Differences of Sex Development

Presentation and Diagnosis of Turner Syndrome

In the prenatal period, fetuses with a 45,X karyotype and its variants frequently have intrauterine growth restriction. Suspicion for Turner syndrome should be raised if the presence of large septate cystic hygromas, nuchal thickening, short femur, total body lymphangiectasia, or cardiac defects is detected by ultrasound. At birth, Turner syndrome babies often have low birth weights; 30% of babies will present with lymphedema of the upper and lower extremities ( Fig. 17.7 ), an extension of the lymphangiectasia in utero , which disappears in the first few months of life. Additionally, the presence of a webbed neck (pterygium colli) (see Fig. 17.6 ) and dysmorphic features including a high-arched palate, low-set prominent ears, low posterior hairline, epicanthal folds, micrognathia, hypoplastic nail beds, or hypoplastic fourth and fifth metacarpals at birth should raise the suspicion for 45,X DSD. Turner syndrome patients may have increased carrying angle of the arms (cubitus valgus), shield-like chest with wide-set nipples, hearing loss, and a higher frequency of cardiovascular disease such as coarctation of the aorta. Furthermore, the incidence of renal anomalies is between 30% and 50%, the most common finding being a horseshoe kidney, followed by abnormal vasculature.

During adolescence, the most common presentation is short stature, amenorrhea, and lack of secondary sexual characteristics. Patients may also have any of the other characteristics mentioned above. 45,X women have normal intelligence. Depending on the degree of GD, up to 30% of Turner patients undergo some degree of spontaneous puberty.

Diagnosis is made by karyotype, which will show in a 45,X, or mosaic variant of cells. If the karyotype appears normal but the presentation is suggestive of Turner syndrome, fluorescent in situ hybridization (FISH), or chromosomal microarray study should be done because there may be a cryptic deletion in the pseudoautosomal region in one of the X chromosomes. Typically patients with 45,X or 46,XX with a chromosomal abnormality in one X chromosome have more prominent phenotypic features than those who are a mosaic.

Management of Turner Syndrome

For most patients with 45,X DSD, the greatest concern is for short stature and secondary sexual development. The treatment of both of these should be carefully titrated. For cases diagnosed in childhood, the use of low-dose anabolic steroids coupled with growth hormone has been shown to increase final adult height with increased beneficial effects compared to those who started therapy late. Optimal duration and timing of the steroid treatment are still under investigation. At the onset of the age of puberty, high-dose estrogen and progesterone should be given to promote the development of secondary sex characteristics. Estrogens and progesterone should be taken throughout life to prevent complications, such as osteoporosis.

In young patients diagnosed with Turner syndrome, spontaneous puberty is a good indicator of the presence of functional ovarian tissue. Overall, it is estimated that 2% to 5% of patients with Turner syndrome have the potential for spontaneous pregnancy.

A frequent question from families concerns fertility. Despite progress in egg preservation techniques, which could provide hope for fertility in women with Turner syndrome, pregnancies (conceived with either autologous or donated oocytes) carry a high risk of life-threatening cardiovascular complications (aortic dissection, severe hypertension). A complete cardiac assessment is recommended prior to consideration of pregnancy, as well as close monitoring during pregnancy and in the postpartum period. Yet, a growing number of centers have developed programs of fertility preservation for Turner syndrome and novel approaches that are still considered experimental such as in vitro maturation of immature oocytes and in vitro activation (IVA) of immature follicles, are promising.

For optimal management of all conditions associated with Turner syndrome and variants, patients should be referred to multidisciplinary centers specializing in their care when available.

Presentation and Diagnosis of Klinefelter Syndrome

Klinefelter syndrome is largely undiagnosed in the general population. In early life, Klinefelter may be diagnosed in boys with behavioral disorders, abnormally small testes, and long legs ( Fig. 17.8 ). The presence of only long lower extremities distinguishes Klinefelter syndrome from the other forms of eunuchoidism that result in equally long upper and lower extremities. In patients with Klinefelter syndrome, IQ typically falls in the normal range; however, it tends to be below that of siblings. ^, Most patients present in adolescence with small firm testes and hypogonadism with varying degrees of androgen deficiency. In later life, many males present at infertility centers with azoospermia.

Diagnosis of Klinefelter syndrome is performed by lymphocyte karyotype or chromosomal microarray. Some mosaic cases will only be detected by karyotype of skin fibroblasts and occasionally of testicular biopsy specimens.

Management of Klinefelter Syndrome

The European Society of Andrology recently published guidelines for the management of Klinefelter syndrome. Early detection allows for early intervention for cognitive and behavioral traits. Current studies are underway exploring the role of androgen replacement therapy in childhood, to help with cognitive and behavioral disabilities. Replacement of androgens also allows for the development of masculine secondary sex characteristics, improved self-esteem, and increased libido, strength, and bone mineral density. Typically, androgen replacement therapy is started if puberty does not start on time or is not maintained. However, recent randomized clinical trials tested whether low doses of androgens (Oxandrolone) given as early as 4 years of age modified the clinical outcome. Positive effects on measures of cardiometabolic health in prepubertal boys with KS were observed, albeit accompanied by lower HDL cholesterol and advanced bone age. Positive effects were shown on visual motor functions and psychosocial function (anxiety, depression, social problems) but not on cognitive function or on hyperactive or aggressive behaviors. Another trial starting at 6 weeks of age (Testosterone cypionate 25 mg intramuscularly monthly for three doses versus no treatment) showed changes in body composition by 5 months of age, with adiposity of untreated infants 15% greater than that of male controls.

With regard to fertility, testicular mosaicism is an important factor in determining spermatogenesis and the potential for fertility. The vast majority of 47,XXY men are azoospermic but, since the advent of surgical testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) technology, have the potential to be fertile. The success rates of TESE are similar to those in other causes of nonobstructive azoospermia if it is performed between 16 and 35 years of age.

It should be noted that there is a higher rate of sex chromosomal hyperploidy and autosomal aneupoloidy in the sperm of Klinefelter patients. However, the risk of passing sex chromosome aneuploidy to the offspring remains unclear. Regardless, preimplantation genetic diagnosis (PGD) or fetal karyotype can identify chromosomal abnormalities in the progeny. Using testicular sperm extraction technology, sperm can be successfully extracted from the testes and injected into a donor oocyte, with a reported fertility success of up to 50%, a dramatic change from the near complete historical infertility of Klinefelter patients.

Etiology and Pathophysiology of Pure/Complete Gonadal Dysgenesis

XY GD is a result of abnormal testis development in utero . There are three types of GD, complete (or pure), partial, or mixed, all of which can be differentiated by the extent of normal testicular tissue within the gonad and karyotype of the individual. In complete GD, individuals have intraabdominal, bilateral, fibrous streaks that do not secrete AMH or testosterone. Phenotypically, pure XY GD individuals are unambiguously phenotypic females (previously known as Swyer syndrome) but usually possess hypoplastic Müllerian structures internally.

Given the primary importance of SRY in human testis determination, it is surprising that mutations in SRY only account for approximately 15% of all 46,XY DSDs with complete GD. ^, This suggests the existence of many other genes involved in primary human sex determination. SRY is a transcription factor, but its mechanism of action remains incompletely understood. For instance, it is still unclear whether it acts as an activator or repressor, and its only confirmed target is SOX9, another transcription factor from the Sox (Sry-box) family of genes. This interaction is mediated by the testis-specific Sox9 enhancer, TESCO, and by TESCO-independent mechanisms. To date, there are more than 50 verified mutations within the SRY gene. Mutations that result in streak gonads primarily occur within the HMG box and cause reduced DNA binding, ^, alterations in DNA bending, or prevent the nuclear import of the SRY protein. Larger cytogenetic deletions of Yp that include SRY have also been implicated in XY GD. ^{, ,}

Steroidogenic factor 1 (SF1, encoded by the gene NR5A1 ) is an orphan nuclear receptor necessary for adrenal and gonadal development. The genital phenotypic spectrum associated with variants in SF1/ NR5A1 is large, including complete or partial GD and azoospermia in 46,XY individuals and testicular or ovotesticular DSD and POI in 46,XX individuals (see Table 17.3 ), suggesting that modifier genes may be responsible for the variability. At least 15% of isolated XY GD can be attributed to SF1 haploinsufficiency. NR5A1 variants are also a frequent cause of 46,XY partial GD (see below), and rare NR5A1 mutations with XY GD and adrenal hypoplasia congenita have been reported, reflecting the role of SF1/ NR5A1 also in adrenal development. ^, Together, mutations or deletions of SRY and NR5A1 account for approximately a third of XY GD, indicating that other mechanisms critical to the testes determination cascade still remain to be discovered.

Interactions between NR5A1 /SF1 and the transcription factors GATA-binding protein 4 (GATA4) and friend of GATA 2 (FOG2) appear to be necessary for SRY expression in developing testes ^, and, in mice, the Gata4 protein has been shown to interact with Nr5a1 /Sf-1, Fog2, and/or Wt1 to regulate the downstream cascade of genes critical for urogenital development, such as Sry , Sox9 , Cyp19a1 , Hsd3b2 or Star. Very rare instances of chromosomal rearrangements leading to disruption of the FOG2/ ZPMF2 gene ^, presented with GD in XY individuals. Exome sequencing of patient cohorts led to reports of large numbers of variants in these two genes in XY DSD cohorts. However, reanalysis and more accurate measurement of the level of activity of the variant proteins have led to the reclassification of many of these variants as likely benign and the role of GATA4 in human DSD has been difficult to pinpoint. So far, only variants in a very specific domain of GATA-4 (N-terminal Zinc Finger domain) have been shown to be associated with DSD, in a condition termed Testicular Anomalies with or without Congenital Heart Disease (TACHD, OMIM # 615542 ).

Mutations in genes involved in testis sex determination (SOX9, WT-1, DHH, DMRT1) and duplications of putative “anti-testis” genes (WNT-4, DAX1 /NR0B1) have also been shown to be responsible for a minority of all XY GD. ^{, ,} Research exome sequencing continues to identify other genes, such as that MAP3K1 ^, or DHX37 ^, as causative genes, which need to be included in sequencing panels for diagnosis.

Etiology and Pathophysiology of Partial or Mixed Gonadal Dysgenesis

Like many developmental disorders, there is a wide range of phenotypic variability in patient presentation. When testes dysgenesis does not involve the entirety of both gonads and the external genital phenotype is ambiguous rather than typical female, the condition is called partial GD . The internal genitalia display varying degrees of Wolffian and Müllerian development, which correspond with the proportion of the gonads that is dysgenetic or “streak.” The medical literature is often confusing in regard to the difference between “partial” and “mixed” GD, which are often used interchangeably. Partial XY GD refers to intermediate stages of dysgenetic testes, between streak gonads and normal testes, and usually has a 46,XY, nonmosaic karyotype. Mixed XY GD typically refers to a situation in which one gonad is a streak gonad while the contralateral gonad is partially dysgenic or a normal testis. Mosaicism for 45,X/46,XY is the most frequent cause of mixed GD, although a minority have a 46,Xi (Yq) karyotype. The evidence of the variable phenotypic spectrum of individuals with 45,X/46,XY mosaicism was reported in a series of 10 45,X/46,XY patients in which four individuals were undervirilized males with bilateral testes, three were diagnosed with mixed GD and genital ambiguity, and three were diagnosed with Turner syndrome. Although not proven, there is preliminary evidence that the percentage of normal testicular tissue and phenotypic “maleness” are correlated with an increase in the proportion of gonadal Y chromosome. ^,

Familial cases of complete and partial 46,XY GD ^{, , ,} have been reported. In some of these cases, SRY mutations present with phenotypic variability in which the father and male relatives are phenotypically normal and fertile, while their XY offspring have GD. ^{, ,} From these cases, it is clear that autosomal genetic modifiers influence the sex determination cascade and ultimate phenotypic appearance of the individual. There are also cases of fathers who are mosaic for mutant and normal SRY transmitting the mutation to their XY female daughters. ^, Presumably, the normal father’s mosaicism reflects a postzygotic mutation event.

Besides SRY , variants in NR5A1 may explain ∼20% of PGD, with DHX37 and DHH a few each but the genetic cause of about half of 46,XY GD remains unknown.

Presentation and Diagnosis of Gonadal Dysgenesis

Complete XY GD presents as a phenotypic female with normal or tall stature, bilateral streak gonads, delayed puberty, amenorrhea, small or normal Müllerian structures, and without signs of Turner syndrome. If patients are not diagnosed at birth (e.g., when in utero karyotype does not match the phenotypic sex at birth), most patients will be diagnosed during adolescence due to pubertal delay and primary amenorrhea. Patients can rarely present with an abdominal or pelvic mass, which is often a gonadoblastoma. Occasionally, XY GD can be part of a constellation of other symptoms, outlined in Table 17.4 .

Individuals with partial or mixed 46,XY GD typically present at birth with varying degrees of masculinization of external and internal genitalia. Depending on the percent of testicular tissue, dysgenetic testes can be found anywhere along the line of testes descent, from the abdomen to the scrotum. However, the streak gonad in mixed 46,XY GD is always abdominally located. Patients with partial 46,XY GD usually have female external genitalia with some degree of virilization, such as clitoromegaly or a bifid scrotum ( Fig. 17.9 ). Uterus and Fallopian tubes are usually well formed but occasionally may be hypoplastic. In mixed GD, the development of Wolffian and Müllerian structures and the virilization of external genitalia correlate with the degree of development of the ipsilateral testis resulting in asymmetric virilization of the external or internal genitalia and unilateral cryptorchidism. Pelvic ultrasound or MRI can often detect the presence or absence of male or female internal genitalia and, in the case of mixed XY GD, asymmetry in the development of the Müllerian and Wolffian structures. Although rare, there are reported cases of patients presenting with premature adrenarche in an otherwise unambiguous female, due to a testosterone-producing gonadal tumor.

Isolated 46,XY pure GD is considered in an adolescent with primary amenorrhea and sexual immaturity with a full female external phenotype, while partial or mixed GD should be considered more likely in the differential diagnosis of a 46,XY patient with ambiguous genitalia. The biochemical changes in complete, partial, and mixed GD are outlined in Table 17.5 .

The major criteria for the diagnosis of pure, partial, or mixed GD are the appearance and histology of both gonads. Therefore, the ultimate diagnosis of any of the forms of GD, particularly the distinction between mixed and partial GD, requires a biopsy of both gonads. Because the risk of gonadoblastoma in these patients is elevated, a precise diagnosis of the type of GD is usually determined after prophylactic or therapeutic gonadectomy. In pure 46,XY GD both gonads are streak gonads. Partial GD is defined by bilateral dysgenetic gonads, whereas mixed GD typically has one streak gonad. Karyotype of peripheral leukocytes shows 46,XY in pure and partial GD, and mosaic 45,X/46,XY is frequent in mixed GD. However, if there is a streak gonad on one side and a normal testis contralaterally, but the peripheral karyotype is 46,XY, cryptic mosaicism can often be revealed within the gonad.

Once a presumptive diagnosis is made in 46,XY patients, FISH for SRY or for Yp can be performed. Only a minority of cases of XY GD can be explained by complete or partial SRY deletion or mutation, or NR5A1 /SF1 mutation. Sequencing of the SRY or NR5A1 /SF1 open reading frames for mutations is only positive in up to 30% of 46,XY pure GD. Certain isolated and syndromic forms of XY GD can be diagnosed molecularly with cytogenetics, chromosomal microarrays, or sequencing of known genes (see Table 17.5 ). For example, 46,XY DSD in conjunction with congenital heart disease may increase the suspicion of a GATA4 mutation. However, next-generation sequencing is now available in the clinical realm to replace this sequential guesswork and should be prioritized as a diagnostic tool to identify etiology.

In addition, whole-genome chromosomal microarray studies have identified many rare copy number variants associated with patients with 46,XY GD and 46,XX testicular and ovotesticular DSDs, including deletions and duplications in and around SOX3 , GATA4 , WWOX , and DMRT1 . ^, Deletions or duplications in the upstream promoter region of SOX9 are associated with isolated and familial cases of 46,XY GD and 46,XX testicular DSDs (see Table 17.3 ). ^,

If a mutation or translocation involving SRY is found, the father should be tested for a possible familial mutation because SRY mutations can result in a full spectrum of phenotypes, from 46,XY fertile males to ambiguous individuals to females with partial or complete GD. Due to potential autosomal modifiers, it is very difficult to predict the risk of recurrence of a GD phenotype in XY offspring.

Management of XY Gonadal Dysgenesis

The major concern in the treatment of patients with XY GD is the risk of gonadoblastoma, a mixed germ-cell, sex-cord tumor. The risk of gonadoblastoma formation in XY GD increases with age and has been estimated to be as high as 30% by 30 years of age. ^{, ,} Due to the high risk of gonadoblastoma formation, patients typically undergo prophylactic or therapeutic gonadectomy in the first decade of life. Patients are followed with regular ultrasounds every 6 months starting at age 2 until gonadectomy can be performed.

In individuals with XY complete GD, genitals and gender identity are unambiguously female. There are no reported cases of gender dysphoria. In patients with partial or mixed GD, the degree of genital masculinization can be used for initial gender assignment. However, issues of gender identity do arise in partial and mixed GD and are outlined further in the management section.

To complete secondary sex development, sex steroid replacement should be initiated at puberty. Sex steroid replacement is essential not only for the development of secondary sex characteristics but also for growth spurts and normal accrual of bone mineral density. Height, weight, and bone density should be monitored regularly. Furthermore, a dual-energy X-ray absorptiometry (DEXA) scan for bone density should be performed prior to induction of puberty with exogenous hormone replacement, and yearly thereafter for the first 2 years. As long as the first three DEXA scans are reported as normal, they can be done every 2 to 3 years.

Because a normal uterus is often present, hysterectomies are not common to preserve childbearing potential with in vitro fertilization methods. Despite this, for unclear reasons, most patients do not carry successful pregnancies. Mixed XY GD individuals with more masculinization of the external genitalia are often raised as males and consequently require lifetime testosterone therapy, through intramuscular injections or transdermal patches or gel.

Differential Diagnosis: Testicular Regression Syndrome

Testicular regression syndrome (TRS), also known as vanishing testes , results from an insult after initial testes determination, most likely vascular thrombosis or testicular torsion. Internal and external genitalia are variably developed, which presumably depends on when the loss of fetal testes occurred. ^{, , ,} Rarely, patients can present in adulthood as females with primary amenorrhea. ^, Typically TRS is characterized by primitive epididymis and the spermatic cord in the presence of a fibrous nodule with hemosiderin deposition rather than the streak or dysgenetic gonads seen in 46,XY GD. ^{, ,} The presence of spermatic cord structures, the absence of Müllerian derivatives, and the typical male external phenotype suggest viable testes existed early in development, and imply a late fetal or early neonatal regression. Diagnostic criteria are outlined in Table 17.5 .

The incidence of TRS has been estimated at 5% of males presenting with cryptorchidism and as high as 12% of cryptorchid patients older than 1 year. ^, Correct diagnosis of TRS versus 46,XY GD is essential because of the significant malignant potential of abdominal or dysgenetic testes. Mutational analysis of SF1 /NR5A1 as a cause of TRS did not find any association between mutations in NR5A1 and TRS and the etiology of TRS remains mostly unknown. However, recent reports have identified variants in the gene DHX37 in cases clinically diagnosed as both GD and TRS, underscoring the difficulty of the differential diagnosis. ^, Although the condition is thought to be rather frequent, optimal management remains unclear. No reports of testicular germ cell tumors in TRS have been published, suggesting that excision of the remnant gonad may be unnecessary.

Congenital Lipoid Adrenal Hyperplasia

Mutations in genes encoding proteins involved in the initial enzymatic regions of steroidogenesis, such as steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc, also known as CYP11A1) , ^, results in global silencing of adrenal and gonadal steroidogenesis. The accumulation of cholesterol in the adrenals and gonads ultimately results in primary adrenal and gonadal failure. StAR protein transports cholesterol across the inner and outer mitochondrial membranes of steroidogenic cells. P450scc catalyzes the initial reaction in all steroidogenic tissues, the conversion of cholesterol to pregnenolone (see Fig. 17.10 ). ^, On adrenal histology, patients with mutations in StAR protein or P450scc have lipid vacuoles. However, patients with STAR mutations have adrenal hyperplasia, whereas patients with P450SCC mutations typically do not exhibit adrenal enlargement.

3β-Hydroxysteroid Dehydrogenase Deficiency

This rare variant of CAH can have a wide spectrum of phenotypes, from classical presentation (salt-wasting, with a range of female external genitalia to ambiguous genitalia in genetic XY individuals) to nonclassical (no salt-wasting and later presentation). Rare defects in adrenal and gonadal 3β-hydroxysteroid dehydrogenase (HSD3B2) affect the synthesis of three major adrenal steroid hormones: cortisol, aldosterone, and testosterone synthesis. The resulting phenotype is adrenal insufficiency in XX and XY but genital ambiguity only in XY.

17α-Hydroxylase/17,20-Lyase Deficiency

Biosynthesis of adrenal and sex hormones requires cytochrome P450 17α-hydroxylase/17,20-lyase (P450C17) encoded by the CYP17A1 gene. Here, a single protein, P450C17, catalyzes two steps in the steroidogenic pathway (see Fig. 17.10 ), both of which are essential for sex steroid and glucocorticoid synthesis. Mutations in this gene ultimately result in decreased cortisol synthesis with shunting of steroidogenesis toward a mineralocorticoid precursor that has mineralocorticoid activity. Isolated cases of 17,20-lyase deficiency are caused by mutations in Cytochrome b5 (CYB5A), a gene that promotes the specific allosteric interaction between CYP17A1 and POR. ^,