Nonsyndromic 46,xx Testicular Disorders Of Sex Development

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2021-01-18

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SOX9, NR0B1, NR5A1, SOX3, SRY, RSPO1, AR, STS, FOXL2, SOX10, CDIPT

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Summary

Clinical characteristics.

Nonsyndromic 46,XX testicular disorders of sex development (46,XX testicular DSD) are characterized by the presence of a 46,XX karyotype; male external genitalia ranging from normal to ambiguous; two testicles; azoospermia; and absence of müllerian structures. Approximately 85% of individuals with nonsyndromic 46,XX testicular DSD present after puberty with normal pubic hair and normal penile size but small testes, gynecomastia, and sterility resulting from azoospermia. Approximately 15% of individuals with nonsyndromic 46,XX testicular DSD present at birth with ambiguous genitalia. Gender role and gender identity are reported as male. If untreated, males with 46,XX testicular DSD experience the consequences of testosterone deficiency.

Diagnosis/testing.

Diagnosis of nonsyndromic 46,XX testicular DSD is based on the combination of clinical findings, endocrine testing, and cytogenetic testing. Endocrine studies usually show hypergonadotropic hypogonadism secondary to testicular failure. Cytogenetic studies at the 550-band level demonstrate a 46,XX karyotype. SRY, the gene that encodes the sex-determining region Y protein, is the principal gene known to be associated with 46,XX testicular DSD. Approximately 80% of individuals with nonsyndromic 46,XX testicular DSD are SRY positive as shown by use of FISH or chromosomal microarray (CMA). Rearrangements in or around SOX9 and SOX3 detected by CMA, or rarely karyotype, have recently been reported in a few cases; at least one more as-yet-unknown gene at another locus is implicated.

Management.

Treatment of manifestations: Similar to that for other causes of testosterone deficiency. After age 14 years, low-dose testosterone therapy is initiated and gradually increased to reach adult levels. In affected individuals with short stature who are eligible for growth hormone therapy, testosterone therapy is either delayed or given at lower doses initially in order to maximize the growth potential. Reduction mammoplasty may need to be considered if gynecomastia remains an issue following testosterone replacement therapy. Treatment for osteopenia is by standard protocols. Providers are encouraged to anticipate the need for further psychological support.

Surveillance: Monitor for testosterone effects during testosterone replacement therapy, including prostate size and prostate-specific antigen (PSA) in adults; routine monitoring of hematocrit, lipid profile, and liver function tests; bone mineral density determination by bone densitometry (DEXA) annually, if osteopenia has been diagnosed.

Agents/circumstances to avoid: Contraindications to testosterone replacement therapy include prostate cancer (known or suspected) and breast cancer; oral androgens such as methyltestosterone and fluoxymesterone should not be given because of liver toxicity.

Genetic counseling.

SRY-positive 46,XX testicular DSD is generally not inherited because it results from de novo abnormal interchange between the Y chromosome and the X chromosome, resulting in the presence of SRY on the X chromosome and infertility. When SRY is translocated to another chromosome or when fertility is preserved, sex-limited autosomal dominant inheritance is observed.

Autosomal dominant inheritance has been documented for familial cases thought to be caused by CNV in or around SOX9.

The mode of inheritance of other SRY-negative 46,XX testicular DSD is not known, but autosomal recessive inheritance has been postulated. Prenatal testing for pregnancies at risk for SRY-positive 46,XX testicular DSD is possible.

Diagnosis

Algorithms have been developed for the evaluation and diagnosis of disorders of sex development (DSD), including nonsyndromic 46,XX testicular DSD [Barseghyan et al 2015] (Figure 2).

Suggestive Findings

The diagnosis of a nonsyndromic 46,XX testicular disorder of sex development (46,XX testicular DSD) may be suggested by the following clinical findings and/or laboratory findings.

Clinical findings

Male external genitalia that range from typical to ambiguous (penoscrotal hypospadias with or without chordee)
Two testicles
No evidence of müllerian structures

Laboratory findings

A 46,XX karyotype using conventional staining methods
Azoospermia
Endocrine studies that demonstrate hypergonadotropic hypogonadism secondary to testicular failure [Pérez-Palacios et al 1981]:
- Basal serum concentration of LH and FSH are moderately elevated (normal range for LH: 1.5-9 mIU/mL in adult males; for FSH: 2.0-9.2 mIU/mL).
- Serum testosterone concentration is usually decreased, typically with serum testosterone concentration below 300 ng/dL in adults (normal range: 350-1,030 ng/dL in adult males).
- Human chorionic gonadotropin (hCG) stimulation test typically shows a low-to-subnormal testosterone response, with little or no elevation of serum testosterone concentration after IM injection of hCG.
Preservation of the hypothalamic-pituitary axis:
- GnRH stimulation testing shows a normal LH and FSH response.
  Note: Such testing is not warranted for diagnosis.
Testicular biopsy that shows a decrease in size and number of seminiferous tubules, peritubular fibrosis, absence of germ cells, and hyperplasia of Leydig cells [de la Chapelle 1981]
Note: Such testing is not warranted for diagnosis.

Establishing the Diagnosis

The diagnosis of a nonsyndromic 46,XX testicular DSD is established in a proband who has the above clinical features and an XX sex chromosome complement. A genetic diagnosis is established if there is evidence of either SRY (the gene encoding the sex-determining region Y) or copy number variants or rearrangements in or around SOX9 or SOX3.

Molecular genetic testing approaches rely on fluorescence in situ hybridization (FISH) for SRY and/or chromosomal microarray (CMA).

FISH for SRY. This is a standard test that is often sent concurrently with karyotype in individuals for whom a DSD diagnosis is being entertained. This test, however, cannot detect copy number variants or rearrangements in or around SOX9 or SOX3.
Chromosome microarray (CMA) can detect SRY and copy number variants or rearrangements in or around SOX9 or SOX3.

SRY-positive 46,XX testicular DSD is established in individuals with evidence of SRY.

SRY-negative 46,XX testicular DSD is established in individuals with no evidence of SRY on CMA or FISH and evidence of copy number variants or rearrangements in or around SOX9 or SOX3:

SOX9. Small duplication or triplication of the promoter region of SOX9, a balanced chromosomal translocation involving the 17q24.3 region, or duplication of SOX9 (in mosaic or nonmosaic form) [Huang et al 1999, Refai et al 2010, Cox et al 2011, Vetro et al 2011, Xiao et al 2013, Lee et al 2014, Kim et al 2015, Vetro et al 2015]
SOX3. Microdeletions just upstream of the open reading frame of SOX3 [Sutton et al 2011] or microduplications in SOX3 [Sutton et al 2011, Moalem et al 2012]

Molecular Genetic Testing Strategy

In an individual with ambiguous genitalia in whom no chromosome study has been performed

One testing option is to perform karyotype with FISH for SRY first. If the karyotype is normal 46,XX and FISH for SRY is negative, proceed to chromosomal microarray (CMA).
An alternative option is to perform CMA first, which will give information about the sex chromosome complement, the presence of SRY, and other copy number variants of clinical relevance.
If the CMA is normal female without any clinically significant copy number variants in or around SRY, SOX9, and SOX3, consider karyotype as a next step to evaluate for balanced rearrangements.

In a phenotypic male or an individual with ambiguous genitalia in whom a 46,XX karyotype is already established

FISH of an SRY probe to metaphase chromosomes should be performed to determine the presence and, if positive, nature of the rearrangement (SRY located on an X chromosome vs SRY located on an autosome). The inheritance patterns and genetic counseling issues are different for each one of these rearrangements.
If SRY by FISH is not positive, CMA should be the next step. CMA may reveal the presence of SRY not detected by FISH, including mosaicism. It will also identify copy number variants in and around SOX9 and SOX3.

Table 1.

Molecular Genetic Testing Used in Nonsyndromic 46,XX Testicular Disorders of Sex Development by Phenotype

Gene ¹	Method	Proportion of 46,XX Testicular DSD Accounted for by Mutation of Gene
Gene ¹	Method	Normal Male Genitalia	Ambiguous Male Genitalia
SRY	CMA or FISH ²	80% ³	Rare
SOX9	CMA	<10% ⁴	<10% ⁴
SOX3	CMA	Rare ⁴	Rare ⁵
Unknown ⁶	NA

1.: See Table A. Genes and Databases for chromosome locus and protein.
2.: The presence of SRY can be determined equally well by CMA or FISH.
3.: Fechner et al [1993], McElreavey et al [1993], Boucekkine et al [1994]
4.: Benko et al [2011], Seeherunvong et al [2012], Chiang et al [2013] , Kim et al [2015], Vetro et al [2015]
5.: Sutton et al [2011], Moalem et al [2012], Vetro et al [2015]
6.: Refai et al [2010], Chiang et al [2013]

Clinical Characteristics

Clinical Description

Approximately 85% of individuals with a nonsyndromic 46,XX testicular disorder of sex development (46,XX testicular DSD) present after puberty with normal pubic hair and normal penile size, but small testes, gynecomastia, and sterility resulting from azoospermia [Zenteno-Ruiz et al 2001]; up to 90% of these individuals are SRY positive [McElreavey et al 1993]. The small testes are usually soft but may become firmer with age. Among these individuals, a minority have cryptorchidism (undescended testes) and/or anterior hypospadias (atypical urethral opening) [Boucekkine et al 1994]. Gender role and gender identity are reported as male for the common, unambiguous presentation, but systematic psychosexual assessment has not been performed on a significant number of individuals with 46,XX testicular DSD.

Approximately 15% of individuals with a nonsyndromic 46,XX testicular DSD present at birth with ambiguous genitalia, typically penoscrotal hypospadias with or without chordee [Zenteno-Ruiz et al 2001]; only a minority of these individuals are SRY positive [Fechner et al 1993, McElreavey et al 1993, Boucekkine et al 1994].

Nonsyndromic 46,XX testicular DSD is not associated with learning disorders or behavioral issues.

The natural history of individuals with nonsyndromic 46,XX testicular DSD, if untreated, is similar to the typical consequences of testosterone deficiency:

Low libido and possible erectile dysfunction
Decrease in secondary sexual characteristics, such as sparse body hair, infrequent need to shave, and reduced muscle mass
Increase in fat mass with lower muscle strength
Increased risk of osteopenia
Increased risk of depression

SRY-positive nonsyndromic 46,XX testicular DSD. Individuals with SRY-positive 46,XX testicular DSD typically present after puberty with the following:

Shorter-than-average stature (mean height: 168.2 cm, compared to normal mean height: 173.5 cm) [de la Chapelle 1972]
Gynecomastia
Small testes
Azoospermia

Individuals with SRY-positive 46,XX testicular DSD rarely present with atypical genitalia and are less likely than individuals with SRY-negative 46,XX testicular DSD to have gynecomastia [Ferguson-Smith et al 1990, Boucekkine et al 1994, Ergun-Longmire et al 2005].

SRY-negative nonsyndromic 46,XX testicular DSD. Individuals with SRY-negative 46,XX testicular DSD tend to present with ambiguous genitalia at birth, such as penoscrotal hypospadias and cryptorchidism, and, if untreated, almost always develop gynecomastia around the time of puberty.

SOX9-related nonsyndromic 46,XX testicular DSD. Of the limited number of individuals with confirmed SOX9-related 46,XX testicular DSD reported to date, none presented in the newborn period with ambiguous genitalia, one was ascertained at age four years with small testes as the sole finding, and three presented as adults with infertility and azoospermia.

A primary presentation of delayed puberty has not been reported.

As gonadal biopsy is not routinely performed, it is unclear what percentage of individuals with copy number variants in and around SOX9 have testicular DSD vs ovotesticular DSD, an allelic disorder.

SOX3-related nonsyndromic 46,XX testicular DSD. Shorter-than-average stature, small testes with azoospermia, and low testosterone are seen in affected individuals.

Only one of five affected individuals presented at birth with atypical male genitalia. One affected individual was diagnosed in adulthood because of infertility. In the other three individuals, SOX3-related 46,XX testicular DSD without genital ambiguity was discovered during consultation for developmental delay or gender dysphoria (see Differential Diagnosis for discussion of syndromic forms of DSD).

Genotype-Phenotype Correlations

In nonsyndromic 46,XX testicular DSD, the presence of SRY is often associated with the presence of normal male external genitalia, whereas the absence of SRY is more often associated with ambiguous genitalia [Grigorescu-Sido et al 2005]. However, genotype-phenotype correlation is not entirely reliable, because a small number of individuals with SRY-negative nonsyndromic 46,XX testicular DSD have typical male external genitalia [Vilain et al 1994, Zenteno et al 1997, Kolon et al 1998, Vernole et al 2000, Abusheikha et al 2001]. This number may increase as larger cohorts of adults undergoing evaluation for fertility problems are tested. A recent study found three individuals with a 46,XX karyotype among 555 infertile adult Taiwainese males; two of the three individuals were SRY positive [Chiang et al 2013].

Due to the small number of individuals reported with SOX9-related 46,XX testicular DSD and SOX3-related 46,XX testicular DSD, genotype-phenotype correlations are not yet available.

Penetrance

Penetrance in those with a known genetic etiology is believed to be 100%, but no data are available.

One report of a small duplication in the SOX9 promoter region in a newborn with a 46,XX karyotype and ambiguous genitalia states that the duplication was found not only in the proband's 46,XY fertile father and phenotypically normal male brothers but also in his paternal 46,XX (fertile) grandmother [Benko et al 2011].

Nomenclature

At an international consensus conference on the management of Intersexuality held in October 2005 under the auspices of the Lawson Wilkins Pediatric Endocrine Society (USA) and the European Society for Pediatric Endocrinology, a multidisciplinary panel of experts proposed that the names "XX male syndrome" and "true hermaphrodite" be replaced by the names "46,XX testicular disorders of sex development" and "46,XX ovotesticular DSD," respectively [Hughes et al 2006, Lee et al 2006].

Prevalence

The prevalence of 46,XX testicular DSD is estimated at one in 20,000 males.

No populations are known to be at greater or lesser risk for this disorder.

Differential Diagnosis

Syndromic forms of 46,XX testicular DSD

Syndromic forms of 46,XX testicular DSD – characterized by palmoplantar keratosis and predisposition to squamous cell carcinoma of the skin – have been shown to be associated with pathogenic variants in R-Spondin 1 (RSPO1) [Parma et al 2006, Tomaselli et al 2008] (OMIM 610644).
46,XX testicular DSD may be associated with microphthalmia and linear skin defects when the X/Y abnormal interchange results in microdeletion of Xp [Lindsay et al 1994, Kotzot et al 2002, Anguiano et al 2003].
Facial dysmorphic features were found in association with 46,XX testicular DSD in the reported cases of balanced translocation involving the SOX9 region, 46,XX,t(12;17)(q14.3;q24.3) [Refai et al 2010] and 46,XX,t(11;17)(p13;q24.3) [Vetro et al 2015].
Developmental delay was associated with 46,XX testicular DSD (without ambiguous genitalia) in the two individuals with de novo large (5.6 and 6 Mb) duplications of the Xq27 region, including SOX3 and several other genes. The most likely etiology for the developmental issues in these individuals is deletion of multiple genes around SOX3.

The most common disorders in the differential diagnosis of nonsyndromic 46,XX testicular DSD can be distinguished by karyotype and by FISH testing.

Sex chromosome abnormalities

Klinefelter syndrome. Klinefelter syndrome (47,XXY) and its variants (48,XXXY, 49,XXXXY, and 46XY/47,XXY mosaicism) are suspected in males with hypogonadism, small testes, and gynecomastia, all of which are also present in individuals with 46,XX testicular DSD. In contrast to 46,XX testicular DSD, Klinefelter syndrome is often characterized by normal or tall stature, speech delay, learning disorders, and behavioral problems.
46,XX/46,XY. Depending on the relative ratio of XX and XY cells, individuals with 46,XX/46,XY chimerism may present with external genitalia ranging from typical male to ambiguous to typical female. In addition, evidence suggests that some XX individuals who are masculinized show some low-level hidden, tissue-specific mosaicism for Y-chromosome-derived sequences [Queipo et al 2002].
45,X/46,XY. Affected individuals often present as male and may have short stature depending on the percentage of 45,X cells. Clinically, this presentation may be indistinguishable from 46,XX testicular DSD; however, the chromosome findings are diagnostic. If the percentage of 45,X cells is very high, the phenotype is likely to be female with classic Turner syndrome.

46,XX ovotesticular DSD. Individuals with ovotesticular DSD (formerly known as "true hermaphrodites") have both testicular and ovarian tissue either as an ovotestis or as an ovary and a contralateral testis, whereas the gonads of individuals with 46,XX testicular DSD consist only of testicular tissue. The type of gonadal tissue can be established by gonadal biopsy. Possible bias of sampling of a gonadal biopsy that may miss the ovarian portion of the gonads needs to be considered. Ovotesticular DSD may be associated with the presence of a uterus or a hemi-uterus; individuals with 46,XX testicular DSD have no müllerian structures. Endocrine investigations may reveal estrogen production in individuals with ovotesticular DSD.

Testicular and ovotesticular DSD may represent the same genetic entity, as both phenotypes may be represented in families with 46,XX males. However, it is critical to differentiate them, as their potential outcomes differ, thus affecting management. The presence of ovarian tissue, however minimal, in a self-identified boy may lead to feminization of physical characteristics (reduced hair, gynecomastia, menstrual flow), a possible indication for surgical excision of the ovarian portion of the gonad. Conversely, the presence of testicular tissue in a self-identified girl could eventually lead to unwanted hirsutism and may increase tumor risk. While 46,XX ovotesticular DSD has been associated with the presence of SRY (most likely in either mosaic or chimeric form) and with copy number variants in and around SOX9, to date it has not been reported in association with SOX3 copy number variants.

Prenatal exposure of 46,XX fetuses to androgens

Congenital adrenal hyperplasia. 21-hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by the adrenal cortex. In classic 21-OHD CAH, excessive adrenal androgen biosynthesis results in virilization in all individuals and salt wasting in some. Virilized females have ambiguous external genitalia and a normal uterus and ovaries. The diagnosis of 21-OHD is established by comparison of baseline and stimulated serum concentrations of the steroid precursor 17-hydroxy progesterone (17-OHP). Molecular genetic testing of CYP21A2 for a panel of nine common pathogenic variants and gene deletions detects approximately 80%-98% of disease-causing alleles in affected individuals and carriers. Inheritance is autosomal recessive. Rarer genetic causes of CAH (less common 21-OHD variants and variants in other genes) exist and testing may also be available for these variants.
Prenatal exposure of a fetus with an XX karyotype to externally administered androgens such as Danazol or to androgens endogenously produced by the mother can cause virilization resulting in an infant with ambiguous genitalia that may look similar to those of a male with 46,XX testicular DSD and ambiguous genitalia.

Management

Evaluations Following Initial Diagnosis

To establish the extent of disease and needs in an individual diagnosed with a 46,XX testicular disorder of sex development (46,XX testicular DSD), the following evaluations are recommended:

Assessment of mood, libido, energy, erectile function, acne, and breast tenderness and size by history and/or physical examination
Dexascan to evaluate for osteopenia
Consultation with a clinical geneticist and/or genetic counselor

Treatment of Manifestations

Testosterone-replacement therapy. Management of individuals with 46,XX testicular DSD with testosterone deficiency is similar to that for other causes of testosterone deficiency. Physicians should check for the most current preparations and dosage recommendations before initiating testosterone replacement therapy.

After age 14 years, low-dose testosterone therapy can be initiated. Note: If an individual has short stature and is eligible for growth hormone therapy, testosterone therapy should be either delayed or given at lower doses initially in order to maximize growth potential.

Testosterone enanthate is given IM every three to four weeks, starting at 100 mg and increasing by 50 mg every six months to 200-400 mg. Initial high doses of testosterone should be avoided to prevent priapism. The treatment should plateau, in adulthood, at the best possible dosage, typically between 50 and 400 mg every two to four weeks.

Injection of testosterone enanthate is the preferred method of replacement therapy because of low cost and easy, at-home regulation of dosage; however, side effects include pain associated with injection and large variations of serum testosterone concentration between injections, resulting in a higher risk of mood swings.

Alternative delivery systems that result in a more stable dosing include transdermal patches (scrotal and non-scrotal) and transdermal gels. Testosterone-containing gels, however, are associated with the risk of interpersonal transfer, which can be reduced by the use of newer hydroalcoholic gels [Kühnert et al 2005].

Gynecomastia. Regression of gynecomastia may occur with testosterone replacement therapy. If it does not, and if it causes psychological distress to the individual, reduction mammoplasty can be offered.

Osteopenia. Depending on the degree of osteopenia, treatment may include: calcium, exercise, vitamin D, biphosphonates, or calcitonin. Referral to an internist, pediatrician, or endocrinologist is recommended.

Psychological support. Sensitivity is necessary when conveying information to individuals with 46,XX testicular DSD about the genetic cause and associated sterility of the disorder. This information must be presented in a manner that helps minimize psychological distress. Providers are encouraged to anticipate the need for further psychological assistance.

Surveillance

Monitoring during testosterone replacement therapy should include the following:

Evaluation of mood, libido, energy, erectile function, acne, and breast tenderness and size.
Measurement of serum testosterone concentration at three-month intervals (prior to the next injection) to evaluate nadir testosterone concentrations. Concentrations lower than 200 ng/dL or higher than 500 ng/dL may require adjustment of total dose or frequency.
In adults, digital rectal examination and measurement of prostate-specific antigen (PSA) prior to treatment and three, six, and 12 months after initiation of therapy to evaluate for the presence of an overt prostate cancer, which would be a contraindication to the treatment. Such testing should then be performed annually.
For individuals on testosterone replacement therapy, evaluation of hematocrit at three, six, and 12 months, then annually because of risk of increased hematocrit with subsequent risk of hypoxia and sleep apnea
Lipid profile and liver function tests, as testosterone may alter lipid profile and liver function
Bone mineral determination by bone densitometry (DEXA) once a year, if osteopenia has been diagnosed
Ongoing psychosocial support

Agents/Circumstances to Avoid

Contraindications to testosterone replacement therapy include prostate cancer (known or suspected) and breast cancer.

Oral androgens such as methyltestosterone and fluoxymesterone should not be given (especially for long-term therapy) because of liver toxicity.

Evaluation of Relatives at Risk

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

Search ClinicalTrials.gov in the US and EU Clinical Trials Register in Europe for information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.