Etiology of Ambiguous Genitalia in the Newborn
Generally, the medical team has a final diagnosis and has determined the etiology of the intersex state by the end of 3 weeks.
In the XX newborn, gonads are not palpable, which classically leads to the diagnosis of female pseudohermaphroditism. Excessive androgen production can be traced back to the mother (excessive maternal androgen production or maternal exposure to androgens or progestins), the placenta (placental aromatase deficiency) or the fetal adrenal gland (CAH) (
Excessive Maternal Androgens
Any maternal source of elevated androgens can induce virilization in the female fetus. Maternal ingestion of androgens, progestagens, or other drugs is a notable cause. For example, several oral progestational compounds, given to prevent spontaneous abortion in the past, such as 19-nortestosterone, have been implicated. Exogenous steroids administered during pregnancy may cause posterior fusion of the labia, clitoridal enlargement and even increased degrees of androgenization. Other drugs, like danazol, which are also used in pregnancy, have been associated with abnormalities of sexual differentiation. Ovarian tumors include luteoma of pregnancy, arrhenoblastoma, hilar-cell tumor, masculinizing ovarian stromal cell tumor, and Krukenberg tumor. Untreated maternal virilizing CAH is another potential cause, though androgen-secreting adrenal tumor in the mother is rare. In both cases, investigation of abnormal androgen production by the mother must be performed immediately after delivery.
A discrepancy between marked virilization in the mother and a minimal androgen effect in female offspring indicates placental aromatase activity, which converts androgens to estrogens, or androgen metabolism, which thus becomes less active.
Excessive Placental Androgens
Female pseudohermaphroditism due to placental aromatase deficiency, which has recently been reported by several investigators, illustrates the critical role of placental aromatization in protecting the fetus from excess androgen exposure. A defect in the placental conversion of androgens to estrogens causes virilization in female offspring. In the absence of aromatase, androgens cannot be converted to estrone, estradiol or estriol, and large quantities of androstenedione and testosterone are transferred to the maternal and fetal circulation, resulting in masculinization of the urogenital sinus and external genitalia of the female fetus. The mother may undergo virilization during gestation as well. Cloning and sequencing of the CYP19 aromatase gene has provided new opportunities for identifying mutations. Detection of mutations in this gene has confirmed the fundamental role of aromatase for the fetal-placental unit and for sexual differentiation of the female fetus.
Management of excessive androgens in the female fetus is somewhat difficult and will differ, for example, if there is a family history of female pseudohermaphroditism or if androgen excess is suspected from ultrasonography.
Excessive Fetal Androgens
CAH is the most frequent cause of androgen excess and ambiguous genitalia in the female newborn and the various forms of CAH are due to defects in the biosynthesis of cortisol, with the subsequent excessive ACTH production leading to an accumulation of adrenal androgens and steroid precursors. Adrenal androgens undergo peripheral conversion to testosterone and dihydrotestosterone, and steroid precursors produce specific findings depending upon the exact enzyme deficiency. The enzymatic defects causing female virilization involve 3β-hydroxysteroid dehydrogenase Δ5–Δ4 isomerase (3β-HSD), P450C21 hydroxylase (21-OH), and P450C11 hydroxylase (11-OH).
The association of ambiguous genitalia and salt loss at birth signals on enzymatic block of the adrenal glands.
The differential diagnosis is made by the marked accumulation of the steroid above the enzymatic block: 17-OHP for 21-OH deficiency, 11-deoxycortisol for 11-OH deficiency and pregnelonone and other Δ5 precursors for 3β-HSD deficiency. The molecular genetics of these defects have been extensively studied. More than 90% of CAH is due to 21-hydroxylase deficiency and, in the newborn period, salt losing is present in 75% of patients. Glucocorticoid treatment should be started as soon as possible and adjusted on the basis of length, weight and steroid levels. Mineralocorticoid should be added if salt losing is documented. 11β-OH deficiency is associated with hypertension. 3β-Hydroxylase dehydrogenase is exceptional.
Prenatal diagnosis of these defects is possible in high-risk families and prenatal treatment can be offered to the mother of a 21-OH-deficient girl for future pregnancies. In preterm infants, differential diagnosis with the exceptional clitoridal hypertrophy associated with a high concentration of adrenal androgen should be considered.
The true hermaphrodite has both testicular and ovarian tissue present in either the same or opposite gonads. Both the external genitalia and the internal duct structures display gradations between male and female. The initial manifestation is ambiguous genitalia in 90% of the cases and, more rarely, isolated clitoromegaly or penile hypospadias is seen. Two thirds of true hermaphrodites are raised as males. Among those raised as females, two thirds will have clitoromegaly.
Virtually all patients have a urogenital sinus and in most cases a uterus is present. The most common peripheral karyotype is 46,XX but mosaicisms are observed (XX/XY). SRY gene is present in 10–30% of patients, suggesting that true hermaphroditism is a heterogeneous condition in terms of its genetic background.
The most critical factor in managing true hermaphroditism is gender assignment. A decision of male sex of rearing should be based on both the findings at laparotomy and the potential for adequate penile length. Another important consideration is that true hermaphrodites have the potential for fertility.
In the XY newborn, gonads are usually palpable and the diagnosis will be male pseudohermaphroditism (
If testosterone rises normally after hCG stimulation, a defect in androgen sensitivity or 5α-RD is indicated. Androgen resistance is the most common cause of ambiguous genitalia in male pseudohermaphroditism.
Complete androgen insensitivity is characterized by an unambiguous female phenotype with a blind vagina pouch and no uterus. Underdevelopment of the clitoris and labia minora may also be observed. The development of an inguinal hernia signals the possibility of complete androgen insensitivity in infancy, whereas this diagnosis is evoked by primary amenorrhea in puberty.
Pubertal breast development is normal or augmented in the majority of cases, however, in contrast to absent or scanty axillary and pubic hair. Patients with complete androgen insensitivity develop female habitus. The major treatment decision for complete androgen insensitivity syndrome primarily concerns the optional timing of gonadectomy. Our group performs gonadectomy before puberty and prescribes estrogens during puberty.
Partial Androgen Insensitivity Syndrome (PAIS)
PAIS covers a wide spectrum of clinical phenotypes, from patients with predominantly female phenotype (i.e., mild clitoromegaly) to an undervirilized male phenotype. In addition, the wolffian duct may develop to a variable extent.
Simple hypospadias or micropenis in children, or undervirilization and gynecomastia in adolescent boys, should also come to medical attention.
Newborns with PAIS have increased LH and testosterone secretion, while estrogen production is also higher: serum SHBG concentrations are intermediate between those of normal male and normal female. The SHBG response to the increase in serum testosterone induced by an hCG stimulation test has been used as an aid in the differential diagnosis between PAIS and other forms of male pseudohermaphroditism.
Androgen binding in genital skin fibroblasts has revealed that defects are heterogeneous since they range between reduced capacity, reduced affinity, thermolability, increased ligand dissociation rate and altered ligand specificity.
No research group, however, has been able to report any consistent correlation between the concentration of AR and the degree of undervirilization.
Since the AR gene was cloned, the tools of molecular biology have made it possible to identify mutations within the gene from patients with different phenotypes of PAIS. Screening procedures with sequencing of the AR gene allow identification of subtle changes responsible for missense or nonsense mutations. Measurements of androgen receptor mRNA have been useful in identifying mutations that cause PAIS by altering the state levels or the size of the mRNA. Transfection of constructs expressing the mutant AR in mammalian ells is the main approach for demonstrating the causative role of the mutation in the development of the androgen insensitivity.
The management of patients with PAIS must be individualized depending on the degree of genital ambiguity, the growth response of the penis to supraphysiologic doses of testosterone and the type of androgen receptor mutation.
Although certain androgen receptor defects may be amenable to androgen therapy, multiple reconstruction of external genitalia and azoospermia are good arguments to prefer a female sex of rearing.
5 α-Reductase Deficiency (5 α-RD)
Patients with 5α-RD are characterized at birth by an undervirilized phenotype: affected newborns exhibit ambiguous genitalia with a hypospadic phallus resembling a clitoris, a bifid scrotum which is labia-like and a urogenital sinus opening on the perineum. It is worth noting that the testes have been found in the inguinal canal, labia majora or scrotum. The clinical presentation can actually range from almost normal female phenotype to a clear-cut male phenotype with isolated hypospadias, but in all cases wolffian ducts have been differentiated normally into vas deferens, epididymis and seminal vesicles.
The main characteristic of patients with 5α-RD is the virilization of the external genitalia that occurs at puberty along with the acquisition of male genetic identity in these patients usually raised as females. Diagnosis should be based on physical examination, pedigree analysis, analysis of basal and posthCG stimulation plasma T and DHT levels, 5β/5α urinary steroid metabolite ratio, measurement of 5α-RD activity in cultured genital skin fibroblasts and finally by analysis of the 5α-R2 gene.
The characteristic endocrine features of 5α-RD are as follows: normal male to high levels of plasma T and low levels of plasma DHT, an elevated ratio of the concentration of plasma T to DHT in adulthood and after stimulation with hCG in childhood, and elevated ratios of urinary 5β- to 5α-metabolites of androgen and C21 steroids. The biological diagnosis of 5α-RD is mainly supported by an increased T/DHT ratio.
From a biochemical point of view, the decrease in 5α-reductase activity in the intact genital skin fibroblasts supports the diagnosis of 5α-RD but enzymatic activity is sometimes in the normal range. The decreased activity in sonicated cell extracts at acidic pH provides strong evidence that the mutation resulted in a loss of type 2 enzyme activity.
Isolation and sequencing of the cDNA encoding 5α-reductase type 2 provides the molecular tools required for definition of the gene abnormalities responsible for 5α-RD. The management of 5α-RD is primarily dependent upon the phenotypic findings and gender at the time of diagnosis. Given the severe defect of the external genitalia, most newborns are raised as female!
Gonadectomy should be performed early to prevent masculinization, along with vaginoplasty and clitoridal reduction. If the diagnosis is made in puberty, one can consider raising such a child as a male.
If testosterone does not rise after the therapeutic trial, either testicular dysgenesis, which is an anomaly in testicular determination, or a disturbance in testicular biosynthesis may be responsible. Genitography is useful for differential diagnosis. When a vagina and uterine cavity are found, the diagnosis of testicular dysgenesis is likely. When a blind vagina without a uterine cavity is found, the diagnosis is instead disturbance in testicular biosynthesis.
Abnormal Testicular Determination
Dysgenetic Testis. Newborns with dysgenetic testis present with bilateral dysgenetic testes, persistent müllerian structures, cryptorchidism, and inadequate virilization. This disorder shows wide clinical heterogeneity. Because the uterus is present, the sex of rearing should be female and gonadectomy is recommended as for mixed gonadal dysgenesis. These patients should be screened routinely for tumor.
Defect of Testis Maintenance. Bilateral vanishing testis (or embryonic testicular regression) is characterized by an XY karyotype and absent or rudimentary testes. The syndrome entails the presence of testes that vanish during embryogenesis, although the etiology is unclear: the regression of the testes in utero may be due to genetic mutation, a teratogen factor or bilateral torsion. Clinically, the syndrome encompasses a spectrum of phenotypes, ranging in severity from genital ambiguity to a male phenotype with an empty scrotum.
Patients with a defect in testis maintenance will be managed according to their position in the clinical spectrum of the disorder. In true agonadism, external genitalia are ambiguous and müllerian derivatives are absent or rudimentary due to complete or partial anti-müllerian hormone secretion, without secretion of testosterone. Patients with rudimentary testis have a male phenotype with micropenis and small atrophic testis with pre-Sertoli and Leydig cells. Some patients present with perineal hypospadias and persistent müllerian duct structures. Congenital anorchia is characterized by the complete absence of testicular tissue at birth, but normal male sexual differentiation without müllerian structures.
Patients with ambiguous genitalia require meticulous assessment to determine the optimal sex of rearing. In the absence of palpable gonads, measurement of basal- or hCG-stimulated testosterone secretion above the female range is informative, but laparotomy or celioscopy followed by histologic analysis of the gonads is absolutely essential. In general, the sex of rearing should be male with testosterone replacement therapy, which will lead to normal puberty and sexual function. Phenotypic males require long-term androgen replacement, beginning at the time of expected puberty.
Leydig Cell Hypoplasia
Male pseudohermaphrodites with Leydig cell hypoplasia have impaired Leydig cell differentiation and testosterone production. The phenotype is usually female, although it may be ambiguous; in all cases, müllerian structures are absent. Inhibiting mutations in the LH receptor gene have recently been reported in these patients.
Defect in Testosterone Synthesis
Decreased androgen production caused by an alteration in the enzymes involved in the testosterone biosynthesis pathway is another cause of male pseudohermaphroditism. Increased fetal production of androgens, on the other hand, results in female pseudohermaphroditism.
Congenital Lipoid Adrenal Hyperplasia (CLAH). CLAH is a rare disease characterized by a defect in the synthesis of the three classes of steroid hormones, resulting in severe salt wasting and female phenotype. Few mutations in humans have been found thus far in the gene encoding for P450scc, which is the principal candidate gene. Recently, the gene responsible for CLAH was cloned and validated by nonsense mutation. This gene encodes for the StAR protein (steroidogenic acute regulatory protein), which is thought to be responsible for the transport of cholesterol to the inner membrane of mitochondria, and thus to the P450scc enzyme complex.
17α-Hydroxylase Deficiency. Defects in P450C17 lead to male pseudohermaphroditism with various degrees of ambiguous genitalia. This is frequently a severe form and is most often diagnosed at puberty, with female phenotype associated with hypertension. Cytochrome P450C17 catalyzes the transformation of progesterone and pregnenolone into 17-OH-progesterone and 17-OHpregnenolone (17α-hydroxylase activity), respectively, and then into dehydroepiandrosterone and Δ4-androstenedione (17,20-lyase activity). The gene encoding for this enzymatic complex, CYP17, is located on chromosome 10q24–25. Several different mutations have been reported in the CYP17 gene leading to either a complete or partial form of the disease.
3β-HSD Deficiency. Defects in the function of 3β-HSD result in 46,XY individuals with male pseudohermaphroditism who sometimes show salt wasting in the classic form. Almost 15 mutations of the type II 3β-HSD enzyme have been reported to date. No mutation of type I has been found, however, which explains how virilization can occur in 46,XX subjects by peripheral, nonsteroidogenic conversion of elevated testosterone precursors.
17β-Hydroxysteroid Dehydrogenase (17β-HSD) Deficiency. Type 3 17β-HSD deficiency is a rare autosomal recessive cause of male pseudohermaphroditism.
The typical subject is a 46,XY male born with female external genitalia and testes located in the inguinal canals or labia majora. This disorder is particularly puzzling: the deficiency in testosterone synthesis and the defect in virilization, both of which are usually more complete during embryogenesis than in later life, contrast with the well-differentiated wolffian duct structures, which suggests that androgen acts in utero by an alternate mechanism in these tissues. Substantial virilization is seen at puberty, in association with elevated levels of androstenedione but low to normal plasma levels of testosterone. As in the case of 5α-RD, pubertal virilization may result from extraglandular testosterone formation due to peripheral conversion of increased testicular androstenedione by unaffected 17β-HSD isoenzymes. Missense and nonsense mutations, splice junction abnormalities and a small deletion resulting in a frame shift have been described, among other gene alterations. Expression of mutant enzymes after site-directed mutagenesis showed that the missense mutations caused almost complete loss of enzymatic activity.
Although affected newborns are generally considered to be female, the choice of sex of rearing will be greatly influenced by family values and cultural background. When female sex of rearing is maintained, we strongly urge that orchidectomy be carried out during infancy or childhood. When diagnosis is not made before puberty, a gender change to male at that time is acceptable.
Male pseudohermaphroditism may in some cases be part of a multiple malformation syndrome. In other cases, etiology cannot be determined, in which case the so-called idiopathic pseudohermaphroditism should raise the suspicion of environmental contamination by pesticides during gestation.
For the 46,XY/XO newborn, the diagnosis is mixed gonadal dysgenesis.
The most common karyotype in mixed gonadal dysgenesis is 45,XO/46,XY, but other mosaics have been reported with structurally abnormal or normal Y chromosome. The characteristics include a unilateral testis that is often intraabdominal, a contralateral streak gonad and persistent müllerian duct structures.
Because affected patients are at great risk of gonadal tumor, the gonads should be removed and the patients can then be reared as females. When the abnormality of the external genitals is minor, however, parents may perceive their child as a boy. Male sex of rearing will then impose life-long surveillance of the gonads.
In any case, mixed gonadal dysgenesis is associated with varying degrees of inadequate masculinization, and such males would be infertile. It should be remembered that the distinction between mixed gonadal dysgenesis and Turner syndrome with Y material is unclear.
Revision date: June 18, 2011
Last revised: by David A. Scott, M.D.