CHAPTER 13 Sexual differentiation
normal and abnormal
Introduction
Sexual differentiation and its control are fundamental to the continuation of most species. The understanding of this process has advanced greatly in recent years, but before abnormalities of these disorders can be discussed, an understanding of normal sexual development is important. At fertilization, the haploid gametes unite and the conceptus contains 46 chromosomes with 22 autosomes derived from each of the gametes (i.e. sperm and ovum). The ovum donates one X chromosome and the sperm donates either one X or one Y chromosome; the axiom of mammalian reproduction is that a 46XX embryo will differentiate into a female, and a 46XY embryo becomes a male. It is, however, the presence or absence of the Y chromosome which determines whether the undifferentiated gonad becomes a testis or an ovary, and therefore the development of a male or female of the species.
Normal Embryological Development of the Reproductive System
Although chromosomal sex is determined at the time of fertilization, gonadal sex results from the differentiation of the indifferent undifferentiated gonad which becomes either a testis or an ovary. This begins during the fifth week of embryological development; at this time, an area of coelemic epithelium develops on the medial aspect of the urogenital ridge, and proliferation leads to the establishment of the gonadal ridge. Epithelial cords then grow into the mesenchyme (primary sex cords), and the gonad now possesses an outer cortex and an inner medulla. In XY individuals, the medulla becomes the testis and the cortex regresses. In embryos with an XX complement, the cortex differentiates to become an ovary and the medulla regresses. The primordial germ cells develop by the fourth week in the endodermal cells of the yolk sac, and during the fifth week, they migrate along the dorsal mesentery of the hindgut to the gonadal ridges, eventually becoming incorporated into the mesenchyme and the primary sex cords by the end of the sixth week (Figure 13.1).
Development of the testis
The primary sex cords become concentrated on the medulla of the gonad and proliferate, and their ends anastomose to form the rete testis. The sex cords become isolated by the development of a capsule called the ‘tunica albuginea’ and the developing sex cords become the seminiferous tubules. Mesenchyme grows between the tubes to separate them (Leydig cells). The seminiferous tubules are composed of two layers of cells: supporting cells (Sertoli cells) derived from the germinal epithelium, and spermatogonia derived from the primordial germ cells (Figure 13.1).
Development of the ovary
The development of the ovary is much slower than that of the testis, and the ovary is not evident until the 10th week. Now, the primary sex cords regress and finally disappear. Around 12 weeks, secondary sex cords arise from the germinal epithelium, and the primordial germ cells become incorporated into these cortical cords. At 16 weeks, these cortical cords break up to form isolated groups of cells called ‘primordial follicles’; each cell contains an oogonium derived from a primordial germ cell, surrounded by follicular cells arising from the cortical cords. These oogonia undergo rapid mitosis to increase the numbers to thousands of germ cells called ‘primary oocytes’. Each oocyte is surrounded by a layer of follicular cells, the structure being called a ‘primary follicle’. The surrounding mesenchyme becomes the stroma.
Genetic control of gonadal development (Figure 13.2)
The pathway which controls the genetic interaction which transforms intermediate mesoderm to the bipotential gonad is beginning to be understood. It is now possible to identify with certainty two genes that are associated with this process. These two genes are Wilms’ tumour gene (WT1) and FTZ1. WT1 has been found on chromosome 11, p13 and regulates DNA transcriptase (Hastie 1994, Dong et al 1997). FTZ1 produces steroidogenic factor 1 (SF-1), and this has been found to be required for differentiation to the bipotential gonad (Wilhelm et al 2007).
As the presence of a Y chromosome is essential for testicular differentiation, the localization of testicular-determining factor (TDF) which was localized at the short arm of the Y chromosome using cloning and DNA sequencing techniques, and the gene determining TDF was sequenced and designated SRY (sex-determining region Y gene) (Guellaen et al 1984, Behlke et al 1993). The second gene which is involved in the differentiation of the testis is known as SOX9, and this gene coexists with the SRY gene in its homeobox and is a regulator of DNA transcription. SOX9 gene expression is increased in male gonadal differentiation and decreased in female gonadal differentiation. SOX9 has also recently been shown to be a regulator of type II collagen which is involved in the formation of cartilage (Lefebvre et al 1997). The DAX1 gene is responsible for development of the receptor on the surface of the undifferentiated gonad, which allows SRY and SOX9 to differentiate the gonad to become a testis. Following the development of Leydig cells and Sertoli cells, SF-1 is involved in the control of steroid hydroxylases, and P450 aromatase causes an increase in the synthesis of testosterone and a reduction in the conversion of androgens to oestrogens, leading to a contribution to testicular regulation (Ogata 2008). In combination with ST1, it is also responsible for Sertoli cells producing anti-Müllerian hormone. Following differentiation, Leydig cells produce insulin-like 3 (INSL3) gene, expression of which leads to testicular descent and shortening of the gubernaculums (Foresta and Ferlin 2004).
The genes involved in ovarian differentiation are much more difficult to define, although DAX1 expression continues during ovarian differentiation. Therefore, it is suggested that DAX1 antagonizes the actions of the SRY gene (Swain et al 1998).
Sex chromosome anomalies
Variations may occur in the number or the structure of chromosomes in a mosaic or a non-mosaic form. The non-mosiac forms are summarized in Box 13.1.
Aneuploidy
Aneuploidy may result from the non-disjunction of either miotic division or in either parent or in an early cleavage of the zygote. Some are more common in the offspring of older women (e.g. 47XX and 47XXY), and the non-disjunction is presumed to arise mainly in the maternal miosis. The occurrence of 45X does not seem to be related to maternal age.
Although there are a wide range of clinical effects, some generalizations can be made:
Kleinfelter’s syndrome and XYY syndrome occur in approximately one in 700 newborn males, the clinical features of which are well recognized. These include hypogonadism, infertility, increased risk of testicular cancer, gynaecomastia, increased risk of male breast cancer, and sparse facial and body hair.
Turner’s syndrome results from a chromosome constitution of a missing X chromosome. The overall incidence of this condition is approximately one in 2500 female births, but many of these have a mosaic pattern with a chromosome constitution of 46XX/45X. Small stature is invariable and gonadal failure is usual. There are other somatic features such as web neck, low hairline, cubitus valgus, pigmented naevi and cardiovascular anomalies, particularly coarctation of the aorta. These individuals do not have systematic impairment of the intellect, but they have some impairment of spacial ability.
XXX syndrome arises in approximately one in 1200 live female births, and no consistent clinical syndrome is associated with this.
Mosaicism
This is more common for sex chromosomes than for autosomes and arises when two cell lines arise from a single zygote due to non-disjunction in an early mitosis. The most common are 46XX and 46XY accompanied by a 45X cell line. The clinical effects are wide ranging depending on the balance of the mosaicism.
Structural abnormalities of X chromosomes
Apart from the number of X chromosomes in mosaicism, there are a number of possible variations within the X chromosome in the female. The X chromosome carries a large number of genes, not all of which have been delineated but some of which are responsible for metabolic and development disorders, and the gene map for the X chromosome is constantly being updated. If there is one normal X chromosome and the other is abnormal, inactivation of the abnormal X chromosome usually occurs in the somatic cells and this tends to diminish the effect of the abnormality, except in the ovary. Although only one X chromosome is sufficient for early ovarian development, germ cell maintenance requires several loci on the second X chromosome and on autosomes (Figure 13.3) (Simpson 1999).
Intersex Disorders
Intersex disorders are best classified into three groups (see Figure 13.4). This classification was suggested by Hughes (2008) as a new simple classification which includes all disorders of intersex.
Female (46XX) disorders of sexual development
Definition
This group of disorders comprises conditions in which masculinization of the external genitalia occurs in patients with a normal 46XX karyotype. The degree of masculinization is variable, ranging from mild clitoromegaly to complete fusion of the labial folds with a penile urethra.
Pathophysiology
The abnormalities occur when a female fetus is exposed to elevated levels of androgens. As the differentiation of the external genitalia to male or female depends on the conversion of testosterone to dihydrotestosterone (DHT) in the tissues of the cloaca, the presence of DHT leads to male-type development. If the female fetus is exposed to low levels of androgen, partial masculinization may occur, leading to ambiguous genitalia; however, if the levels are sufficiently high, complete male external genital development may occur, although the testes are naturally absent. If androgen exposure is delayed until after 12 weeks, virilization is limited to clitoral enlargement with no effect on the already differentiated labia. Androgens have no effect on internal sexual development, and therefore the ovaries, uterus and upper vagina are normally formed and functional.
Congenital adrenal hyperplasia
Pathophysiology
This is the most common cause of female pseudohermaphroditism and is an autosomal-recessive disorder resulting in enzyme deficiency in the biosynthesis of cortisol in the adrenal gland. Cortisol production occurs in the zona fasciculata and zona reticularis (Figure 13.5), and is controlled by adrenocorticotrophic hormone (ACTH) secreted by the pituitary gland. Adrenal androgen production occurs in the same area and is influenced by ACTH. A deficiency in any enzyme in the pathway results in decreased production of cortisol with resultant elevated levels of ACTH. This leads to increased steroid production by the adrenal reticularis and consequent hyperplasia. The stimulation by ACTH elevates the levels of circulating androgens, and this results in virilization of the female fetus.
There are three adrenal enzyme deficiencies which result in masculinization: 21-hydroxylase, 11-hydroxylase and 3β-dehydrogenase deficiency.
21-Hydroxylase deficiency
This accounts for 90% of all cases of congenital adrenal hyperplasia. The deficiency results in an increase in progesterone and 17α-hydroxyprogesterone, and this substrate is therefore converted to androstenedione and subsequently to testosterone. Failure of 21-hydroxylase to convert progesterone to 11-deoxycorticosterone may result in aldosterone deficiency; this occurs in approximately two-thirds of cases and is the so-called ‘salt-losing’ type of congenital adrenal hyperplasia.
Aetiology
Deficiency of 21-hydroxylase is an autosomal-recessive disorder. The link between human leukocyte antigen (HLA) type and 21-hydroxylase deficiency was established by Dupont et al (1977), and this allowed mapping of the gene which was located on the short arm of chromosome 6. It is located between the HLA-B and HLA-DR loci, and subgroups of HLA-B have been closely linked to congenital adrenal hyperplasia type: HLA-BW47 is linked to salt-losing congenital adrenal hyperplasia and HLA-BW51 is linked to the simple virilizing form. Studies by Donohoue et al (1986) have shown that there are two 21-hydroxylase genes: 21-OHA and 21-OHB. Only one is active (21-OHB) and they both lie between the fourth components of complement C4A and C4B. A variety of mutations have been reported, including gene deletions of 21-OHB, gene conversions and point mutations.
Epidemiology
The incidence of 21-hydroxylase deficiency is between one in 5000 and one in 15,000, based on neonatal screening programmes (Cacciari et al 1983), although a higher incidence (one in 700) has been reported in specific populations of Eskimos (Pang et al 1982). The incidence of the non-classic form of the disease, when androgenization fails to appear before late childhood or puberty, is much more common; approximately one in 300 in the White population and one in 30 in European Jews (Pang et al 1988).
Presentation
Affected females are born with an enlarged clitoris, fused labioscrotal folds and a urogenital sinus which may become a phallic urethra. There is often great variation in the degree of masculinization of the external genitalia; this is classified according to Prader (1958).
The internal genitalia develop normally as they are not influenced by androgens.
In salt-losing congenital adrenal hyperplasia, the infants develop dehydration, hypotension and hyponatraemia between 7 and 28 days of age, known as a ‘salt-losing crisis’. Non-salt-losing congenital adrenal hyperplasia tends to cause less severe masculinization than the salt-losing type. In general, all children born with ambiguous genitalia, including cryptorchidism and hypospadias, should be screened for congenital adrenal hyperplasia. Without treatment, severe salt-losing disease is fatal.
When an infant is born with ambiguous genitalia, the management of the parents is very important. It is helpful to reassure them that the infant is healthy and that there is a developmental anomaly of the genitalia. If the initial examination of the child fails to identify palpable gonads, it is most likely that the child is female and the parents should be informed as such; the likelihood of congenital adrenal hyperplasia may be raised. The diagnosis must then be made with as much haste as possible to alleviate parental anxiety.
Investigation
The initial investigations are karyotyping, pelvic ultrasound and endocrine studies. The karyotype may be performed on a sample of cord blood or on a venous sample, and rapid results obtained. Pelvic ultrasound to discover the presence of a uterus and vagina will confirm the diagnosis. The specific diagnosis is made by measuring 17α-hydroxyprogesterone in serum, although a 24-h urinary estimate of 17-ketosteroids will also confirm the diagnosis.
Treatment
This is divided into four parts.
Acute salt-losing crisis
This involves correcting the electrolyte imbalance and replacing the cortisol deficiency with deoxycorticosterone acetate (DOCA), 1 mg/24 h. For the majority of cases, 9α-fludrocortisol is used at a dose of 0.1–0.2 mg/day added to the oral feed, and the dosage of DOCA or fludrocortisol is adjusted against the electrolyte levels.
Long-term cortisol replacement therapy
Although previous reports suggested that mineralocorticoid therapy could be discontinued after infancy (Newns 1974), more recent data suggest that it is essential to continue therapy for life (Hughes et al 1979).
Surgical correction
Once the sex of rearing has been established as female, some attempt to feminize the genitalia may be made, usually within the first 18 months of life. If the clitoris is enlarged, a reduction clitoridectomy can be performed and the perineal region modified (Edmonds 1989). There are major surgical problems associated with severe virilization in congenital adrenal hyperplasia. The urogenital sinus has been formed by labial fold fusion and the folds are usually thin, but may be thick with associated narrowing of the lower vagina, especially in salt-losers. If the labial folds are thin, division by a simple posterior incision may be performed around 3 or 4 years of age. However, thick perineal tissue should be left until after puberty, and no attempt at surgery should be made until the girl is physically and mentally sexually mature. The operation, when it is performed, involves a flap vaginoplasty with a pedicle graft of labia used to recreate the vagina. Alternatively, a Williams vaginoplasty may be used.
Mulaikal et al (1987) reviewed the fertility rates in 80 women with 21-hydroxylase deficiency; of 25 with the simple form who attempted pregnancy, 15 were successful, whereas only one of the salt-losers who tried to become pregnant succeeded. There is no doubt that a major reason for the failure of the salt-losing group is the disappointing results of surgery and subsequent lack of adequate sexual function.
Prenatal diagnosis
If the parents are heterozygous carriers of 21-hydroxylase deficiency, the fetus has a one in four chance of being affected. Thus, prenatal diagnosis is important, either by amniocentesis to measure levels of 17-hydroxyprogesterone in amniotic fluid, HLA typing of amniotic cells, or chorionic villus sampling and the use of specific DNA probes. Once the diagnosis has been made, the option is available to treat the pregnant woman with oral dexamethasone, which crosses the placenta and suppresses the secretion of ACTH and thus the circulating androgen levels.
11-Hydroxylase deficiency
This is the hypertensive form of congenital adrenal hyperplasia which accounts for approximately 5–8% of all cases (Zachmann et al 1983). The absence of 11-hydroxylase leads to elevated levels of 11-deoxycorticosterone (DOC), and although this means a decreased amount of aldosterone, DOC has salt-retaining properties, leading to hypertension. Androstenedione levels are also elevated and this can result in ambiguous genitalia.
The genetics, however, are rather different. There is no HLA association with 11-hydroxylase deficiency, but the use of a DNA probe has located the gene on the long arm of chromosome 8 (White et al 1985).
3β-Dehydrogenase deficiency
This rare form of congenital adrenal hyperplasia results in a block of steroidogenesis very early in the pathway, giving rise to severe salt-losing adrenal hyperplasia. The androgen most elevated is dehydroepiandrosterone, an androgen which causes mild virilization. The diagnosis rests on the measurement of elevated dehydroepiandrosterone. The gene encoding 3β-dehydrogenase has not yet been cloned but it is not linked to HLA.
Androgen-secreting tumours
Androgen-secreting tumours are rare in pregnancy, but may arise in the ovary or the adrenal gland. They cause fetal virilization. When they occur in the non-pregnant woman, anovulation is induced.
Ovary
A number of androgen-secreting tumours have been reported, including luteoma (Hensleigh and Woodruff 1978, Cohen et al 1982), polycystic ovaries (Fayez et al 1974), mucinous cystadenoma (Novak et al 1970, Post et al 1978), arrhenoblastoma (Barkan et al 1984) and Krukenberg tumours (Connor et al 1968, Forest et al 1978). Not all female fetuses will be affected and there is no association with gestation and exposure. The fetus may be partly protected by the conversion of the maternally derived androgen to oestrogen in the placenta, and thus the degree of virilization is variable.
Adrenal gland
There are only two reports of adrenal adenomas causing fetal masculinization (Murset et al 1970, Fuller et al 1983). These tumours may be responsive to human chorionic gonadotrophin, and thus levels of androgen may be higher in pregnancy than in the non-pregnant state, leading to androgenization of the fetus.
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