). UpToDate. (Accessed May 2017).

(2.) Ahmad SF, Achermann JC, Arlt W, Balen A, Conway G, Edwards Z, et al. Society for Endocrinology UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development (Revised 2015). Clin Endocrinol 2016;84:771-8.

(3.) Mendonca BB, Batista RL, Domenice S, Costa EM, Arnhold IJ, Russell DW, Wilson JD. Reprint of "Steroid 5[alpha]-reductase 2 deficiency". J Steroid Biochem Mol Biol 2017;165: 95-100.

(4.) Koyama Y, Homma K, Hasegawa T. Urinary steroid profiling: a powerful method for the diagnosis of abnormal steroidogenesis. Expert Rev Endocrinol Metab 2014;9:273-82.

(5.) Nowakowski H, Lenz W. Genetic aspects in male hypogonadism. Recent Prog Horm Res 1961;17:53-95.

(6.) Imperato-McGinley J, Guerrero L, Gautier T, Peterson RE. Steroid 5alpha-reductase deficiency in man: an inherited form of male pseudohermaphroditism. Science 1974; 186:1213-5.

(7.) Walsh PC, Madden JD, Harrod MJ, Goldstein JL, MacDonald PC. Familial incomplete male pseudohermaphroditism type 2. Decreased dihydrotestosterone formation in pseudovaginal perineoscrotal hypospadias. N Engl J Med 1974;18:944-9.

(8.) Maimoun L, Philibert P, Cammas B, Audran F, Bouchard P, Fenichel P, et al. Phenotypical, biological, and molecular heterogeneity of 5[alpha]-Reductase deficiency: an extensive international experience of 55 patients. J Clin Endocrinol Metab 2011;96: 296-307.

(9.) Chan AO, But BW, Lee CY, Lam YY, Ng KL, Tung JY, et al. Diagnosis of 5[alpha]-reductase 2 deficiency: is measurement of dihydrotestosterone essential? Clin Chem 2013;59: 798-806.

(10.) Thigpen AE, Davis DL, MilatovitchA, Medndonca BB, Imperato-McGinley J, Griffin JE, et al. Molecular genetics of steroid 5 alpha-reductase 2 deficiency. J Clin Invest 1992;90:799-809.

Nilika Wijeratne, [1,2,3] * Alan R. McNeil, [1] James C.G. Doery, [2,3] Elizabeth McLeod, [4] Philip B. Bergman, [5,6] and Joseph Montalto [1]

[1] Department of Biochemistry, Dorevitch Pathology, Heidelberg, Victoria, Australia; [2] Monash Pathology, [3] Department of Medicine, Monash University, [4] Department of Pediatric Surgery, Monash Children's, and [5] Department of Pediatric Endocrinology and Diabetes, Monash Children's, Monash Health, Clayton, Victoria, Australia, [6] Department of Pediatrics, Monash University.

* Address correspondence to this author at: Dorevitch Pathology, 18 Banksia street, Heidelberg, Victoria 3084, Australia. E-mail

Received May 25, 2017; accepted August 31, 2017.

DOI: 10.1373/clinchem.2017.277046

[7] Nonstandard abbreviations: hCG, human chorionic gonadotropin; DHT, dihydrotestosterone; USP, urine steroid profile; CAIS, complete androgen insensitivity syndrome; DSD, disorders of sex development; CAH, congenital adrenal hyperplasia; 5ARD, 5areductasetype 2 deficiency.

[8] Human genes: SRY, sex determining region Y; SRD5A1, steroid 5 [alpha]-reductase 1; SRD5A2, steroid 5 [alpha]-reductase 2.

Caption: Fig. 1. Synthesis and metabolism of steroid hormones.

Steroid names in conventional script are steroid hormones and precursors; those in italics are their urinary metabolites. The major enzymes are in rectangular boxes. Cofactor enzymes are not included in this figure. Nonstandard abbreviations: StAR, steroidogenic acute regulatory protein; CYP11A1, P450 side-chain cleavage enzyme; CYP11B1, 11[beta] hydroxylase; CYP11B2, aldosterone synthase; CYP17A1, 17 [alpha]-hydroxylase/ 17,20-lyase; CYP21A2,21-hydroxylase; HSD3B2, 3[beta]-hydroxysteroid dehydrogenase type 2; HSD11B1, 11[beta]-hydroxysteroid dehydrogenase type 1; HSD11B2, 11[beta]-hydroxysteroid dehydrogenase type 2; HSD17B, 17 [beta]-hydroxysteroid dehydrogenase; SRD5A2, 5[alpha]-reductase type 2; CYP19A1, P450 Aromatase; SULT2A1, sulfotransferase 2A1.
Table 1. Patient's serum hormone results.

                                   Concentration at the
Analyte                               age of 9 years

Testosterone, ng/dL (nmol/L)           <8.7 (<0.3)
FSH, mlU/mL (IU/L)                        1 (1)
LH, mIU/mL (IU/L)                       0.3 (0.3)
DHT (using RIA), ng/dL (nmol/L)         <12 (<0.4)
Androstenedione, ng/dL (nmol/L)          40(1.4)
DHEAS, ug/dL (umol/L)                       --
Estradiol, pg/mL (pmol/L)                   --

                                   Concentration at the
Analyte                              age of 13 years

Testosterone, ng/dL (nmol/L)            412 (14.3)
FSH, mlU/mL (IU/L)                         4(4)
LH, mIU/mL (IU/L)                         7 (7)
DHT (using RIA), ng/dL (nmol/L)         14.5 (0.5)
Androstenedione, ng/dL (nmol/L)          172 (6)
DHEAS, ug/dL (umol/L)                   173 (4.7)
Estradiol, pg/mL (pmol/L)                 8(29)

                                   Reference interval
Analyte                            (prepubertal, male)

Testosterone, ng/dL (nmol/L)           <14 (<0.5)
FSH, mlU/mL (IU/L)                       <3 (<3)
LH, mIU/mL (IU/L)                        <1(<1)
DHT (using RIA), ng/dL (nmol/L)     14.5-55(0.5-1.9)
Androstenedione, ng/dL (nmol/L)       14-85(0.5-3)
DHEAS, ug/dL (umol/L)               18-122 (0.5-3.3)
Estradiol, pg/mL (pmol/L)              <5.5 (<20)

FSH, follicle stimulating hormone; LH, luteinizing hormone; RIA,
radioimmuno assay; DHEAS, dehydroepiandrosterone sulphate.
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Title Annotation:Clinical Case Study
Author:Wijeratne, Nilika; McNeil, Alan R.; Doery, James C.G.; McLeod, Elizabeth; Bergman, Philip B.; Montal
Publication:Clinical Chemistry
Date:Jun 1, 2018
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