Clinical and biological assessments of the undervirilized male

Authors


M. Nicolino, Division of Pediatric Endocrinology and Metabolism Hôpital Debrousse, 21 rue Sœur Bouvier 69322 Lyon Cedex 05, France.
e-mail: marc.nicolino@chu-lyon.fr

INTRODUCTION

The first step in managing the undervirilized male is to make a causal diagnosis as soon and as accurately as possible, to determine the different elements that must be considered in deciding to rear the patient as a male or a female. This requires a strategy of clinical and biological investigations, including molecular biological techniques and specific imaging.

Schematically, it is mandatory to make an anatomical description of the external and internal genitalia as completely as possible (pictures, imaging, etc.). In the newborn the first step is to exclude a steroid enzyme deficiency that could lead to a severe salt-losing syndrome at a few days old. This is the situation seen in the quite rare cases of 46XY individuals with inborn errors of testosterone biosynthesis, which are also associated with nonvirilizing congenital adrenal hyperplasia (CAH).

The second step is to investigate the quality of testicular synthesis (from Leydig and Sertoli cells) and equally important to evaluate the action of testosterone on its target organs, the external genitalia in particular. In this review, only the situations in infants are discussed, while prenatal diagnostic assessments will not be detailed.

DEFINITION

The undervirilized male is an individual whose genetic constitution is either ‘normal’ (i.e. 46XY karyotype), or includes some specific Y-chromosome fragments or genes, whose gonadal tissue is more or less differentiated into a testis and who presents with incomplete virilization of his external and/or internal genitalia.

CLASSIFICATION

An incomplete masculinization of genetically XY males may be the consequence of several intersex disorders that can be divided schematically into three major categories.

  • (i) Defects of gonadal determination (i.e. differentiation and development of the testis is abnormal).
  • (ii) Defects of male differentiation (i.e. the testes develop normally but there is an impaired synthesis or action of either androgens or anti-Müllerian hormone, AMH). These patients with such anomalies were traditionally called ‘male pseudohermaphrodites’.
  • (iii) Hermaphroditism (i.e. presence of both ovarian and testicular tissue in the same ‘bisexual’ gonad, or a testis on one side and an ovary on the other side); see Appendix 1.

HISTORY, PHYSICAL EXAMINATION AND ‘TALKING’ WITH THE PARENTS

An adequate history should concentrate on the search for:

  • (i) Parental consanguinity, history of salt-losing, unexplained infant deaths, or genital ambiguity in some relatives. These elements may indicate autosomal recessive genetic disorders associated with disturbed steroidogenesis. By contrast, an X-linked recessive mode of inheritance is suggestive of androgen insensitivity syndrome (AIS), which is a relatively frequent cause of undervirilization.
  • ii) Maternal ingestion of drugs or exposure to specific environmental factors able to inhibit virilization of the fetus during the pregnancy. Interestingly, many of undervirilized males are born small for age, with no clear explanation to date.

Physical examination

In the undervirilized male there can be any degree of sexual ambiguity; thus phenotypes range from predominantly male genotype (i.e. almost normal male appearance) to complete female phenotype, through varying degrees of ambiguous genitalia (Fig. 1). For example, infants presenting with AIS can have any of these phenotypes (as classified by Quigley et al.[1]) according to the severity of the molecular defect. It is also mandatory to recognize specific symptoms in the rare salt-losing male infants with non-virilizing CAH, because if ignored there can be tragic consequences of the subsequent presentation, e.g. life-threatening hyponatraemia, acidosis, collapse, and/or death.

Figure 1.

Undervirilized males with varying degrees of severity in genital ambiguity: (a) Moderate undervirilization in a 46XY infant presenting with a micropenis with minimal scrotal development and bilateral cryptorchidism (final diagnosis was testicular regression syndrome); (b) Marked undervirilization in a 46XY infant presenting with a markedly bent penis with perineal hypospadias and a bifid scrotum containing two gonads (final diagnosis, idiopathic male pseudohermaphroditism); (c) Severe undervirilization in a 46XY infant. There is minimal phallic growth, partial labial fusion with no scrotal development, a single urogenital opening at the root of the phallic structure. Gonads were palpable in the inguinal regions (final diagnosis, idiopathic male pseudohermaphroditism); (d) Complete sex reversal with complete female phenotype (of external genitalia) in a 46XY infant. On the left side, the gonad was palpable in the labia majora (final diagnosis, complete form of AIS).

A detailed physical examination is necessary to evaluate the degree of undervirilization, and which gives the paediatrician a precise description of the anatomy of the external genitalia, which is preferable to a simple classification according to the stages defined by Prader (see [2]) or Quigley et al.[1]. The first step is a careful palpation. In most of the patients one or two gonads are palpated either in the scrotum, the inguinal regions, or in the labioscrotal folds. In those presenting with apparently normal female external genitalia, bilateral hernias containing testes (more rarely uterus or tubes) should be sought. In any case, if gonads are palpated externally, there will invariably be testes (ovaries tend to remain in the pelvic position) or occasionally ovotestes, and there is at least a testis-determining factor present, usually on a Y chromosome (see above).

The remaining physical examination includes: (i) a careful measurement of the phallus (stretched dorsal length and diameter), and the presence or absence of a chordee; (ii) the location of two (urethral and vaginal) or one orifice (urethral or urogenital sinus) that opens at the base or at the inner face of the phallic structure; (iii) the description of the degree of the labioscrotal fold fusion, i.e. no scrotal development, a posterior fusion of labia majora, a partially fused hemiscrotum, or completely fused scrotum with a bifid or normal appearance. The nature of the skin (shape, texture, excessive pigmentation) should also be noted.

Asymmetry of the external genitalia (labioscrotal folds appearing clearly of a different size, or a gonad palpable only on one side) indicates two specific causes of genital ambiguity, i.e. mixed gonadal dysgenesis and hermaphroditism, respectively (Fig. 2).

Figure 2.

Ambiguous external genitalia in a 46XY/45X0 infant with mixed gonadal dysgenesis initially reared as a male, later re-assigned to female after surgical reconstruction. Note the asymmetric appearance of the scrotum.

In some instances the genital malformations are less evident, in patients presenting with: (i) isolated cryptorchidism and/or micropenis, for instance in testicular regression syndromes (severe micropenis, absence of testis, undetectable testosterone and AMH levels), or lack of gonadotrophic stimulation or action; (ii) complete female phenotype with or with no inguinal mass, as observed in AIS (complete form), steroid enzyme deficiencies (20,22-desmolase, 17α-hydroxylase, 17β-hydroxysteroid dehydrogenase), or pure gonadal dysgenesis; (iii) cryptorchidism and mild hypospadias. Isolated glanular or coronal hypospadias with normal penis growth, absence of chordee and apparently normal testis in the scrotum are not usually clinical features of undervirilization.

In patients presenting with persistent Müllerian duct syndrome (PMDS) the external genitalia have a normal male appearance (although there may be frequently a history of hernia, and bilateral maldescended testes), while internal genitalia are abnormal.

To make a fully documented diagnosis associated malformations or other symptoms should also be sought (Fig. 3). In particular, short stature and/or Turner-like dysmorphisms are evocative of mixed gonadal dysgenesis with a karyotype including a 45X0 cell line. This condition is associated with a severe growth failure and this might be an additional argument for choosing the female sex of rearing.

Figure 3.

Algorithm for the causal diagnosis in undervirilized male infants.

Talking with the parents

At each step of the procedure the parents must be informed that the biological and genetic process will take time, of the various difficulties to determine the sex of rearing, and on the different therapeutic options. If there is uncertainty because the phallus is too small and the degree of testosterone responsiveness is not yet known, the decision on sex of rearing should be postponed, even if gonads are present in the labioscrotal folds. This must be explained to the parents, who must delay the registration of the sex of the child, because further investigations are needed.

IMAGING INVESTIGATIONS

The strategies of investigation include ultrasonography of the abdomen to determine the presence or not of Müllerian remnants, and initially a uterus. This finding is important for deducing that AMH activity is present or not. Ultrasonography is also helpful to investigate the presence and aspect of the gonads, and the integrity of the kidneys and the urinary tract.

The anatomy of the internal genitalia is also well assessed by genito-urethrography (retrograde contrast medium examination). This may provide useful information on the presence or not of a vagina (a cervical imprint on the uterine cavity) and to locate the implantation of an eventual vaginal cavity on the urethra; this last point is useful for choosing a therapeutic option (see below).

KARYOTYPING

A karyotype determination is mandatory and should be used at any age. Until the results are available the presence of a Y chromosome or SRY gene can be tested rapidly by using PCR. The search for the presence of two X chromosomes, by studying the presence of Barr bodies, is practically obsolete.

LABORATORY INVESTIGATIONS

Initial studies in the newborn

In the rare 46XY males with nonvirilizing CAH, salt loss can occur, typically not until 7–10 days of age or even later. If suspected, serum electrolytes and plasma renin activity should be monitored. Before 24 h of life, 3–4 mL of blood should be taken to measure baseline levels of testosterone, dihydrotestosterone (DHT), and other steroid precursors, e.g. progesterone, dehydroepiandrosterone, its sulphate, Δ4-androstenedione, 17α-hydroxyprogesterone and 17α-hydroxypregnenolone, as there is a physiologically high level of testosterone secretion at birth [3] (Fig. 4).

Figure 4.

The postnatal testosterone peak. Personal data from MG Forest. Illustration adapted from [3].

After the first 2 weeks of life, basal levels of testosterone, LH, FSH and AMH are very helpful, as there is an activation of the hypothalamic-pituitary-gonadal axis with spontaneous secretion of androgens in this period, characterized by a peak at 1–2 months of life [3] (Fig. 4). At this specific period of life (i.e. ≈ 6 weeks) basal measurements can sometimes be as informative as the hCG stimulation test.

Indeed, schematically:

(i) In presence of low levels of testosterone 

elevated levels of plasma LH (and FSH) indicate a testicular dysfunction, while undetectable levels of LH (and FSH) indicate a hypogonadotrophic hypogonadism. When testosterone precursors are in the normal range for age, normal levels of AMH are suggestive of Leydig cell hypoplasia/agenesis, while low levels of AMH are suggestive of gonadal dysgenesis. Very high levels of specific precursor(s) and testosterone indicate a testosterone biosynthesis defect.

(ii) In the presence of normal levels of testosterone a decrease in DHT is supposed to indicate abnormal 5α-reductase activity. However, there are two 5α-reductase enzyme activities, type 1 being quite active in infancy and type 2 only responsible for 5α-reductase deficiency. Thus, the testosterone/DHT ratio is not always helpful for a definitive diagnosis in infancy. Low levels of AMH suggest gonadal dysgenesis but in some patients testosterone values can decline with time and should be re-evaluated. High levels of both testosterone and LH are suggestive of AIS. In the absence of any hormonal abnormality, environmental factors may be the cause of the sexual ambiguity.

In any case, a hCG test should be performed.

An hCG stimulation test is required to assess Leydig cell function. In particular this is the best method to identify testosterone biosynthesis defects. Serum testosterone and its precursors, and DHT before and after hCG stimulation, can be evaluated at any age. As yet there is no general consensus on how to stimulate testosterone production [4]. We consider that the best procedure is to use a long hCG test, because of our long experience with it, and because the normal response of testosterone precursors has been established according to age [5,6]. Our protocol consists of giving 1500 IU of hCG intramuscular on days 0, 2, 4, 6, 8, 10 and taking blood samples on day 11.

Normal or low levels of testosterone in response to hCG should be interpreted in relation to LH, FSH and AMH values, as detailed above. In cases with testosterone biosynthesis defects, the absolute increase of testosterone precursors (and eventually their ratio) indicates the level of enzyme deficiency (Table 1).

Table 1.  There are five enzymes involved in testosterone biosynthesis. Steroid 5α-reductase is involved in testosterone metabolism. Each of these six enzymes can be impaired and lead to a specific steroid enzyme deficiency
Enzyme deficiencyBest steroid markerNote
  1. HSD, hydroxysteroid dehydrogenase; 17-OHPregn, 17α-hydroxypregnenolone; DHA, dehydroepiandrosterone; P, progesterone; 17-OHP, 17α-hydroxyprogesterone; Δ4-A, Δ4-androstenedione; T, testosterone; DHT, dihydrotestosterone.

20,22-desmolase 3β-HSD type IIAll steroid very low 17-OHPregn ‰‰, DHA ‰in all, testosterone
17α-hydroxylaseP and corticosterone ‰is
17,20-desmolase17-OHP ‰ DHAÊΔ4-AÊabnormally
17β-HSD type3Δ4-A ‰low
5α-reductase type2DHT low, T/DHT ‰T is normal

Other hormone measurements

  • (i) GnRH test. 
  • After the first 2–3 months of life, basal levels of testosterone, LH and FSH are no longer helpful because their values are low and inconclusive. A GnRH test can be used to exclude pituitary dysfunction, and to evaluate LH sensitivity to GnRH. In case of AIS there may be a high response.

  • (ii) Iterative determination of AMH and/or inhibin B levels may provide useful information about Sertoli cell function. In physiological conditions, AMH concentrations are high in boys, especially during the first 9 years of life. Abnormally low levels are specifically found in testicular dysgenesis, findings usually associated with the presence of a uterus on ultrasonography and with deficient spermatogenesis that will result in infertility later in life.
  • (iii) The sex hormone-binding globulin response to oral anabolic steroids 
  • can be used to evaluate the degree of androgen insensitivity; there is no or insufficient decline of these globulin levels in AIS, a finding easier to interpret in complete forms of AIS.

  • (iv) The ACTH test is required when any hormone abnormalities appear to suggest nonvirilizing CAH. In particular, deficiencies in 20,22-desmolase associated with salt loss, through a mutation of steroidogenic acute regulatory protein [7], 3β-hydroxysteroid dehydrogenase usually associated with salt loss [8], or 17α-hydroxylase/17,20-desmolase (associated with hypertension only in the pure form of 17α-hydroxylase deficiency).

TEST OF SENSITIVITY TO ANDROGENS

This test is very useful not only for diagnostic purposes but also to provide arguments for making the decision about the sex of rearing (see below). Failure to respond indicates a complete resistance to the action of androgens, while a positive response is a clear increased length or diameter of the penis.

When the male sex of rearing has been decided, the aim of the test is also to normalize the infant's penile size before surgical repair.

There is no consensus about the dosage, mode of administration, timing and duration of androgen treatment. Our protocol consists of 4 intramuscular injections with 100 mg/m2 of depot-testosterone every 2 weeks. Better results are obtained in infancy and in very early childhood. We are reluctant to use this test after 4–5 years of age, because it can advance bone-age maturation too rapidly.

In older patients there are two options; either to use larger doses of depot testosterone (intramuscular) at the normal age of puberty, or to locally apply a DHT gel for several weeks (if applied by the mothers, they must wear gloves), but the response in terms of growth of the penis is generally less satisfactory, and not fully evaluated.

MOLECULAR ANALYSES

After making all biological assessments several genes can be analysed. By order of frequency, and in accordance with the results of the biological investigations, the search for mutations includes the following genes: androgen receptor, steroid enzyme genes, LH and its receptor, AMH and its receptor, and genes of testicular development (SRY, SOX-9, WT-1, DAX-1, SF-1) [9] (Appendix).

THE DECISION ABOUT THE SEX OF REARING

The decision to rear an undervirilized neonate as male or female depends on several factors. What are the arguments that help the physician to make a decision? A full discussion should be made by a multidisciplinary and experienced team and must include the parents. It is important to keep in mind that there is no perfect choice of sex of rearing and the most reasonable outcome should be considered (Table 2).

Table 2.  Arguments for the choice of sex of rearing
In favour of
female gendermale gender
Small size of the clitoral structure (1–2 cm)Size of the phallic structure >2 cm
Presence of uterusNormal scrotum containing testis
Bilateral cryptorchidismNo vagina or small prostatic utricle
Gonads to be removedGood/normal response to hCG
Vaginal pouch >2 cmAndrogen sensitivity
Low testosterone response to hCGPrediction of normal stature
Androgen resistanceParental adherence
Prediction of small stature
Parental adherence

CONCLUSION

The undervirilized male presents a complex and difficult problem for the clinician. Clinical and biological management are a great diagnostic challenge and in most patients with normal testes the cause of the undervirilization remains ‘idiopathic’. A crucial issue is that this subgroup of individuals is becoming more frequent. The role of fetal endocrine disruptors of environmental origin that might interfere at critical stages of genital development remains to be determined and evaluated [10].

APPENDIX

Classification of abnormal sex differentiation

I. Undervirilized male: Incomplete virilization of XY male

A. Defective testicular development

1. Defective testicular determination = defective determination of the bipotential gonad

mutations of SF-1

mutations of WT-1 (Denys–Drash syndrome, Frasier syndrome)

2. Defective testicular differentiation = defective testis formation or maintenance

(a) With 46,XY karyotype

Pure gonadal dysgenesis (i.e. absence of gonads or presence of streak gonads on both sides):

mutations of SRY

mutations of SOX-9

mutations of DAX-1

mutations of WNT4

mutations of DMRT1 and DMRT2

associated with chromosomal deletions (2q32.1–35; 10qter)

associated with multiple anomaly congenital syndromes

idiopathic

Mixed gonadal dysgenesis (i.e. one testis is present on one side which may be defective, and the other is represented by a streak gonad)

Bilateral dysgenetic testis

Testicular regression syndromes

(b) With abnormal karyotype (i.e. mosaicism of the sex chromosomes: 46XY/45X0, 46XY/46XX, 47XXY/45X0, XYY or XXY males; Y chromosome anomalies)

Pure gonadal dysgenesis

Mixed gonadal dysgenesis

Seminiferous tubule dysgenesis (Klinefelter syndrome and variants)

B. Defective male differentiation of external and/or internal genitalia

1. Impaired testosterone production

(a) Impaired gonadotropic or somatotropic action

Leydig cells hypoplasia or agenesis (LH receptor mutations)

LH deficiency (LH mutations, Kallman syndrome, GnRH receptor mutations)

Hypopituitarism (may be part of a syndrome)

(b) Inborn error of cholesterol biosynthesis (Smith–Lemli–Opitz syndrome)

(c) Inborn errors of testosterone biosynthesis

With nonvirilizing CAH:

congenital lipoid adrenal hyperplasia (20,22-desmolase deficiency, mutations of StAR)

3β-hydroxysteroid dehydrogenase deficiency

17α-hydroxylase deficiency

Without nonvirilizing CAH:

17,20-desmolase deficiency

17α-hydroxysteroid dehydrogenase deficiency

2. Impaired metabolism of testosterone = steroid 5α-reductase deficiency type 2

3. Impaired action of androgens

Androgen insensitivity syndrome (complete or partial forms due to mutations of androgen receptor)

4. Persistent Müllerian duct syndrome

Impaired AMH production; impaired action, due to mutation in AMH receptor

5. Other causes of abnormal male differentiation

Associated with multiple congenital anomaly syndromes

Iatrogenic (maternal ingestion of antiandrogens during pregnancy)

Idiopathic

II. Hermaphroditism

A With 46XX karyotype (60%)

B With 46XY karyotype (10%)

C With mosaicism of sex chromosomes (30%)

III. Virilized female: virilization of XX female

Ancillary