Ambiguous genitalia
The newborn with abnormal genital development presents a difficult diagnostic and treatment challenge. It is important that a definitive diagnosis be determined as quickly as possible so that an appropriate treatment plan can be established to minimize medical, psychological, and social complications. The purpose of this is to identify which newborns among those with abnormal genital development need to be screened for inter-sexuality, which need further investigations, and which require referral to a center with experience in the management of these disorders.1
Gonadal development
Normal sexual differentiation is a complex mechanism, depending on genetic and hormonal control. The bipotent gonad arises at the genital ridge under the control of autosomal genes, which are also involved in the formation of other organs. Progression towards testicular differentiation is mediated through both autosomal and gonosomal genes, leading to alignment of Sertoli cells and Leydig cells. Within the latter, androgen formation is induced by human chorionic gonadotrophin and luteinizing hormone. The influence of testosterone and dihydrotestosterone leads to differentiation of epidydimis, vasa deferentia, prostate and external genitalia. Androgens mediate their action through the androgen receptor, a nuclear transcription factor controlling the regulation of so far unknown target genes. Genetic variations within this pathway interrupt normal male development and will lead to malformation of external and/or internal genitalia.2
The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. The basic underlying principle of sexual development is that genetic sex--determined at fertilization by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program that leads to male sexual differentiation. In the absence of testicular hormones, the female pathway of sexual differentiation occurs.3
In response to a signal from a dominant-acting gene on the Y chromosome, primordial cells in the embryonic gonad ridge differentiate into Sertoli cells and affect newly migrated germ cells to differentiate as spermatogonia, thus creating a testis. The cells of the embryonic testis secrete hormones that lead to the development of most, if not all, male secondary sexual characteristics. The Sertoli cells secrete mullerian inhibitory factor (MIF), causing regression of the mullerian ducts and of stray oogonia. The Leydig cells secrete testosterone, causing differentiation and growth of the wolffian duct structures. Dihydrotestosterone, created by metabolism of testosterone, causes growth of the prostate and phallus and fusion of the labioscrotal folds. In the absence of SRY, Sertoli cell differentiation does not occur. Rather germ cells migrating into the primordial gonad differentiate as oogonia and cause interstitial cells to differentiate as granulosa cells. In the absence of MIF and testosterone, the mullerian ducts differentiate and grow as female internal genitalia and the external genitalia are feminized. Several genes have been identified that control testis determination. These include SRY, WT1, SOX9, SF1, XH2, and DAX1. Most of these genes were discovered by analysis of rare cases of sex reversal (genetic sex of one type, gonadal sex of the other type).4 Androgens are essential for male sexual differentiation, which may be impaired due to defects in synthesis of the androgens (testosterone and/or dihydrotestosterone) or due to defects in the androgen receptor itself. Defects in the development of the male phenotype can range from complete testicular feminization to men with hypospadia and gynecomastia (Reifenstein syndrome) and to men with infertility and/or minor degrees of undervirilization.5
Abnormalities of gonadal differentiation.
Gonadal differentiation involves a complex interplay of developmental pathways. The sex determining region Y (SRY) gene plays a key role in testis determination, but its interaction with other genes is less well understood. Abnormalities of gonadal differentiation result in a range of clinical problems. 46,XY complete gonadal dysgenesis is defined by an absence of testis determination. Subjects have female external genitalia and come to clinical attention because of delayed puberty. Individuals with 46,XY partial gonadal dysgenesis usually present in the newborn period for the valuation of ambiguous genitalia. Gonadal histology always shows an abnormality of seminiferous tubule formation. A diagnosis of 46,XY true hermaphroditism is made if the gonads contain well-formed testicular and ovarian elements. Despite the pivotal role of the SRY gene in testis development, mutations of SRY are unusual in subjects with a 46,XY karyotype and abnormal gonadal development. 46,XX maleness is defined by testis determination in an individual with a 46,XX karyotype. Most affected individuals have a phenotype similar to that of Klinefelter syndrome. In contrast, subjects with 46,XX true hermaphroditism usually present with ambiguous genitalia. The majority of subjects with 46,XX maleness have Y sequences including SRY in genomic DNA. However, only rare subjects with 46,XX true hermaphroditism have translocated sequences encoding SRY.6 Mosaicism and chimaerism involving the Y chromosome can also be associated with abnormal gonadal development. However, the vast majority of subjects with 45,X/46,XY mosaicism have normal testes and normal male external genitalia.7 Anti-Müllerian hormone (AMH, Müllerian inhibitory substance, MIS), a glycoprotein secreted by the testes causes regression of the Müllerian structures including the uterus, fallopian tubes and vagina in the male fetus. Persistent Müllerian duct syndrome (PMDS) is associated with a phenotypic male who has normal virilization, but a uterus and fallopian tubes are discovered at surgery for an inguinal hernia. In PMDS, AMH may be measurable in some cases, but not others.8
Defects in the androgen pathway
Inactivating mutations in the luteinizing hormone (LH) receptor gene cause complete absence of the LH receptor expression in the Leydig and thecal cells causing testicular and ovarian resistance, respectively, to LH. The male phenotype has primary hypogonadism and varying degrees of male pseudohermaphroditism; Leydig cells are absent, hypoplastic or unresponsive.9 The female phenotype has partial ovarian failure characterized by defective folliculogenesis, anovulation, absence of a luteal phase, delay or incomplete feminization at puberty, amenorrhea and infertility, because LH normally stimulates the thecal cells to produce androgen precursors that are aromatized to estradiol by the granulosa cells which promotes follicular maturation and ovulation, formation of the corpus luteum and stimulates progesterone secretion. Specifically, Arg554stop (TGA) and Ser616Tyr are described. Deficiency of 5-alpha-reductase, which normally converts testosterone to dihydrotestosterone (DHT), causes male pseudohermaphroditism,10 because the prostate, seminal vesicles, scrotum, and penis, which normally develop in response to DHT, fail to develop.11 Because AMH secretion is normal, regression of Müllerian structures occurs, and internal organs are male, but external genitalia are female. Affected members are usually raised as females until puberty, at which time testosterone production causes virilization and often a gender, change from female to male. Mutations in the 5-alpha-reductase 2 gene, but not the 5-alpha-reductase 1 gene are described.12 Although DHT is synthesized in the peripheral tissues (essentially a paracrine hormone) and serum concentrations are not an accurate reflection of 5a-reductase activity, the diagnosis can be confirmed by low serum DHT (<25 ng/dL, normal range = 8-58 ng/dL) or elevated testosterone:DHT ratio (>20, normal range = 9-20). Defects in the X-linked androgen receptor gene are phenotypically expressed in the hemizygous XY individuals as disorders of male sexual development. Lack of androgen action is compatible with life but causes easily ascertainable development abnormalities in the male, which are more common than defects in other members of the steroid/thyroid receptor gene family. Some patients with complete testicular feminization show complete or partial deletion of the androgen receptor gene by Southern blot analysis. Most patients with defective ligand binding show single base pair nucleotide substitutions, causing abnormal splicing or, more commonly, the introduction of premature termination codons or conformational changes that result in impaired receptor stability or defective ligand binding. In approximately one-sixth of patients with androgen resistance, base pair substitutions occur in the DNA binding region of the androgen receptor gene and no qualitative or quantitative abnormality in ligand binding is detectable. No clear-cut relationship is evident between the phenotype and the types of mutation.
The DAX-1 gene and adrenal hypoplasia congenita
The X-linked cytomegalic form of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) are due to mutations in the DAX-1 gene, a member of the nuclear hormone receptor gene superfamily. Because a single gene is responsible, all boys with a mutation involving the DAX-1 locus should not only be treated with glucocorticoid and mineralocorticoid replacement, but should be considered for testosterone or pulsatile gonadotropin-releasing hormone to achieve pubertal growth and sexual maturity.13 Single base pair deletions are detectable by PCR and sequencing and submicroscopic deletions are detectable by FISH.14 The DAX-1 gene has been involved in the dosage sensitive sex reversal (DSS) phenotype, a male-to-female sex-reversal syndrome due to the duplication of a small region of human chromosome Xp21. Dax-1 and Sry have been shown to act antagonistically in the mouse system, where increasing expression of the former leads to female development and increasing activity of the latter to male development. The DAX-1 protein, an unusual member of the nuclear hormone receptor, may act as a transcriptional repressor. It has been shown to both repress transcriptional activators by direct protein-protein interactions and to bind DNA hairpin structures and repress target genes.15
Congenital adrenal hyperplasia
The diagnostic term congenital adrenal hyperplasia (CAH) applies to a family of inherited disorders of steroidogenesis caused by an abnormality in one of the five enzymatic steps necessary in the conversion of cholesterol to cortisol. The enzyme defects are translated as autosomal recessive traits, with the enzyme deficient in more than 90% of CAH cases being 21-hydroxylase. In the classical forms of CAH (simple virilizing and salt wasting), owing to 21-hydroxylase deficiency (21-OHD), androgen excess causes external genital ambiguity in newborn females and progressive postnatal virilization in males and females. The gene for adrenal 21-hydroxylase, CYP21, is located on chromosome 6p in the area of HLA genes. Specific mutations may be correlated with a given degree of enzymatic compromise and the clinical form of 21-OHD. Prenatal treatment of 21-OHD with dexamethasone has been utilized for a decade, has been safe for both the mother and the fetus, and has been effective in preventing ambiguous genitalia in the affected female newborn. Dexamethasone administered at or before 10 weeks of gestation in affected female fetuses was effective in reducing virilization. This treatment spares the affected female the consequences of genital ambiguity, of genital surgery, sex misassignment and gender confusion.16
Aromatase deficiency
Our knowledge of the physiologic roles of estrogen in women and men has been advanced by recent descriptions of mutations disrupting estrogen biosynthesis and action. Aromatase deficiency results from autosomal recessive inheritance of mutations in the CYP19 gene. It gives rise to ambiguous genitalia in 46,XX individuals. At puberty, affected girls have hypergonadotropic hypogonadism, fail to develop secondary sexual characteristics, and exhibit progressive virilization. The affected 46,XY individuals have normal male sexual differentiation and pubertal maturation. These men are extremely tall and have eunuchoid proportions with continued linear growth into adulthood, lack of epiphyseal closure, and osteoporosis due to estrogen deficiency.17
