The Chromosomal Basis of Sex Determination
The sex of the fetus normally is determined at fertilization. The cells of normal females contain 2 X chromosomes; those of normal males contain 1 X and 1 Y. During meiotic reduction, half of the male gametes receive a Y chromosome and the other half an X chromosome. Because the female has 2 X chromosomes, all female gametes contain an X chromosome. If a Y-bearing gamete fertilizes an ovum, the fetus is male; conversely, if an X-bearing gamete fertilizes an ovum, the fetus is female.
Arithmetically, the situation described previously should yield a male/female sex ratio of 100—the sex ratio being defined as 100 times the number of males divided by the number of females. However, for many years, the male/female sex ratio of the newborns in the white population has been approximately 105. Apparently the sex ratio at fertilization is even higher than at birth; most data on the sex of abortuses indicate a preponderance of males.
Abnormalities of Meiosis and Mitosis
The discussion in this section is limited to anomalies of meiosis and mitosis that result in some abnormality in the sex chromosome complement of the embryo.
Chromosome studies in connection with various clinical conditions suggest that errors in meiosis and mitosis do indeed occur. These errors result in any of the following principal effects: (1) an extra sex chromosome, (2) an absent sex chromosome, (3) 2 cell lines having different sex chromosomes and arising by mosaicism, (4) 2 cell lines having different sex chromosomes and arising by chimerism, (5) a structurally abnormal sex chromosome, and (6) a sex chromosome complement inconsistent with the phenotype.
By and large, an extra or a missing sex chromosome arises as the result of an error of disjunction in meiosis I or II in either the male or the female. In meiosis I, this means that instead of each of the paired homologous sex chromosomes going to the appropriate daughter cell, both go to 1 cell, leaving that cell with an extra sex chromosome and the daughter cell with none. Failure of disjunction in meiosis II simply means that the centromere fails to divide normally.
A variation of this process, known as anaphase lag, occurs when 1 of the chromosomes is delayed in arriving at the daughter cell and thus is lost. Theoretically, chromosomes may be lost by failure of association in prophase and by failure of replication, but these possibilities have not been demonstrated.
Persons who have been found to have 2 cell lines apparently have experienced problems in mitosis in the very early stage of embryogenesis. Thus, if there is nondisjunction or anaphase lag in an early (first, second, or immediately subsequent) cell division in the embryo, mosaicism may be said to exist. In this condition, there are 2 cell lines; 1 has a normal number of sex chromosomes, and the other is deficient in a sex chromosome or has an extra number of sex chromosomes. A similar situation exists in chimerism, except that there may be a difference in the sex chromosome: 1 may be an X and 1 may be a Y. This apparently arises by dispermy, by the fertilization of a double oocyte, or by the fusion, very early in embryogenesis, of 2 separately fertilized oocytes. Each of these conditions has been produced experimentally in animals.
Structural abnormalities of the sex chromosomes—deletion of the long or short arm or the formation of an isochromosome (2 short arms or 2 long arms)—result from injury to the chromosomes during meiosis. How such injuries occur is not known, but the results are noted more commonly in sex chromosomes than in autosomes—perhaps because serious injury to an autosome is much more likely to be lethal than injury to an X chromosome, and surviving injured X chromosomes would therefore be more common.
The situation in which there is a sex chromosome complement with an inappropriate genotype arises in special circumstances of true hermaphroditism and XX males (see later sections).
The X Chromosome in Humans
At about day 16 of embryonic life, there appears on the undersurface of the nuclear membrane of the somatic cells of human females a structure 1 μm in diameter known as the X-chromatin body. There is genetic as well as cytogenetic evidence that this is 1 of the X chromosomes (the only chromosome visible by ordinary light microscopy during interphase). In a sense, therefore, all females are hemizygous with respect to the X chromosome. However, there are genetic reasons for believing that the X chromosome is not entirely inactivated during the process of formation of the X-chromatin body. In normal females, inactivation of the X chromosome during interphase and its representation as the X-chromatin body are known as the Lyon phenomenon (for Mary Lyon, a British geneticist). This phenomenon may involve, at random, either the maternal or the paternal X chromosome. Furthermore, once the particular chromosome has been selected early in embryogenesis, it is always the same X chromosome that is inactivated in the progeny of that particular cell. Geneticists have found that the ratio of maternal to paternal X chromosomes inactivated is approximately 1:1.
The germ cells of an ovary are an exception to the X inactivation concept in that X inactivation does not characterize the meiotic process. Apparently, meiosis is impossible without 2 genetically active X chromosomes. Although random structural damage to 1 of the X chromosomes seems to cause meiotic arrest, oocyte loss, and therefore failure of ovarian development, an especially critical area necessary for oocyte development has been identified on the long arm of the X. This essential area involves almost all of the long arm and has been specifically located from Xq13 to Xq26. If this area is broken in 1 of the X chromosomes as in a deletion or translocation, oocyte development does not occur. However, a few exceptions to this rule have been described.
It is a curious biologic phenomenon that if 1 of the X chromosomes is abnormal, it is always this chromosome that is genetically inactivated and becomes the X-chromatin body, regardless of whether it is maternal or paternal in origin. Although this general rule seems to be an exception to the randomness of X inactivation, this is more apparent than real. Presumably, random inactivation does occur, but the disadvantaged cells—ie, those left with a damaged active X—do not survive. Consequently, the embryo develops only with cells with a normal active X chromosome (X-chromatin body) (Fig. 3–5).
Relation of X-chromatin body to the possible sex chromosome components.
If there are more than 2 X chromosomes, all X chromosomes except 1 are genetically inactivated and become X-chromatin bodies; thus, in this case, the number of X-chromatin bodies will be equal to the number of X chromosomes minus 1. This type of inactivation applies to X chromosomes even when a Y chromosome is present, eg, in Klinefelter's syndrome.
Although the X chromosomes are primarily concerned with the determination of femininity, there is abundant genetic evidence that loci having to do with traits other than sex determination are present on the X chromosome. Thus, in the catalog of genetic disorders given in the 10th edition of Mendelian Inheritance in Man, 320 traits are listed as more or less definitely X-linked. Substantial evidence for X linkage has been found for about 160 of these traits; the rest are only suspected of having this relationship. Hemophilia, color blindness, childhood muscular dystrophy (Duchenne's dystrophy), Lesch-Nyhan syndrome, and glucose-6-phosphate dehydrogenase deficiency are among the better known conditions controlled by loci on the X chromosome. These entities probably arise from the expression of a recessive gene due to its hemizygous situation in males.
X-linked dominant traits are infrequent in humans. Vitamin D-resistant rickets is an example.
At least 1 disorder can be classified somewhere between a structural anomaly of the X chromosome and a single gene mutation. X-linked mental retardation in males is associated with a fragile site at q26, but a special culture medium is required for its demonstration. Furthermore, it has been shown that heterozygote female carriers for this fragile site have low IQ test scores.
The Y Chromosome in Humans
Just as the X chromosome is the only chromosome visible by ordinary light microscopy during interphase, the Y chromosome is the only chromosome visible in interphase, after exposure to quinacrine compounds, by fluorescence microscopy. This is a very useful diagnostic method.
In contrast to the X chromosome, few traits have been traced to the Y chromosome except those having to do with testicular formation and those at the very tip of the short arm, homologous with those at the tip of the short arm of the X. Possession of the Y chromosome alone, ie, without an X chromosome, apparently is lethal, because such a case has never been described.
Present on the Y chromosome is an area that produces a factor that allows for testicular development. This factor is termed testis-determining factor (TDF). Without the presence of TDF, normal female anatomy will develop. When TDF is present, testicular development occurs with subsequent differentiation of Sertoli cells. The Sertoli cells in turn produce a second factor central to male differentiation, müllerian-inhibiting factor (MIF), also termed antimüllerian factor (AMF). The presence of MIF causes the regression of the müllerian ducts and thereby allows for the development of normal internal male anatomy.
In addition to sex determination function, human Y chromosome also has a role in spermatogenesis controlled by multiple genes along proximal Yq. The locus for spermatogenesis is on the euchromatic part of Yq (Yq11) called azoospermic factor (AZF). The AZF region is divided into three nonoverlapping regions AZFa, AZFb, and AZFc. The term "microdeletion" means that the size of the deleted segment is not visualized on karyotyping but must be discerned through molecular biology technique. There is no specific phenotype–genotype correlation between the degree of spermatogenic failure and type of Yq microdeletion. Complete deletion of AZFa and AZFb regions is associated with Sertoli cell-only syndrome and spermatogenic arrest, respectively. However, partial deletions of AZFa or AZFb or complete/partial deletions of AZFc are associated with a variable degree of spermatogenic failure ranging from oligozoospermia to Sertoli cell-only syndrome. There are reports of progressive impairment of spermatogenesis over time in patient with AZFc deletion. The fourth AZFd region, which was earlier proposed, does not exist based on the Y chromosome sequencing. There are many candidate genes within the deleted regions that are responsible for impaired spermatogenesis. The extensively studied genes are DAZ on AZFc region, RBMY1A1 on AZFb region, and USP9Y, DBY, and UTY on AZFa region. Because the deleted genes are expressed mainly in testes, men carrying the deletions have no abnormalities other than spermatogenic failure.
The incidence of Yq microdeletions in infertile men varies from 1–55% depending on study design. The most frequently deleted region is AZFc (~60%), whereas the deletion of the AZFa region is extremely rare (5%). The identification of Yq microdeletion has a prognostic value for the chance of successful testicular sperm retrieval. Men with complete deletion of AZFa and AZFb regions have almost no chance of having sperm recovered from surgical testicular sperm retrieval procedure, and no treatment is presently available for their fertility problem besides the use of donor sperm.
In the past, the majority of cases of Yq microdeletions have been de novo in infertile men during embryogenesis or from meiotic error in the germline of the fertile father. However, with the advent of assisted reproductive technologies, these infertile men can conceive genetic offspring with intracytoplasmic sperm injection (ICSI) technique, so Yq microdeletion can pass from generation to generation. A few studies show that when a Yq microdeletion is present in infertile men, ICSI-derived sons will inherit the same deletion. In view of genetic counseling, although Yq microdeletion is transmitted to the male offspring, the phenotype of male offspring regarding the degree of spermatogenesis is unpredictable due to the influence of the presence or absence of environmental factors that could affect spermatogenesis and the period of lifetime when spermatogenesis is assessed.
In 1938, Turner described 7 girls 15–23 years of age with sexual infantilism, webbing of the neck, cubitus valgus, and retardation of growth. A survey of the literature indicates that "Turner's syndrome" means different things to different writers. After the later discovery that ovarian streaks are characteristically associated with the clinical entity described by Turner, "ovarian agenesis" became a synonym for Turner's syndrome. After discovery of the absence of the X-chromatin body in such patients, the term ovarian agenesis gave way to "gonadal dysgenesis," "gonadal agenesis," or "gonadal aplasia."
Meanwhile, some patients with the genital characteristics mentioned previously were shown to have a normally positive X-chromatin count. Furthermore, a variety of sex chromosome complements have been found in connection with streak gonads. As if these contradictions were not perplexing enough, it has been noted that streaks are by no means confined to patients with Turner's original tetrad of infantilism, webbing of the neck, cubitus valgus, and retardation of growth but may be present in girls with sexual infantilism only. Since Turner's original description, a host of additional somatic anomalies (varying in frequency) have been associated with his original clinical picture; these include shield chest, overweight, high palate, micrognathia, epicanthal folds, low-set ears, hypoplasia of nails, osteoporosis, pigmented moles, hypertension, lymphedema, cutis laxa, keloids, coarctation of the aorta, mental retardation, intestinal telangiectasia, and deafness.
For our purposes, the eponym Turner's syndrome will be used to indicate sexual infantilism with ovarian streaks, short stature, and 2 or more of the somatic anomalies mentioned earlier. In this context, terms such as ovarian agenesis, gonadal agenesis, and gonadal dysgenesis lose their clinical significance and become merely descriptions of the gonadal development of the person. At least 21 sex chromosome complements have been associated with streak gonads (Fig. 3–6), but only about 9 sex chromosome complements have been associated with Turner's syndrome. However, approximately two-thirds of patients with Turner's syndrome have a 45,X chromosome complement, whereas only one-fourth of patients without Turner's syndrome but with streak ovaries have a 45,X chromosome complement.
The 21 sex chromosome complements that have been found in patients with streak gonads.
Karyotype–phenotype correlations in the syndromes associated with ovarian agenesis are not completely satisfactory. Nonetheless, if gonadal development is considered as 1 problem and if the somatic difficulties associated with these syndromes are considered as a separate problem, one can make certain correlations.
With respect to failure of gonadal development, it is important to recall that diploid germ cells require 2 normal active X chromosomes. This is in contrast to the somatic cells, where only 1 sex chromosome is thought to be genetically active, at least after day 16 of embryonic life in the human, when the X-chromatin body first appears in the somatic cells. It is also important to recall that in 45,X persons no oocytes persist, and streak gonads are the rule. From these facts, it can be inferred that failure of gonadal development is not the result of a specific sex chromosome defect but rather of the absence of 2 X chromosomes with the necessary critical zones.
Karyotype–phenotype correlations with respect to somatic abnormalities are even sketchier than the correlations with regard to gonadal development. However, good evidence shows that monosomy for the short arm of the X chromosome is related to somatic difficulties, although some patients with long-arm deletions have somatic abnormalities.
History of Gonadal Agenesis
The histologic findings in these abnormal ovaries in patients with gonadal streaks are essentially the same regardless of the patient's cytogenetic background (Fig. 3–7).
Gonadal streaks in a patient with the phenotype of Turner's syndrome. (Redrawn and reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
Fibrous tissue is the major component of the streak. It is indistinguishable microscopically from that of the normal ovarian stroma. The so-called germinal epithelium, on the surface of the structure, is a layer of low cuboid cells; this layer appears to be completely inactive.
Tubules of the ovarian rete are invariably found in sections taken from about the midportion of the streak.
In all patients who have reached the age of normal puberty, hilar cells are also demonstrated. The number of hilar cells varies among patients. In those with some enlargement of the clitoris, hilar cells are present in large numbers. These developments may be causally related. Nevertheless, hilar cells are also found in many normal ovaries. The origin of hilar cells is not precisely known, but they are associated with development of the medullary portion of the gonad. Their presence lends further support to the concept that in ovarian agenesis the gonad develops along normal lines until just before the expected appearance of early oocytes. In all cases in which sections of the broad ligament have been available for study, it has been possible to identify the mesonephric duct and tubules—broad ligament structures found in normal females.
The newborn with streak ovaries often shows edema of the hands and feet. Histologically, this edema is associated with large dilated vascular spaces. With such findings, it is obviously desirable to obtain a karyotype. However, some children with streak ovaries—particularly those who have few or no somatic abnormalities—cannot be recognized at birth.
The arresting and characteristic clinical finding in many of these patients is their short stature. Typical patients seldom attain a height of 1.5 m (5 ft) (Fig. 3–8). In addition, sexual infantilism is a striking finding. As mentioned earlier, a variety of somatic abnormalities may be present; by definition, if 2 or more of these are noted, the patient may be considered to have Turner's syndrome. Most of these patients have only 1 normal X chromosome, and two-thirds of them have no other sex chromosome. Patients of normal height without somatic abnormalities may also have gonadal streaks. Under these circumstances, there is likely to be a cell line with 2 normal sex chromosomes but often a second line with a single X. The internal findings are exactly the same as in patients with classic Turner's syndrome, however.
Patient with Turner's syndrome. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
An important finding in patients of any age—but especially after expected puberty, ie, about 12 years—is elevation of total gonadotropin production. From a practical point of view, ovarian failure in patients over age 15 cannot be considered a diagnostic possibility unless the serum follicle-stimulating hormone level is more than 50 mIU/mL and luteinizing hormone level is more than 90 mIU/mL.
Nongonadal endocrine functions are normal. Urinary excretion of estrogens is low, and the maturation index and other vaginal smear indices are shifted well to the left.
Substitution therapy with estrogen is necessary for development of secondary characteristics.
Therapy with growth hormone will increase height. Whether ultimate height will be greater than it otherwise would be is uncertain, but current evidence suggests that it will be.
The incidence of malignant degeneration is increased in the gonadal streaks of patients with a Y chromosome, as compared with normal males. Surgical removal of streaks from all patients with a Y chromosome is recommended.
By classic definition, true hermaphroditism exists when both ovarian and testicular tissue can be demonstrated in 1 patient. In humans, the Y chromosome carries genetic material that normally is responsible for testicular development; this material is active even when multiple X chromosomes are present. Thus, in Klinefelter's syndrome, a testis develops with up to 4 Xs and only 1 Y. Conversely (with rare exceptions), a testis has not been observed to develop in the absence of the Y chromosome. The exceptions are found in true hermaphrodites and XX males, in whom testicular tissue has developed in association with an XX sex chromosome complement.
No exclusive features clinically distinguish true hermaphroditism from other forms of intersexuality. Hence, the diagnosis must be entertained in an infant with any form of intersexuality, except only those with a continuing virilizing influence, eg, congenital adrenal hyperplasia. Firm diagnosis is possible after the onset of puberty, when certain clinical features become evident, but the diagnosis can and should be made in infancy.
In the past, most true hermaphrodites have been reared as males because they have rather masculine-appearing external genitalia (Fig. 3–9). Nevertheless, with early diagnosis, most should be reared as females.
External genitalia of a patient with true hermaphroditism. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
Almost all true hermaphrodites develop female-type breasts. This helps to distinguish male hermaphroditism from true hermaphroditism, because few male hermaphrodites other than those with familial feminizing hermaphroditism develop large breasts.
Many true hermaphrodites menstruate. The presence or absence of menstruation is partially determined by the development of the uterus; many true hermaphrodites have rudimentary or no development of the müllerian ducts (Fig. 3–10).
Internal genitalia of a patient with true hermaphroditism. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
A few patients who had a uterus and menstruated after removal of testicular tissue have become pregnant and delivered normal children.
Sex Chromosome Complements
Most true hermaphrodites have X-chromatin bodies and karyotypes that are indistinguishable from those of normal females. In contrast to these, a few patients who cannot be distinguished clinically from other true hermaphrodites have been reported to have a variety of other karyotypes—eg, several chimeric persons with karyotypes of 46,XX/46,XY have been identified.
In true hermaphrodites, the testis is competent in its müllerian-suppressive functions, but an ovotestis may behave as an ovary insofar as its müllerian-suppressive function is concerned. The true hermaphroditic testis or ovotestis is as competent to masculinize the external genitalia as is the testis of a patient with the virilizing type of male hermaphroditism. This is unrelated to karyotype.
Deletion mapping by DNA hybridization has shown that most (but not all) XX true hermaphrodites have Y-specific sequences. Abnormal crossover of a portion of the Y chromosome to the X in meiosis may explain some cases. This latter statement is further supported by the finding of a positive H-Y antigen assay in some patients with 46,XX true hermaphroditism.
In general, the clinical picture of true hermaphroditism is not compatible with the clinical picture in other kinds of gross chromosomal anomalies. For example, very few true hermaphrodites have associated somatic anomalies, and mental retardation almost never occurs.
The principles of treatment of true hermaphroditism do not differ from those of the treatment of hermaphroditism in general. Therapy can be summarized by stating that surgical removal of contradictory organs is indicated, and the external genitalia should be reconstructed in keeping with the sex of rearing. The special problem in this group is how to establish with certainty the character of the gonad. This is particularly difficult in the presence of an ovotestis, because its recognition by gross characteristics is notoriously inaccurate, and one must not remove too much of the gonad for study. In some instances, the gonadal tissue of 1 sex is completely embedded within a gonadal structure primarily of the opposite sex.
This condition, first described in 1942 by Klinefelter, Reifenstein, and Albright, occurs only in apparent males. As originally described, it is characterized by small testes, azoospermia, gynecomastia, relatively normal external genitalia, and otherwise average somatic development. High levels of gonadotropin in urine or serum are characteristic.
By definition, this syndrome applies only to persons reared as males. The disease is not recognizable before puberty except by routine screening of newborn infants. Most patients come under observation at 16–40 years of age.
Somatic development during infancy and childhood may be normal. Growth and muscular development may also be within normal limits. Most patients have a normal general appearance and no complaints referable to this abnormality, which is often discovered during the course of a routine physical examination or an infertility study.
In the original publication by Klinefelter and coworkers, gynecomastia was considered an essential part of the syndrome. Since then, however, cases without gynecomastia have been reported.
The external genitalia are perfectly formed and in most patients are quite well developed. Erection and intercourse usually are satisfactory.
There is no history of delayed descent of the testes in typical cases, and the testes are in the scrotum. Neither is there any history of testicular trauma or disease. Although a history of mumps orchitis is occasionally elicited, this disease has not been correlated with the syndrome. However, the testes are often very small in contrast to the rest of the genitalia (about 1.5 × 1.5 cm).
Psychological symptoms are often present. Most studies of this syndrome have been performed in psychiatric institutions. The seriousness of the psychological disturbance seems to be partly related to the number of extra X chromosomes—eg, it is estimated that about one-fourth of XXY patients have some degree of mental retardation.
One of the extremely important clinical features of Klinefelter's syndrome is the excessive amount of pituitary gonadotropin found in either urine or serum assay.
The urinary excretion of neutral 17-ketosteroids varies from relatively normal to definitely subnormal levels. There is a rough correlation between the degree of hypoleydigism as judged clinically and a low 17-ketosteroid excretion rate.
Histologic & Cytogenetic Findings
Klinefelter's syndrome may be regarded as a form of primary testicular failure.
Several authors have classified a variety of forms of testicular atrophy as subtypes of Klinefelter's syndrome. Be this as it may, Klinefelter believed that only those patients who have a chromosomal abnormality could be said to have this syndrome. Microscopic examination of the adult testis shows that the seminiferous tubules lack epithelium and are shrunken and hyalinized. They contain large amounts of elastic fibers, and Leydig cells are present in large numbers.
Males with positive X-chromatin bodies are likely to have Klinefelter's syndrome. The nuclear sex anomaly reflects a basic genetic abnormality in sex chromosome constitution. All cases studied have had at least 2 X chromosomes and 1 Y chromosome. The most common abnormality in the sex chromosome constitution is XXY, but the literature also records XXXY, XXYY, XXXXY, and XXXYY, and mosaics of XX/XXY, XY/XXY, XY/XXXY, and XXXY/XXXXY. In all examples except the XX/XXY mosaic, a Y chromosome is present in all cells. From these patterns, it is obvious that the Y chromosome has a very strong testis-forming impulse, which can operate in spite of the presence of as many as 4 X chromosomes.
Thus, patients with Klinefelter's syndrome will have not only a positive X-chromatin body but also a positive Y-chromatin body.
The abnormal sex chromosome constitution causes differentiation of an abnormal testis, leading to testicular failure in adulthood. At birth or before puberty, such testes show a marked deficiency or absence of germinal cells.
By means of nursery screening, the frequency of males with positive X-chromatin bodies has been estimated to be 2.65 per 1000 live male births.
There is no treatment for the 2 principal complaints of these patients: infertility and gynecomastia. No pituitary preparation has been effective in the regeneration of the hyalinized tubular epithelium or the stimulation of gametogenesis. Furthermore, no hormone regimen is effective in treating the breast hypertrophy. When the breasts are a formidable psychological problem, surgical removal may be a satisfactory procedure. In patients who have clinical symptoms of hypoleydigism, substitution therapy with testosterone is an important physiologic and psychological aid. Donor sperm may be offered for treatment of infertility.
A few cases of adult males with a slightly hypoplastic penis and very small testes but no other indication of abnormal sexual development have been reported. These males are sterile. Unlike those with Klinefelter's syndrome, they do not have abnormal breast development. They are clinically very similar to patients with Del Castillo's syndrome (testicular dysgenesis). Nevertheless, the XX males have a positive sex chromatin and a normal female karyotype. These may be extreme examples of the sex reversal that usually is partial in true hermaphroditism.
The finding of more than 1 X-chromatin body in a cell indicates the presence of more than 2 X chromosomes in that particular cell. In many patients, such a finding is associated with mosaicism, and the clinical picture is controlled by this fact—eg, if 1 of the strains of the mosaicism is 45,X, gonadal agenesis is likely to occur. There also are persons who do not seem to have mosaicism but do have an abnormal number of X chromosomes in all cells. In such persons, the most common complement is XXX (triplo-X syndrome), but XXXX (tetra-X syndrome) and XXXXX (penta-X syndrome) have been reported.
An additional X chromosome does not seem to have a consistent effect on sexual differentiation. The body proportions of these persons are normal, and the external genitalia are normally female. A number of such persons have been examined at laparotomy, and no consistent abnormality of the ovary has been found. In a few cases, the number of follicles appeared to be reduced, and in at least 1 case the ovaries were very small and the ovarian stroma poorly differentiated. About 20% of postpubertal patients with the triplo-X syndrome report various degrees of amenorrhea or some irregularity in menstruation. For the most part, however, these patients have a normal menstrual history and are of proved fertility.
Almost all patients known to have multiple-X syndromes have some degree of mental retardation. A few have mongoloid features. (The mothers of these patients tended to be older than the mothers of normal children, as is true with Down syndrome.) Perhaps these findings are in part circumstantial, as most of these patients were discovered during surveys in mental institutions. The important clinical point is that mentally retarded infants should have chromosomal study.
Uniformly, the offspring of triplo-X mothers have been normal. This is surprising, because theoretically in such cases meiosis should produce equal numbers of ova containing 1 or 2 X chromosomes, and fertilization of the abnormal XX ova should give rise to XXX and XXY individuals. Nevertheless, the triplo-X condition seems selective for normal ova and zygotes.
The diagnosis of this syndrome is made by identifying a high percentage of cells with double X-chromatin bodies in the buccal smear and by finding 47 chromosomes with a karyotype showing an extra X chromosome in all cells cultured from the peripheral blood. It should be noted that in the examination of the buccal smear, some cells have a single X-chromatin body. Hence, based on the chromatin examination, one might suspect XX/XXX mosaicism. Actually, in triplo-X patients, only a single type of cell can be demonstrated in cultures of cells from the peripheral blood. The absence of the second X-chromatin body in some of the somatic cells may result from the time of examination of the cell (during interphase) and from the spatial orientation, which could have prevented visualization of the 2 X-chromatin bodies (adjacent to the nuclear membrane). In this syndrome, the number of cells containing either 1 or 2 X-chromatin bodies is very high—at least 60–80%, as compared with an upper limit of about 40% in normal females.
Female Hermaphroditism Due to Congenital Adrenal Hyperplasia
- Female pseudohermaphroditism, ambiguous genitalia with clitoral hypertrophy, and, occasionally, persistent urogenital sinus.
- Early appearance of sexual hair; hirsutism, dwarfism.
- Urinary 17-ketosteroids elevated; pregnanetriol may be increased.
- Elevated serum 17-hydroxyprogesterone level.
- Occasionally associated with water and electrolyte imbalance—particularly in the neonatal period.
Female hermaphroditism due to congenital adrenal hyperplasia is a clearly delineated clinical syndrome. The syndrome has been better understood since the discovery that cortisone may successfully arrest virilization. The problem usually is due to a deficiency of a gene required for 21-hydroxylation in the biosynthesis of cortisol.
If the diagnosis is not made in infancy, an unfortunate series of events ensues. Because the adrenals secrete an abnormally large amount of virilizing steroid even during embryonic life, these infants are born with abnormal genitalia (Fig. 3–11). In extreme cases, there is fusion of the scrotolabial folds and, in rare instances, even formation of a penile urethra. The clitoris is greatly enlarged so that it may be mistaken for a penis (Fig. 3–12). No gonads are palpable within the fused scrotolabial folds, and their absence has sometimes given rise to the mistaken impression of male cryptorchidism. Usually, there is a single urinary meatus at the base of the phallus, and the vagina enters the persistent urogenital sinus as noted in Figure 3–13.
External genitalia of a female patient with congenital virilizing adrenal hyperplasia. Compare with Figure 3–12. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
External genitalia of a female patient with congenital virilizing adrenal hyperplasia. This is a more severe deformity than that shown in Figure 3–11.
Sagittal view of genital deformities of increasing severity (A–E) in congenital virilizing adrenal hyperplasia. (Redrawn and reproduced, with permission, from Verkauf BS, Jones HW Jr. Masculinization of the female genitalia in congenital adrenal hyperplasia. South Med J 1970;63:634–638.)
During infancy, provided there are no serious electrolyte disturbances, these children grow more rapidly than normal. For a time, they greatly exceed the average in both height and weight. Unfortunately, epiphyseal closure occurs by about age 10, and as a result, these people are much shorter than normal as adults (Fig. 3–14).
Untreated adult with virilizing adrenal hyperplasia. Note the short stature and the relative shortness of the limbs. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
The process of virilization begins at an early age. Pubic hair may appear as early as age 2 years but usually somewhat later. This is followed by growth of axillary hair and finally by the appearance of body hair and a beard, which may be so thick as to require daily shaving. Acne may develop early. Puberty never ensues. There is no breast development. Menstruation does not occur. During the entire process, serum adrenal androgens and 17-hydroxyprogesterone levels are abnormally high.
Although our principal concern here is with this abnormality in females, it must be mentioned that adrenal hyperplasia of the adrenogenital type may also occur in males, in whom it is called macrogenitosomia precox. Sexual development progresses rapidly, and the sex organs attain adult size at an early age. Just as in the female, sexual hair and acne develop unusually early, and the voice becomes deep. The testes are usually in the scrotum; however, in early childhood they remain small and immature, although the genitalia are of adult dimensions. In adulthood, the testes usually enlarge and spermatogenesis occurs, allowing impregnation rates similar to those of a control population. Somatic development in the male corresponds to that of the female; as a child, the male exceeds the average in height and strength, but (if untreated) as an adult he is stocky, muscular, and well below average height.
Both the male and the female with this disorder—but especially the male—may have the complicating problem of electrolyte imbalance. In infancy, it is manifested by vomiting, progressive weight loss, and dehydration and may be fatal unless recognized promptly. The characteristic findings are an exceedingly low serum sodium level, low CO2-combining power level, and high potassium level. The condition is sometimes misdiagnosed as congenital pyloric stenosis.
A few of these patients have a deficiency in 11-hydroxylation that is associated with hypertension in addition to virilization.
The adrenal changes center on a reticular hyperplasia, which becomes more marked as the patient grows older. In some instances, the glomerulosa may participate in the hyperplasia, but the fasciculata is greatly diminished in amount or entirely absent. Lipid studies show absence of fascicular and glomerular lipid but an abnormally strong lipid reaction in the reticularis (Fig. 3–15).
Normal adrenal architecture and adrenal histology in congenital virilizing adrenal hyperplasia. Note the great relative increase in the zona reticularis.
The ovarian changes can be summarized by stating that in infants, children, and teenagers, there is normal follicular development to the antrum stage but no evidence of ovulation. With increasing age, less and less follicular activity occurs, and primordial follicles disappear. This disappearance must not be complete, however, because cortisone therapy, even in adults, usually results in ovulatory menstruation after 4–6 months of treatment.
Developmental Anomalies of the Genital Tubercle & Urogenital Sinus Derivatives
The phallus is composed of 2 lateral corpora cavernosa, but the corpus spongiosum is normally absent. The external urinary meatus is most often located at the base of the phallus (Fig. 3–11). An occasional case may be seen in which the urethra does extend to the end of the clitoris (Fig. 3–12). The glans penis and the prepuce are present and indistinguishable from these structures in the male. The scrotolabial folds are characteristically fused in the midline, giving a scrotumlike appearance with a median perineal raphe; however, they seldom enlarge to normal scrotal size. No gonads are palpable within the scrotolabial folds. When the anomaly is not severe (eg, in patients with postnatal virilization), fusion of the scrotolabial folds is not complete, and by gentle retraction it is often possible to locate not only the normally located external urinary meatus but also the orifice of the vagina.
An occasional patient has no communication between the urogenital sinus and the vagina. In no case does the vagina communicate with that portion of the urogenital sinus that gives rise to the female urethra or the prostatic urethra. Instead, the vaginal communication is via caudal urogenital sinus derivatives; thus, fortunately, the sphincter mechanism is not involved, and the anomalous communication is with that portion of the sinus that develops as the vaginal vestibule in the female and the membranous urethra in the male. From the gynecologist's point of view, it is much more meaningful to say that the vagina and (female) urethra enter a persistent urogenital sinus than to say that the vagina enters the (membranous [male]) urethra. This conclusion casts some doubt on the embryologic significance of the prostatic utricle, which is commonly said to represent the homologue of the vagina in the normal male.
Important and specific endocrine changes occur in congenital adrenal hyperplasia of the adrenogenital type. The ultimate diagnosis depends on demonstration of these abnormalities.
The progressive virilization of female hermaphrodites caused by adrenal hyperplasia would suggest that estrogen secretion in these patients is low, and this hypothesis is further supported by the atrophic condition of both the ovarian follicular apparatus and the estrogen target organs. Actually, the determination of urinary estrogens, both fluorometrically and biologically, indicates that it is elevated.
The development of satisfactory radioimmunoassay techniques for measuring steroids in blood serum has resulted in an increased tendency to measure serum steroids rather than urinary metabolites in diagnosing the condition and monitoring therapy. Serum steroid profiles of many patients with this disorder show that numerous defects in the biosynthesis of cortisol may occur. The most common defect is at the 21-hydroxylase step. Less frequent defects are at the 11-hydroxylase step and the 3β-ol-dehydrogenase step. Rarely, the defect is at the 17-hydroxylase step. In the most common form of the disorder—21-hydroxylase deficiency—the serum 17-hydroxyprogesterone level and, to a lesser extent, the serum progesterone level are elevated. This is easily understandable when it is recalled that 17-hydroxyprogesterone is the substrate for the 21-hydroxylation step (Fig. 3–16). Likewise, in the other enzyme defects, the levels of serum steroid substrates are greatly elevated.
Enzymatic steps in cortisol synthesis. Localization of defects in congenital adrenal hyperplasia.
Pathogenesis of Virilizing Adrenal Hyperplasia
The basic defects in congenital virilizing adrenal hyperplasia are 1 or more enzyme deficiencies in the biosynthesis of cortisol (Fig. 3–16). With the reduced production of cortisol, normal feedback to the hypothalamus fails, with the result that increased amounts of adrenocorticotropic hormone (ACTH) are produced. This excess production of ACTH stimulates the deficient adrenal gland to produce relatively normal amounts of cortisol—but also stimulates production of abnormally large amounts of estrogen and androgens by the zona reticularis. In this overproduction, a biologic preponderance of androgens causes virilization. These abnormal sex steroids suppress the gonadotropins so that untreated patients never reach puberty and do not menstruate.
Therefore, the treatment of this disorder consists in part of the administration of sufficient exogenous cortisol to suppress ACTH production to normal levels. This in turn should reduce overstimulation of the adrenal so that the adrenal will cease to produce abnormally large amounts of estrogen and androgen. The gonadotropins generally return to normal levels, with consequent feminization of the patient and achievement of menstruation.
The pathogenesis of the salt-losing type of adrenal hyperplasia involves a deficiency in aldosterone production.
Hermaphroditism due to congenital adrenal hyperplasia must be suspected in any infant born with ambiguous or abnormal external genitalia. It is exceedingly important that the diagnosis be made at a very early age if undesirable disturbances of metabolism are to be prevented.
All patients with ambiguous external genitalia should have an appraisal of their chromosomal characteristics. In all instances of female pseudohermaphroditism due to congenital hyperplasia, the chromosomal composition is that of a normal female. A pelvic ultrasound in the newborn to determine the presence of a uterus is very helpful and, if positive, strongly suggests a female infant.
The critical determinations are those of the urinary 17-ketosteroid and serum 17-hydroxyprogesterone levels. If these are elevated, the diagnosis must be either congenital adrenal hyperplasia or tumor. In the newborn, the latter is very rare, but in older children and adults with elevated 17-ketosteroids, the possibility of tumor must be considered. One of the most satisfactory methods of making this different diagnosis is to attempt to suppress the excess androgens by administration of dexamethasone. In an adult or an older child, a suitable test dose of dexamethasone is 1.25 mg/45 kg (100 lb) body weight, given orally for 7 consecutive days. In congenital adrenal hyperplasia, there should be suppression of the urinary 17-ketosteroids on the seventh day of the test to less than 1 mg/24 h; in the presence of tumor, either there will be no effect or the 17-ketosteroid levels will rise.
Determination of urinary dehydroepiandrosterone (DHEA) or serum dehydroepiandrosterone sulfate (DHEAS) levels can also be helpful in differentiating congenital adrenal hyperplasia from an adrenal tumor. Levels in patients with congenital adrenal hyperplasia may be up to double the normal amount, whereas an adrenal tumor is usually associated with levels that are much higher than double the normal level.
Determination of the serum sodium and potassium levels and CO2-combining power is also important to ascertain whether electrolyte balance is seriously disturbed.
The treatment of female hermaphroditism due to congenital adrenal hyperplasia is partly medical and partly surgical. Originally, cortisone was administered; today, it is known that various cortisone derivatives are at least as effective. It is most satisfactory to begin treatment with relatively large doses of hydrocortisone divided in 3 doses orally for 7–10 days to obtain rapid suppression of adrenal activity. In young infants, the initial dose is about 25 mg/d; in older patients, 100 mg/d. After the output of 17-ketosteroids has decreased to a lower level, the dose should be reduced to the minimum amount required to maintain adequate suppression. This requires repeated measurements of plasma 17α-hydroxyprogesterone in order to individualize the dose.
It has been found that even with suppression of the urinary 17-ketosteroids to normal levels, the more sensitive serum 17-hydroxyprogesterone level may still be elevated. It seems difficult and perhaps undesirable to suppress the serum 17-hydroxyprogesterone values to normal because to do so may require doses of hydrocortisone that tend to cause cushingoid symptoms.
In the treatment of newborns with congenital adrenal hyperplasia who have a defect of electrolyte regulation, it is usually necessary to administer sodium chloride in amounts of 4–6 g/d, either orally or parenterally, in addition to cortisone. Furthermore, fludrocortisone acetate usually is required initially. The dose is entirely dependent on the levels of the serum electrolytes, which must be followed serially, but it is generally 0.05–0.1 mg/d.
In addition to the hormone treatment of this disorder, surgical correction of the external genitalia is usually necessary.
During acute illness or other stress, as well as during and after an operation, additional hydrocortisone is indicated to avoid the adrenal insufficiency of stress. Doubling the maintenance dose is usually adequate in such circumstances.
Female Hermaphroditism Without Progressive Masculinization
Females with no adrenal abnormality may have fetal masculinization of the external genitalia with the same anatomic findings as in patients with congenital virilizing adrenal hyperplasia. Unlike patients with adrenogenital syndrome, patients without adrenal abnormality do not have elevated levels of serum steroids or urinary 17-ketosteroids, nor do they show precocious sexual development or the metabolic difficulties associated with adrenal hyperplasia as they grow older. At onset of puberty, normal feminization with menstruation and ovulation may be expected.
The diagnosis of female hermaphroditism not due to adrenal abnormality depends on the demonstration of a 46,XX karyotype and the finding of normal levels of serum steroids or normal levels of 17-ketosteroids in the urine. If fusion of the scrotolabial folds is complete, it is necessary to determine the exact relationship of the urogenital sinus to the urethra and vagina and to demonstrate the presence of a uterus by rectal examination or ultrasonography or endoscopic observation of the cervix. When there is a high degree of masculinization, the differential diagnosis between this condition and true hermaphroditism may be very difficult; an exploratory laparotomy may be required in some cases.
Patients with this problem may be seen because of a variety of conditions.
Maternal ingestion of androgen
Maternal androgenic tumor
Luteoma of pregnancy
Adrenal androgenic tumor
Idiopathic: No identifiable cause.
Special or nonspecific: The same as condition 2 except that it is associated with various somatic anomalies and with mental retardation.
Familial: A very rare anomaly.
Persons with abnormal or ectopic testes may have external genitalia so ambiguous at birth that the true sex is not identifiable (Fig. 3–17). At puberty, these persons tend to become masculinized or feminized depending on factors to be discussed. Thus, the adult habitus of these persons may be typically male, ie, without breasts, or typically female, with good breast development. In some instances, the external genitalia may be indistinguishable from those of a normal female; in others, the clitoris may be enlarged; and in still other instances, there may be fusion of the labia in the midline, resulting in what seems to be a hypospadiac male. A deep or shallow vagina may be present. A cervix, a uterus, and uterine tubes may be developed to varying degrees; however, müllerian structures are often absent. Mesonephric structures may be grossly or microscopically visible. Body hair may be either typically feminine in its distribution and quantity or masculine in distribution and of sufficient quantity as to require plucking or shaving if the person is reared as a female. In a special group, axillary and pubic hair is congenitally absent. Although there is a well-developed uterus in some instances, all patients so far reported have been amenorrheic—in spite of the interesting theoretic possibility of uterine bleeding from endometrium stimulated by estrogen of testicular origin. There is no evidence of adrenal malfunction. In the feminized group, and less frequently in the nonfeminized group, there is a strong familial history of the disorder. Male hermaphrodites reared as females may marry and be well adjusted to their sex role. Others, especially when there has been equivocation regarding sex of rearing in infancy, may be less than attractive as women because of indecisive therapy. Psychiatric studies indicate that the best emotional adjustment comes from directing endocrine, surgical, and psychiatric measures toward improving the person's basic characteristics. Fortunately, this is consonant with the surgical and endocrine possibilities for those reared as females, because current operative techniques can produce more satisfactory feminine than masculine external genitalia. Furthermore, the testes of male hermaphrodites are nonfunctional as far as spermatogenesis is concerned. Only about one-third of male hermaphrodites are suitable for rearing as males.
External genitalia in male hermaphroditism. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
Since about 1970, considerable progress has been made in identifying specific metabolic defects that are etiologically important for the various forms of male hermaphroditism. Details are beyond the scope of this text. Nevertheless, it is important to point out that all cases of male hermaphroditism have a defect in either the biologic action of testosterone or the MIF of the testis. Furthermore, it now seems apparent that nearly all—if not all—of these defects have a genetic or cytogenetic background. The causes and pathogenetic mechanisms of these defects may vary, but the final common pathway is 1 of the 2 problems just mentioned; in the adult a study of the serum gonadotropins and serum steroids, including the intermediate metabolites of testosterone, can often pinpoint a defect in the biosynthesis of testosterone. In other cases, the end-organ action of testosterone may be defective. In children, the defect is sometimes more difficult to determine before gonadotropin levels rise at puberty, but one may suspect a problem by observing abnormally high levels of steroids that act as substrates in the metabolism of testosterone. A working classification of male hermaphroditism is as follows:
Male hermaphroditism due to a central nervous system defect
Abnormal pituitary gonadotropin secretion
No gonadotropin secretion
Male hermaphroditism due to a primary gonadal defect
Identifiable defect in biosynthesis of testosterone
Pregnenolone synthesis defect (lipoid adrenal hyperplasia)
3β-Hydroxysteroid dehydrogenase deficiency
17β-Ketosteroid reductase deficiency
Unidentified defect in androgen effect
Defect in duct regression (Figs. 3–18 and 3–19)
Familial gonadal destruction
Leydig cell agenesis
Bilateral testicular dysgenesis
Male hermaphroditism due to peripheral end-organ defect
Androgen insensitivity syndrome (Fig. 3–20)
Androgen-binding protein deficiency
Unidentified abnormality of peripheral androgen effect
Male hermaphroditism due to Y chromosome defect
Y chromosome mosaicism (asymmetric gonadal differentiation) (Fig. 3–21)
Structurally abnormal Y chromosome
No identifiable Y chromosome
External genitalia in male hermaphroditism. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
Internal genitalia of the patient whose external genitalia are shown in Figure 3–18.
Androgen insensitivity syndrome.
Internal genitalia in asymmetric gonadal differentiation. (Reproduced, with permission, from Jones HW Jr, Scott WW. Hermaphroditism, Genital Anomalies and Related Endocrine Disorders. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1971.)
Differential Diagnosis in Infants with Ambiguous Genitalia
Accurate differential diagnosis is possible in most patients with ambiguous genitalia (Table 3–8). This requires a complex history of the mother's medication use, a complex sex chromosome study, rectal examination for the presence or absence of a uterus, measurement of serum steroid levels, pelvic ultrasonography, and information about other congenital anomalies. The following disorders, however, do not yield to differentiation by the parameters given in Table 3–8: (1) idiopathic masculinization, (2) the "special" forms of female hermaphroditism, (3) 46,XX true hermaphroditism, and, occasionally, (4) the precise type of male hermaphroditism. For these differentiations, laparotomy may be necessary for diagnosis and for therapy.
Table 3–8. Differential Diagnosis of Ambiguous External Genitalia. ||Download (.pdf)
Table 3–8. Differential Diagnosis of Ambiguous External Genitalia.
Special or nonspecific
46,XX; 46,XY; etc
+ or −
XX or other
+ or −
XY or other
45,X; 46,XX; 46,XY; etc
+ or −
XO or other
Treatment of Hermaphroditism
The sex of rearing is much more important than the obvious morphologic signs (external genitalia, hormone dominance, gonadal structure) in forming the gender role. Furthermore, serious psychological consequences may result from changing the sex of rearing after infancy. Therefore, it is seldom proper to advise a change of sex after infancy to conform to the gonadal structure of the external genitalia. Instead, the physician should exert efforts to complete the adjustment of the person to the sex role already assigned. Fortunately, most aberrations of sexual development are discovered in the newborn period or in infancy, when reassignment of sex causes few problems.
Regardless of the time of treatment (and the earlier the better), the surgeon should reconstruct the external genitalia to correspond to the sex of rearing. Any contradictory sex structures that may function to the patient's disadvantage in the future should be eradicated. Specifically, testes should always be removed from male hermaphrodites reared as females, regardless of hormone production. In cases of testicular feminization, orchiectomy is warranted because a variety of tumors may develop in these abnormal testes if they are retained, but the orchiectomy may be delayed until after puberty in this variety of hermaphroditism.
In virilized female hermaphroditism due to adrenal hyperplasia, suppression of adrenal androgen production by the use of cortisone from an early age will result in completely female development. It is no longer necessary to explore the abdomen and the internal genitalia in this well-delineated syndrome. The surgical effort should be confined to reconstruction of the external genitalia along female lines.
Patients with streak gonads or Turner's syndrome, who are invariably reared as females, should be given exogenous estrogen when puberty is expected. Those hermaphrodites reared as females who will not become feminized also require estrogen to promote the development of the female habitus, including the breasts. In patients with a well-developed system, cyclic uterine withdrawal bleeding can be produced even though reproduction is impossible. Estrogen should be started at about age 12 and may be given as conjugated estrogens, 1.5 mg/d orally (or its equivalent). In some patients, after a period of time, this dosage may have to be increased for additional breast development. In patients without ovaries who have uteri and in male hermaphrodites in the same condition, cyclic uterine bleeding can often be induced by the administration of estrogen for 3 weeks of each month. In other instances, this may be inadequate to produce a convincing "menstrual" period; if so, the 3 weeks of estrogen can be followed by 3–4 days of progestin (eg, medroxyprogesterone acetate) orally or a single injection of progesterone. Prolonged estrogen therapy increases the risk of subsequent development of adenocarcinoma of the corpus, so periodic endometrial sampling is mandatory in such patients.
Reconstruction of Female External Genitalia
The details of the operative reconstruction of abnormal external genitalia are beyond the scope of this chapter. However, it should be emphasized that the procedure should be carried out at the earliest age possible so as to enhance the desired psychological, social, and sexual orientation of the patient and to facilitate adjustment by the parents. Sometimes the reconstruction can be done during the neonatal period. In any case, operation should not be delayed beyond the first several months of life. From a technical point of view, early operation is possible in all but the most exceptional circumstances.
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