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SEXUAL
DIFFERENTIATION
Sexual Differentiation
1
Normal and Abnormal Sexual Differentiation
At conception, the embryo has bisexual potential. Each gonad can develop into either a testis or an
ovary. Internal ductal primordia are present for both Mullerian and Wolffian systems and the external
genitalia can either feminize or masculinize. An orderly sequence of embryonic events is required for
normal sexual differentiation of the gonads, internal ductal system. and the external genitalia. The
timetables of normal male and female sexual differentiation (I) are provided to Figs. 16.1 and 16.2. For
recapitulation of the species, this is probably the most important embryologic process. Our genetic
blueprint ensures that sexual differentiation in utero occurs flawlessly time and again such that
abnormalities of sexual differentiation are only rarely noted at birth.
A wide spectrum of intersex disorders has been described (Table 16.1), with separate defects occurring
at nearly every step in the process depicted in Figs. 16.1 and 16.3 (1). These defects result in very
predictable abnormalities of the gonads. internal ductal systems, and external genitalia. When faced
with the clinical dilemma of a child with ambiguous genitalia. the clinician can work backward. By
determining the nature and location of external genitalia, internal ductal systems, and gonads, the
physician can often make an initial differential diagnosis with a minimum of ancillary testing.
Despite the fact that most intersex disorders are associated with some degree of ambiguity and sterility,
the psychosocial considerations involved have potentially far more deleterious ramifications than these
physical changes. Delivering a child with ambiguous genitalia should be considered a medical
emergency. Expeditious evaluation and concomitant counseling, early diagnosis and assignment of
sex-of-rearing, and initiation of treatment during the birthing hospitalization is essential for meeting all of the
emotional and medical needs of these individuals and families. The age of molecular medicine has allowed
for rapid testing and decision making during the first 24 hours of life. Furthermore, prenatal
ultrasound screening can identify at-risk fetuses even before delivery. However. none of this technology can
substitute for the informed and experienced counseling that is necessary in the care of these infants from the
time of identification through attainment of adulthood at the conclusion of puberty (2).
NORMAL SEXUAL DIFFERENTIATION
At the time of conception, the embryo has the potential to develop into either a male or a female. The
gonads can differentiate to the medullary region along testicular lines or in the cortical areas as ovaries.
Primordia for both the Wolffian and Mullerian systems are present from the beginning of sexual
Sexual Differentiation
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differentiation. The Mullerian system will continue to grow and develop unless it is ablated by the action
of Mullerian-inhibiting substance (MIS). The Wolffian system, in contrast. will ultimately require
androgen stimulation for completion of its development. The external genitalia of any fetus can either
masculinize or feminize.
Two principal differences exist between the development of male and female fetuses. First, testicular
determinant genes must be present and active in order for testicular development to begin. In contrast,
ovarian determinant genes need only be present at later stages of ovarian development. In the absence
of testicular determinants and absence of other major ovarian genetic stimuli, ovarian development will
begin. This occurs independently of the genetic sex of the embryo. The second principal difference
between male and female sexual development is that the testis is necessarily an endocrine organ
during sexual differentiation while the developing ovary is relatively endocrine quiescent. In the male
the character of the internal and external genitalia depends on testicular steroid (i.e., testosterone and
dihydrotestosterone [DHT]) and nonsteroid (e.g., MIS) hormone production and secretion. For the
female. internal and external genital development occurs in the absence of ovarian hormones.
Male sexual differentiation
The process of male sexual differentiation m utero is well characterized (see Fig. 16.1). A
cascade of molecular events muse occur to permit development of the testes, the Wolffian
system, and the external genitalia. Genes controlling the major steps in male sexual
differentiation and the ultimate fate of each of these systems in male reproductive
morphogenesis are largely characterized. The initial switch for these events appears to be the
Y -chromosomal SRY gene (3,4). A gathering body of data supports the idea that lust as has
been characterized for invertebrates, a host of genes downstream m expression from SRY
exist and act in concert for gonadal morphogenesis. That other genes exist is supported by the
faces that most 46.XY sex-reversed females do not have SRY mutations. some 46,XX
sex-reversed males do not have SRY present, and nearly all 46,XX true hermaphrodites lack
SRY It appears that the protein produce of SRY is a transcription factor that acts as the initial
switch for turning on a number of other genes (5). The protein products of these genes that
likely act downstream in expression to SRY are required for formation of the testis and perhaps
regulation of internal ductal development. Candidates for such downstream genes include MIS
(6), dosage-sensitive sex-reversal gene (DSS) (7), SOX9 (8,9), and the Wilms' tumor or WT-I
Sexual Differentiation
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gene. The SRY protein has been observed to bind with the MIS promotor. DSS represents a
gene locus on the short arm of the X chromosome which, when duplicated in 46,XY
individuals. is associated with sex reversal. Mice with deletions of WT-1 do not form mesoderm
for gonads and this may represent an upstream rather than a downstream gene in expression
to SRY. Individuals with the 46,XY karyotype and autosomally inherited campomelic dysplasia
present with sex reversal m addition to having a broad spectrum of bone abnormalities (8.9).
The translocation breakpoints of three unrelated patients with this syndrome have all mapped
to 17q24.3-q25.1, a locus referred to as the autosomal sex-reversal locus, SRA1. SOX9 is an
SRY-related gene that has been mapped to this region of 17q. Patients with campomelic
dysplasia harbor mutations in this locus (8,9). It appears that this gene is involved both m
testicular formation and in bone morphogenesis.
Concomitant with the initial genetic signaling for testicular morphogenesis, the primordial germ cells
travel by amoeboid motion from the yolk sac over the dorsal mesentery to the genital ridge. These
primordial germ cells increase m number by mitosis and become rapidly enveloped by the developing
seminiferous tubules. At the base of these tubules. the Sertoli cells appear. They have many functions
and, like the granulosa cells in the ovaries, assume the guardianship of the germ cells. They produce
the first hormone of the testis. MIS. This protein has many functions, including arrest of the
development of the Mullerian system, in part by apoptosis (10). Shortly thereafter, fetal Leydig cells
appear and produce the second set of hormones, the androgens. Steroidogenesis in the fetal Leydig
cells is initially stimulated by human chorionic gonadotropin (hCG) and then by fetal luteinizing hormone (LH).
The androgen receptor-hormone dissociation constant is lower for DHT than for testosterone.
This ensures a longer time spent on the androgen receptor for DHT and significantly greater potency as
compared to testosterone. However, during early pregnancy, the human Wolffian system does not have
activity for the steroidogenic enzyme, 5α-reductase, m contrast to the external genitalia (11).
Testosterone stimulates completion of Wolffian development and DHT is responsible for
masculinization of the external genitalia (13). The effects of MIS and testosterone on their respective
Mullerian and Wolffian systems appear to be local and unilateral. In a set of classic fetal rabbit
experiments, just et al. (13) demonstrated that unilateral removal of a testis from a developing male
rabbit resulted in development of a Mullerian system on that side and a Wolffian system on the side of
the remaining gonad. Similarly, transplantation of a fetal testis from a male to a position adjacent to a
developing ovary in a fetal female rabbit caused regression of the Mullerian system and concomitant
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Sexual Differentiation
stimulation of the Wolffian system on that side. For humans. these same observations have been
demonstrated by natural experiments of nature, that is, the existence of patients with discordant gonads
such as those with 43,X/46,XY gonadal dysgenesis and some true hermaphrodites.
External genital development probably begins by 9 weeks' gestation and is completed during
the early part of the middle trimester. It involves labioscrotal fusion concomitant with placement
of the urethral groove onto the genital tubercle and subsequent elongation for development of
a penis. Testicular descent occurs around 32 weeks gestation and results from development of
the gubernaculum and its developmental "pulling" of the testes toward the scrotum. In addition,
testosterone appears to be a required hormonal stimulus for descent and some believe that
MIS may be involved as well (10).
Female sexual differentiation
Ernbryogenesis for female sexual differentiation has been similarly well characterized (see Fig. 16.2).
While molecular events must mediate growth and development of all female genitalia, it would appear
that these genes are broad-spectrum developmental genes and not, as in the male, step-dependent
loci. Ovarian development begins in the absence of testicular or ovarian determinant genes. Mullerian
development begins and continues unless arrested by MIS, and the external genitalia will feminize in
the absence of androgens. Ovarian function during development appears to be exocrine rather than
endocrine. Hormone production is negligible and insignificant. More importantly, mitosis of germ cells
occurs at a much greater rate in the developing ovary than for the testis. A maximum ovarian
endowment of nearly 6 million germ cells is attained by 20 weeks' gestation. Unlike the male in whom
germ cell replenishment occurs throughout adult life, the germ cell mass in females undergoes only
depletion, beginning during fetal life. The most critical step for ovarian morphogenesis that appears to
depend on ovarian determinant genes is preservation of ,,arm cells within the developing ovary. This
requires the surrounding of the primordial germ cells by a primitive granulosa cell mantle forming a
primitive follicle and she initiation and arrest of meiosis I at prophase (14). These required ovarian
determinant genes are located on both arms of the X chromosome and the autosomes as well. Fetuses
with the 45,X karyotype of Turners syndrome have as many germ cells at midgestation as do 46,XX
fetuses. Most of their germ cells are incompletely surrounded by- the granulosa cell mantle and some
have been observed to be physically herniating through these defects of the follicular wall (14). It is also
possible that atresia and germ cell loss may be accelerated to them by the lack of sufficient granulosa
cell-produced meiosis inhibitor(s). Sisters with the 46,XX karyotype who are concordant for gonadal
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Sexual Differentiation
dysgenesis have been repeatedly reported, suggesting that other genes important to the preservation
of the germ cell mass exist upstream or downstream in expression to the X-located ovarian
determinants (2).
In summary once the primordial germ cells arrive at the undifferentiated gonadal ridge, male sexual
development requires 1) the presence and expression of SRY and related testicular genes. 2) the
presence of bilateral MIS production. 3) the presence of bilateral testicular testosterone and DHT
production, and 4) competent androgen receptors to translate the hormone signal to the end-organ
effect of masculinization. Each of the major genes for these steps has been cloned and sequenced,
and mutations producing predictable abnormalities have been found. These genes include SPY, MIS,
the MIS receptor gene, all of the enzyme genes in steroid biosynthesis, and the androgen receptor (AR)
gene. In contrast, once the primordial germ cells arrive at the genital ridge, female sexual differentiation
requires only ovarian determinant gene action at a later time in development. Not one of the genes
regulating female sexual differentiation has been identified. While male sexual differentiation appears to
be a very dependent developmental event, female sexual differentiation occurs relatively
independently. Nature has seemingly put into place a fail-safe mechanism such that failures of male
sexual differentiation default to a female phenotype.
CLASSIFICATION OF PATIENTS WITH ABNORMALITIES OF
SEXUAL DIFFERENTIATION
Abnormalities can occur at each of the steps involved in normal sexual differentiation (see Figs. 16.1
and 162) (1). Each of these abnormalities may present with predictable alterations of the gonads,
internal and eternal genitalia, and pubertal development. An understanding of the molecular nature of
many of these defects has broadened the spectrum of presenting phenotypic changes identified for
them.
Two classifications of patients having abnormalities of sexual differentiation are outlined in Table 16.1
(1). The standard classification system uses the terms pseudohermaphrodite and hermaphrodite. A
pseudohermaphrodite is an individual whose external genitalia is contradictory to the karyotype. For example.
a male pseudohermaphrodite is a 46,XY individual whose external genitalia is
undermasculinized. A female pseudohermaphrodite has a 46.XX karyotype and masculinized external
genitalia. A true hermaphrodite is an individual possessing both ovarian and testicular tissue and
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manifesting varying degrees of genital ambiguity. Gonadal tissue must demonstrate ovarian follicles
and seminiferous tubules.
Another classification presented in Table 16.1 demonstrates a degree of overlap between some of
these syndromes and is based purely on the karyotype of the patient (1). The following summary of
these abnormalities follows this newer classification.
Deletion syndromes
Patients with Turner's syndrome present with an abnormality of sexual differentiation because of a sex
chromosomal deletion syndrome that results in abnormal gonadal development (see Table 16.1). A
normal endowment of oocytes is initially present in the developing ovaries of these patients (14). However,
they are missing X-chromosomal material with important ovarian determinant genes, which are
necessary for preservation of ovarian follicles. These genes are probably located on both arms of both
X chromosomes, as well as the autosomes. Privation of any or all ovarian determinant genes appears
to be associated with incomplete formation of the primitive follicular mantle of granulosa cells that
surrounds the oocyte (14). Unarrested meiotic activity results and the oocytes appear to be able to
herniate through the poorly formed granulosa cell membrane; ovarian streaks relatively devoid of
follicles are formed. The classic karyotype associated with Turners syndrome is a single 45,X cell line.
Most patients with Turner's syndrome, however, have mosaicism with a 45,X cell line accompanied by
another cell line in which the X or Y chromosomes may be structurally normal or abnormal (i.e.,
45,X/46,XY; 45,X/46,XX; or 45,X/46,X,1[Xq]) (2). The most common form of mosaicism involves the
45,X/46,XY karyotype (2).
Patients with Turner's syndrome classically present with bilateral streak gonads and normal Mullerian
and external genital development. Turner's stigmata and associated cardiovascular and renal
abnormalities (i.e., coarctation or dilatation of the aorta and horseshoe kidney) variably occur for them.
A wide spectrum of phenotypic findings for Turner's stigmata and genitalia is associated with the
45,X/46,XY karyotype (2,15,16). Gonadal development for these individuals may range from bilateral
streaks through unilateral streak/contralateral immature testis to bilateral immature testes. The testes
may be located infra-abdominally or within the labioscrotum. Mixed gonadal dysgenesis or asymmetric
gonadal dysgenesis refers to those 45,X/46.XY patients who have " mixed" gonadal structures: a
unilateral streak gonad and contralateral testis (15,16). These patients are Jostian experiments of
nature in that the accessory genital structures depend on the nature of the accompanying gonad.
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Streak gonads are accompanied by a Mullerian system. Immature testes are associated with Wolffian
derivatives. The spectrum of phenotypic findings for these patients also includes the external genitalia.
For patients with bilateral streak gonads, their external genitalia is unquestionably female (15,16).
Varying degrees of external genital masculinization occur in utero and at puberty for those patients who
harbor an XY cell line and at least one testis. Minimal masculinization (i.e., clitoromegaly) usually
occurs for patients with a unilateral infra-abdominal testis. Ambiguity is usually found in individuals with
a unilateral descended testis. A normal male phenotype may be associated with bilateral descended
testes. These latter males are usually diagnosed during an infertility evaluation for azoospermia. Recent
studies suggested that a number of 45.X/46,XY males not only have a normal phenotype, but also
may have normal spermatogenesis (17). Gonadal tumor formation has been reported ` to occur in 15"/o
to 25°/ of patients with -45.X,t46,XY ;gonadal dysgenesis and infra-abdominal ,gonads/streaks if they
are left in place (18,19). The most common tumor is a gonadoblastoma. Any germ cell tumor. commonly
dysgerminoma and rarely others including yolk sac tumors and choriocarcinoma. may be found
(2.20). The risk for tumor formation in individuals with a normal male phenotype and descended
bilateral testes, however, appears to be very small and potentially nonexistent (17). All patients with
45,Xi46,XY gonadal dysgenesis need gonadal extirpation, irrespective of the age at diagnosis. except
for the few phenotypically normal men with apparently bilaterally descended testes. This latter group
may be followed. Some individuals suggest testicular biopsy after puberty. Patients with ambiguity at
birth and given a female sex-of-rearing should have corrective surgery as early as possible and
preferably during the birthing hospitalization. Except for the phenotypically normal males with bilateral
testes. a male sex-of-rearing should be avoided for these patients. Penile reconstructive surgery is
often extensive and rarely satisfactory, the patients are infertile as men, and the risk for gonadal
neoplasia exists.
46,XY Abnormalities of sexual differentiation
Abnormalities of sexual differentiation can occur to individuals with single 46,XY cell lines and be
manifested with varying degrees of undermasculinization (see Table 16.1). Abnormalities resulting in
one of these syndromes have been identified at each of the steps for normal male sexual differentiation
(see Fig. 16.1).
This syndrome results from an error that occurs at one of the earliest steps in male gonadal
morphogenesis (see Fig. 16.1) (21). It was previously theorized for some of these patients that the
germ cells failed to migrate to the genital ridge. Recent studies indicated that for others, mutations may
occur in one of the genes involved in testicular differentiation (23,24). Approximately 15°/u of women
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with Swyer’s syndrome harbor mutations in SPY (23-25). As most patients with Swyer's syndrome
appear to have normal SPY sequences and some present with an X-linked form, mutations must also
occur in autosomal or X-chromosomal genes that are downstream to SRY in the cascade of molecular
events (2,22). No matter what this early defect is, the 46,XY germ cells are not directed appropriately to
begin testicular differentiation. Streak gonads form. The absence of MIS production and androgen
biosynthesis is associated with the development of a normal M611erian system and female external
genitalia.
46,XY Gonadal dysgenesis (Swyer's syndrome) is usually identified because of delayed pubertal
development (2). These women are otherwise phenotypically normal. They are usually taller than their
peers because of the presence of probable Y-chromosomal statural genes. Also their epiphyses remain
open longer during their adolescent years in the absence of sex steroid production. Gonadotropin levels
are elevated and subsequent chromosomal analysts demonstrates a 46.XY cell line. These women are
at the highest risk (i.e., 25%u-35%) for genital ridge tumors of any patients with dysgenetic testes and
Y-cell lines (?,18). Rarely, these germ cell tumors are capable of steroid biosynthesis and may produce
varying amounts of estrogen or testosterone. This has been reported for not only patients with Swyer's
syndrome. but also others with -h.X/46,XY gonadal dysgenesis (26,27). It is for this reason that such
patients have signs of masculinization or feminization at puberty. Gonadal extirpation is necessary at
the time the diagnosis is made.
Mullerian inhibiting substance deficiency
There are 46,XY individuals to whom germ cell migration occurs and SRY is expressed. Testicular
development is initiated. However, there appears to be absent MIS production or a lack of its effect
(see Fig. 16.1) (10:38.29). Testicular development and androgen production are otherwise normal. Patients
unable to produce or express MIS are usually identified as phenotypic males with unilateral or
bilateral cryptorchidism. or an inguinal hernia (2&). At the time of surgery. the hernia or cryptorchid
testes are found to contain Mullerian elements. Also called the persistent Mullerian duct syndrome, this
disorder was previously hypothesized to result from mutations in either the MIS gene or the MIS
receptor gene (28,29). The initial molecular studies of such males uncovered Mutations to the MIS
gene (29,30). No doubt, a host of mutations that either prevent transcription or produce a protein with
abnormal or absent function will be identified. The MIS receptor gene recently was cloned and a
mutation identified for a male with this syndrome who was found to have normal MIS gene sequences.
For these 46,XY patients, testicular development arid its steroid biosynthesis are normal. Mullerian
regression does not occur because an abnormal MIS or MIS receptor protein is produced. Either or
Sexual Differentiation
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both of these otherwise normal testes may be infra-abdominal or inguinal in location and associated
with bilateral fallopian tubes, a uterus, and an upper vagina. Androgen biosynthesis appears normal for
these individuals both in utero and at puberty, with normal penile development resulting. Sterility may
result from variable development of the vas deferens at the time of persistent Mullerian development or
unavoidable damage to the Wolffian system during, surgical removal of the Mullerian system.
Agonadia (testicular regression syndrome)
Phenotypic female patients are rarely identified at puberty arid, like women with Swyer s syndrome, do
not go through the normal pubertal changes. Similarly. they have elevated gonadotropins levels and a
46.XY karyotype. Unlike the patients with 46.XY gonadal dysgenesis, however, these women have a
blind vaginal pouch and both gonads as well as all internal genitalia are absent. These 46,XY
individuals typically have germ cell migration. normal expression of testicular determining factor (TDF)
and testicular development, and normal MIS production and Mullerian regression. It is hypothesized
that a defect in sexual differentiation occurs after the elaboration of MIS and before androgen
biosynthesis (see Fig. 16.1). An environmental insult or potentially a vascular accident may be
associated with the disappearance or regression of the developing testes after the Mullerian system
has regressed. The Wolffian system is not stimulated to continue development because androgens are
not subsequently produced. These patients have also been given the diagnosis of "the empty pelvis
syndrome" because of the absence of all internal genitalia, as demonstrated by laparoscopy. A
spectrum of this disorder exists and variable phenotypes have been described. Testicular regression
can occur at any time during sexual differentiation (31,32). Internal and eternal genital development
depends on the precise timing of the testicular regression.
Defects in androgen biosynthesis
Patients with the 46,XY karyotype and steroid enzyme deficiency have normal expression of TDF
genes and normal MIS production. As a result, these individuals develop normal appearing testes and
Mullerian regression appropriately occurs. However, as hCG stimulates steroidogenesis within the feral
Leydig cells. sub optimal levels of testosterone or DHT, or both, are produced because of an enzyme
deficiency at one of the steps in androgen biosynthesis (Figs. 16.1 and 16.3). Normally, Wolffian
system development is stimulated by testosterone and not DHT The 5a-reduction of testosterone to
DHT is absent m the Wolffian primordia during early embryogenesis. External genital development,
however, depends on the presence of 5a-reductase activity and androgen receptor stimulation by
DEFT Deficiencies of both testosterone and DHT adversely affect both Wolffian and external genital deSexual Differentiation
10
velopment. Deficiency of only DHT adversely affects only external genital development. External genital
ambiguity to a 46,XY individual may include under masculinization of the genital tubercle or scrotum
and abnormal testicular descent. Many of these patients have a blind vaginal pouch that represents the
remnant of the under stimulated prostatic utricle.
Some of these 46,XY patients with enzyme deficiencies produce negligible. if any, androgens, and
therefore, eternal genital masculinization is totally absent. These are individuals with deficiencies of
enzymes necessary for the very early steps of androgen biosynthesis such as cholesterol side-chain
cleavage enzyme deficiency (33-38). 17α - hydroxylase deficiency (38-44), and 17,20-lyase deficiency
(see Fig. 16.3) (45-50). Partial defects can exist for these enzyme-deficient states and produce under
masculinization and ambiguity. A number of other 46,XY patients with enzyme-deficient states predictably
have minimal androgen production associated with varying degrees of testicular descent and
genital ambiguity. For example, 3β-hyroxysteroid dehydrogenase deficiency results in the production or
accumulation of the weak △5 androgens (i.e., 5 dehydroepiandrosterone (DHEA) and in tremendous
under masculinization and external genital ambiguity (see Fig. 16.3) (51-60). Patients with
17-hydroxysteroid dehydrogenase (17-ketoreductase) deficiency produce and accumulate both weak
△5 and weak △4 androgens (i.e., DHEA and androstenedione),and similarly present with tremendous
under masculinization and ambiguity (61-66). Sex-of-rearing has most commonly been female for these
patients because of absent or minimal masculinization associated with such high blocks in the steroid
pathway. In contradistinction, testosterone production may be normal or high for patients with
5α-reductase deficiency, whose block is at the end of the steroid pathway (i.e. the enzyme chat
converts testosterone to DHT) (see Fig. 163) (67-79). Testosterone stimulates the same androgen
receptor as does DHT However, its dissociation constant is much higher (i.e., is moves away from the
receptor much more quickly). The high levels of testosterone produced in patients with 5a-reductase
deficiency allow for normal Wolffian development. Despite minimal DHT production, these high
testosterone levels stimulate external genital development. Although ambiguous, these infants are
more masculinized than are infants with high steroid pathway blocks. Most of these patients have also
been given a female sex-of-rearing at birth.
Patients with enzyme deficiencies may have associated abnormalities that appear after the newborn
period. Some of these conditions have been associated with adrenal failure and others with
hypertension. Adrenal failure is an almost certain occurrence for the untreated newborn with cholesterol
side-chain cleavage enzyme deficiency. It is very likely to occur, although with less consistency, for the
untreated infant with 3β-hyroxysteroid dehydrogenase deficiency. Hypokalemic hypertension occurs in
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Sexual Differentiation
17α-hydroxylase-deficient patients because of accumulation of mineralocorticoid precursors to
17α-hydroxyprogesterone (see Fig. 16.3). The risk of tumor formation exists for all of these 46,XY
patterns with enzyme deficiencies, but is probably no higher for them than it is for otherwise normal
males having only cryptorchidism. Finally, varying degrees of masculinization or feminization, or both,
may occur at puberty for 46.XY patients with androgen-deficient states whose gonads have been left in
place. The pubertal phenotype best characterized is that of 5a-reductase deficiency. A very large
kindred has been studied and reported from the Dominican Republic (77.78). Affected individuals
became known as "guevedoces” meaning penis at 12 (years of age). Characteristically given the
female sex-of-rearing at birth, these patients had tremendous masculinization at puberty and many
developed a male gender identity and changed sex-of-rearing. While they are unable to convert
testosterone to DHT, the high and sustained testosterone levels at the androgen receptor are seemingly
sufficient to stimulate the receptor-mediated masculinization. Additionally, such masculinization at
puberty may be explained by the fact that two genes encode 5n-reductase (84-83). It is the
5α-reductase 2 gene that is responsible for genital production of DHT. Mutations in this gene have
been found to be associated with the intersex syndrome. It has been suggested that in these individuals
the 5a-reductase 1 enzyme may convert testosterone to DHT as an alternative pathway and at other
locations mediating masculinization. Another associated abnormality of some androgen enzyme
deficiencies includes gynecomastia. This occurs in 17 hydroxysteroid dehydrogenase deficiency
because of conversion of weak androgen precursors (i.e., androstenedione) to estrogens (i.e., estrone).
Patients with 17α-hydroxylase deficiency also may develop gynecomastia. Breasts develop for them
because of the lack of the usual androgenic inhibition of breast maturation.
It should be remembered that because these same enzyme deficiencies are usually the result of
autosomal recessive mutations, they also occur in 46,XX individuals (49,50). Since normal female
sexual differentiation is not dependent on in utero sex steroid production, these patients have a normal
female phenotype at birth. The abnormality of sexual differentiation becomes apparent at birth only for
newborns with an adrenal crisis or salt wasting, and cholesterol side-chain cleavage enzyme or 3βhyroxysteroid dehydrogenase deficiency. It may become apparent at puberty for patients with an
enzyme deficiency that precludes estrogen production (i.e., commonly 17a-hydroxylase deficiency).
Diagnosis may also become apparent because of the development of hypokalemic hypertension in the
latter patients.
All of the steroid enzyme genes have been cloned. As of this writing. gene mutations have not been
identified within the cholesterol side-chain cleavage gene for deficient patients. Two genes have been
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Sexual Differentiation
identified to encode 3β-hyroxysteroid dehydrogenase: type I and type II genes. Mutations have been
identified to the 3β-hyroxysteroid dehydrogenase type ll gene for patients with this deficiency (38). A
single gene chat mediates both 17α-hydroxylation and 17,20-lyase functions has been cloned and
sequenced (84). Mutations that seemingly cause either 17α-hydroxylase deficiency or 17,20-(lyase
deficiency have been identified within this single gene (38.85-88). Dependent on the type of mutation,
these patients produce varying amounts of testosterone and DHT The 17-hydroxysteroid
dehydrogenase gene has been cloned. However. gene defects have not yet been identified to explain
the phenotypic findings in patients with this deficiency state. Two genes exist with 5α-reductase activity:
5α-reductase 1 gene and 5a-reductase 2 gene (83.89-93). The 5α-reductase 2 gene codes for the
major isoenzyme present in genital skin` Deletion of this gene has been found in males with
5α-reductase deficiency and a number of unique point mutations have been identified in other kindred
(83,92,93).
Androgen receptor defects (androgen insensitivity syndrome)
Patients with the 46,XY karyotype who have androgen resistance syndromes have normal expression
of TDF genes and testicular development. normal MIS production and Mullerian regression, and normal
testicular androgen biosynthesis (94,95). Mutations of the X-located AR gene cause an absent or
abnormally functioning androgen receptor and prevent the end-organ effect of the androgen signal for
masculinization to utero and at puberty This abnormality of sexual differentiation occurs at the final step
in male sexual morphogenesis (,see Fig. 16.1). The syndromes of androgen resistance have presented
with a wide spectrum of phenotypic findings in 46.XY individuals (Fig. 16.4). Heterozygote carriers
(46,XX individuals) may also have findings related to androgen resistance, although very subtle (i.e.,
sparse pubic and axillary hair).
Complete androgen insensitivity syndrome (CAIS) has been associated with absent masculinization in
utero, the presence of a vaginal pouch, and spontaneous breast development at puberty associated
with absent or diminished pubic hair (94,95). All women have androgen receptors in breast tissue, and
during normal pubertal development, ovarian and adrenal androgens will limit their ultimate breast size.
However, patients `with CAPS often develop very abundant breasts, characteristically composed of
more adipose than glandular tissue, because of their own resistance to androgens. Additionally, some
of their testicular androgens ate converted to estrogens to aid to this breast development. The
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presence of Y-chromosomal statural genes results in a tall linear body habitus for CAPS patients. Many
of these women have modeling careers because of the abundant breasts and tall stature.
Sexual Differentiation
Patients with CAIS have normal-appearing testes, except for incomplete or absent spermatogenesis.
The testes may be intra-abdominal, inguinal, or labioscrotal in location (9496). A relatively high
incidence of neoplasia, ranging from 2%to 22%, is reported to occur in these testes if left in place after
puberty. The occurrence of these rumors, usually seminomas, is probably more related to their
cryptorchid location than to a molecular predisposition (96,97). Testicular rumors rarely develop prior to
puberty. Because of the abundant breast development that these patients experience spontaneously,
the testes are best left in place until such development is completed unless the testes are located in the
inguinal or labioscrotal regions. It has been said that patients with androgen insensitivity syndrome
whose testes are removed prior to puberty do not achieve the same abundant breast development from
exogenously given steroids. In retrospect. this information comes from patients who likely had inguinal
or labioscrotal gonads removed prepubertally and incomplete forms of androgen insensitivity syndrome
associated with some degree of androgen effect. They. therefore, do nor have complete androgen
resistance and after gonadectomy, their adrenal androgens limit breast development during exogenous
estrogen supplementation similar to normal women.
Incomplete androgen insensitivity (IAIS) is far cater than the complete form. Except for the presence of
partial fusion of the labioscrotal folds and usually some degrees of clitoromegaly at birth, this syndrome
is very similar to the complete form and infants have usually been given a female sex-of-rearing
(9.1,95). Pubic hair and clitoral enlargement occur at puberty as does breast development, although
typically not as abundant as in CAIS. A vaginal pouch is usually present and associated with
underdevelopment of the Wolffian system.
The spectrum of androgen insensitivity includes phenotypic males as well (see Fig. 16.-1). Reifenstein’s
syndrome is presently used to describe most of the syndromes that previously
included Reifenstein’s, Lubbs, and Gilbert-Dreyfus syndromes. The spectrum of phenotypic findings for
these syndromes is extremely variable and wide ranging. Usually these syndromes
present with a male phenotype. However, both phenotypic male and females have been affected in the
same sibship. The spectrum ranges from undermasculinized men with varying
degrees of hypospadias to near-normal phenotypic men with gynecomastia or infertility Axillary and
pubic hair is often normal for these individuals, while chest and facial hair is minimal. Recent evidence
14
suggests that the spectrum for androgen-resistant syndromes also includes the infertile,
normal-appearing male (i.e., normal male phenotype associated with azoospermia),as well as the
under virilized fertile male (94,96,98).The latter syndrome is described in males with a normal but small
Sexual Differentiation
penis, decreased male-pattern hair, gynecomastia, and normal semen (99). Furthermore, the spectrum
of androgen insensitivity syndrome may include heterozygote carriers often AR gene mutation.
Females with the -46,XX karyotype who have a normal gene on one X chromosome and a mutant gene
on the other chromosome may present with decreased axillary and pubic hair as their only finding (see
Fig. 16.-1.)
Previous studies of these individuals were limited to androgen receptor analysis. Androgen receptor
studies demonstrated absent androgen binding, qualitatively abnormal androgen binding, decreased
androgen binding, and normal androgen binding for patients with these various syndromes (94,95).
Most patients with CAIS have absent androgen binding. Nearly 50% of all families with androgen
insensitivity syndrome have qualitatively abnormal androgen binding. Only 10% of androgen-resistant
states present with normal androgen binding.
Molecular studies of the AR gene have been extremely informative to our understanding of this
spectrum of syndromes (94,95,100-1Od).The AR gene has eight exons. Mutations have been isolated
to all eight exons, and except for two unrelated families with the same point mutations. all of the
mutations identified to date have been unique to a single family (94,95,104).This makes simple
screening for mutations impossible. Large gene deletions are rare. Point mutations, however, comprise
95% of mutations identified. These mutations are largely located in the steroid binding domain of the
gene (i.e., the region that codes for the portion of the receptor responsible for binding to the androgen
molecule) and rarely in the DNA binding domain. Absent or nearly absent androgen binding has been
associated with large-scale mutations and single nucleotide substitutions in the coding sequences.
Patients with normal androgen binding have the most consistent DNA findings because their AR gene
mutations are all located in the DNA binding domain. For these patients the results of androgen
receptor studies are normal because the steroid binding domain is normal and androgens can bind to
the receptor. However, the androgen-androgen receptor complex cannot bind to the DNA to elicit a
response. Several mutations have been identified in patients with qualitatively abnormal androgen
binding and chose with decreased androgen binding.
Non-endocrine genital ambiguity
Isolated defects of the genitalia that do not appear to be related to interruption of the usual mechanisms
15
for sexual differentiation rarely occur. Rather, these defects demonstrate isolated absence of one of the
units of the reproductive system (e.g., absence of gonads or absence of penis), disorganized growth of
Sexual Differentiation
one of these units (e.g. duplicate phallus, epispadias, and cloaca] formation), or abnormal placement
(e.g., scrotum located above the phallus) in the 46,XY infant. Environmental insults, major
chromosomal defects, and developmental gene abnormalities have all been hypothesized as potential
etiologies. Amniotic band syndrome could potentially cause isolated absence of the penis. Congenital
rubella, for example. has been associated with 46,XY genital ambiguity. This suggests that viral infection
in utero may cause significant disorganization of testicular or external genital growth. Autosomal
anomalies also have been associated with numerous somatic abnormalities including the external
genitalia. D/G Chromosome groups are most commonly involved. In particular, deletion of the long arm
of chromosome 13 (e.g., 46,XYdel[13][q]) produces the most consistent eternal genital abnormalities
(Fig. 16.5).
True hermaphroditism
The karyotypes of true hermaphrodite patients are usually 46,XX, less frequently 46,XX/46,XY, and
rarely 46,XY (see Table 16.1) (105.106). The latter two of these groups of patients present with similar
findings, in that bilateral gonadal hermaphroditism is most frequent: unilateral ovary and contralateral
testes (106). Patients with 46,XX/46,XY true hermaphroditism are chimeras: the presence of two or
more cell lines originating from different embryos. Several mechanisms have been proposed to explain
the presence of the two separate 46,XX and 46,XY cell lines m these individuals: 1) whole body fusion
of separate 46.XX and 46,XY fetuses, and 2) fertilization of the secondary oocyte or the ovum concurrently
with fertilization of the first or second polar body-, respectively (107). The opposing nature of the
gonads (i.e., ovary and testis) is understandable given the opposing nature of 46,XX and 46,XY cell
lines. Because the 46,XY true hermaphrodites present with phenotypes similar to those of the
46,XX/46.XY true hermaphrodites, it has been suggested that they may well have undetected 46,XX
cell lines. Recently, one 46,XY true hermaphrodite was described with a mutation in SRY of gonad
only: a somatic cell mutation rather than a germ line mutation (108). While the diagnosis for this patient
is questionable and the second gonad was not described, it would seem possible that an otherwise
normal male with an SPY mutation in one gonad could result in the development of ovotestis on that
side only, suboptimal total androgen production. and ambiguous genitalia.
The 46,XK true hermaphrodites usually present with unilateral gonadal hermaphroditism: a unilateral
ovotestis with contralateral ovary- or testis (106). It has been proposed that these patients may have
16
gene mutations in one of the TDF genes. Unlike the 46,XX~sex-reversed males, interchange of Y material
onto the X chromosome is the exception rather than the rule for the 46,XX true hermaphrodite
Sexual Differentiation
(109.110). In fact, very few of these true hermaphrodites harbor Y-DNA and the description of these
patients is subject to skepticism. The fact that most, if not all, of them have testicular tissue in the
absence of the SRY gene is further evidence that other genes intimately involved in testicular
morphogenesis must exist.
The ovarian component of true hermaphrodite gonads is typically better developed histologically and is
endocrinologicallv and exocrinologically more functional than is the testicular component (106). The
location of the testes and ovotestes varies for these patients and may be scrotal. inguinal, or
abdominal. The nature of the internal ductal systems (i.e.. Mullerian versus Wolffian) depends on the
associated gonad and its degree of differentiation. Most true hermaphrodites have at least a unilateral
Mullerian system accompanying an ovary or ovotestes, which, if left intact. will function at puberty. The
Wolffian system may accompany a testis or ovotestis and occasionally is on the same side as the
Mullerian system. The development of Mullerian and Wolffian derivatives on the side of an ovotestis
depends on the amount of MIS and testosterone produced. The external genitalia are ambiguous and
usually markedly under masculinized because of suboptimal testosterone production. In previous years,
these infants were reared as males because the masculinization, although poor, was often associated
with a descended gonad. However, pubertal masculinization secondary to endogenously produced
androgens or exogenously given testosterone was also poor and often resulted in gender identity
problems. In tact, true hermaphrodites diagnosed aver puberty demonstrate more feminization than
virilization. Breast development is common. Cyclic menstruation is usual. While only a few normal germ
cells are identified in the testicular component of these gonads. numerous primordial follicles usually
are present. The ovarian exocrine activity is often associated with normal gametogenesis. At least Six
true hermaphrodites raised as females have achieved pregnancy and have been reported in the world
literature. As a result, the infant diagnosed at birth with true hermaphroditism should be raised as a
female and every effort should be made to preserve ovarian and Mullerian systems if normal.
46,XX abnormalities of sexual differentiation
A number of disorders of sexual differentiation occur in individuals .,with a normal 46,XX karyotype (see
Table 16.1). For these patients, varying degrees of masculinization occur, ranging from minimal clitoral
enlargement to a normal male phenotype. Such abnormalities are the result of the superimposition of
endogenously produced or exogenously given androgens on normal female sexual differentiation. In
17
some of these patients the anomalous expression of TDF genes and subsequent testicular
development result in a male phenotype that is contradictory to the 46,XX karyotype.
Sexual Differentiation
True hermaphroditism
As has been previously described, true hermaphrodites usually have a normal 46,XX karyotype. Almost
all of these patients studied to date were devoid critic SKY gene or other Y-chromosomal DNA
sequences studied (111,113). This provides further evidence that genes other than the SPY gene must
be involved in testicular morphogenesis. The end result of anomalous expression of these yet to be
identified testicular determinants is the organization of undifferentiated genital ridges along both
testicular and ovarian lines with suboptimal testicular formation.
Sex-reversed male
46,XX Sex reversal is an intersex disorder in which male sexual differentiation occurs in an individual
with a contradictory 46,XX karyotype. The X and Y chromosomes normally pair during meiosis, and
interchange of this material rarely occurs. The presence of Y DNA sequences of varying lengths
translocated to the X chromosome appears to be the most common cause of 46,XX sec reversal. At
least 60% of these patients have SRY present (110,113). The undifferentiated genital ridge develops
along testicular lines and testicular endocrine function is more normal for these males than it is for the
46.XX true hermaphrodites (114-116). Accessory structures are Wolffian and external male genital
development is complete or nearly so. However, subsequent spermatogenesis is absent. During the Y
to X chromosome interchange, only SKY was translocated and not the other Y -located genes
controlling sperm production. Phenotypically. these men are unquestionably stale and the diagnosis is
usually nor made until after puberty or at the time of infertility evaluation because secondary sexual
characteristic abnormalities may be subtle. The adult testes are usually small and firm and occasionally
cryptorchid. The penis may be small with hypospadias present in fewer than 10% of these men. Pubic
hair may have a female distribution.
Congenital adrenal hyperplasia
The most common and potentially serious abnormality of sexual differentiation is manifested in the
46,XX individual with an enzyme defect for cortisol biosynthesis (38, I 17). The most frequent of these
deficiencies is 21-hydroxylase (21-OH) deficiency. with an incidence in Europe and the United States
ranging between 1 in 5000 and 1 in 15,000 population (I 1 7). The greatest incidence is noted in the
Alaskan Yupik Eskimos. at 1 in 684 (117). Less commonly 11β-hydroxylase, and 3β-hydroxysteroid
18
dehydrogenase deficiencies are reported. For patients with CAH, cortisol production and secretion are
limited or prevented. Chronic cortisol deficiency results in a chronic state of adrenocoticotropin
Sexual Differentiation
hormone (ACTH) overproduction and secretion. because of the negative feedback control between
cortisol and ACTH production and secretion (117-120). Constant adrenal stimulation results in an
accumulation of cortisol precursors and androgens (see Fig. 16.3). The fetal adrenal begins to function
by the third month of development. By the time adrenal androgen levels are significantly elevated. the
ovaries are undergoing normal exocrine activity: the Wolffian system has already regressed, and the
Mullerian system is completing its development. High fetal adrenal androgen levels adversely influence
development of the external genitalia in the female fetus but not the otherwise normally developing
male fetus with CAH. Labioscrotal fusion begins posteriorly to anteriorly, covering over the vaginal
vestibule and forming a urogenital sinus (Fig. 16.6). The extent of virilization of the 46,XX fetus is
variable. even within the same family, and probably depends on the androgen levels achieved and the
embryologic time of exposure. A spectrum of anatomic changes may be present, ranging From minimal
clitoromegaly and mild labioscrotal fusion, through varying degrees of hypospadias (117-1?0). In
patients with extreme androgen overproduction, marked clitoral hypertrophy- occurs. In some the
urethral orifice is displaced onto the genital tubercle and the clitoris looks like a normal penis. Any newborn
identified with bilateral undescended gonads associated either with a normal-appearing penis or
with varying degrees of penile underdevelopment must be considered to have CAH until proved
otherwise. In contradistinction, the ambiguous infant with one or two gonads in their respective
labioscrotal pouches does not have CAH. While both -46.XX and -46.XY infants may be affected with
CAH. only the 46,XX CAH infants swill present with ambiguity Normal ovaries may herniate, but do not
descend into the labioscrotum. Descended gonads have at least some androgen producing testicular
elements present and when accompanied by ambiguity denote one of the other forms of intersex.
Both 46,XX and 46,XY patients who are diagnosed with CAH and untreated will continue to masculinize
after birth. The excess androgens produced will be converted to estrogens and accelerate childhood
growth and cause premature epiphyseal fusion. While tall as children, these individuals will be short
adults. Treated CAH patients are potentially fertile. Skin hyperpigmentation is commonly- associated
with these defects and the degree of pigmentation may reflect the severity of the enzyme deficiency.
The negative feedback stimulation of the hypothalamus and pituitary by cortisol deficiency, produces
increased levels of pro-opiomelanocortin. This precursor peptide is subsequently split into the ACTH
and β-lipoprotein fragments. α-Melanocyte-stimulating hormone (MSH) is a by-product of the ACTH
19
intermediate and (3-MSH is a (3-lipotropin subfragment. Levels of MSH are equally as high as are
those of ACTH in patients with cortisol deficiency, and hyperpigmentation results.
Two forms of classic 21-OH deficiency exist: salt-wasting and simple virilization (117-120). Non-classic
CAH also occurs. Both types of classic 21-OH deficiency are manifested at birth or in the newborn. In
Sexual Differentiation
females, classic CAH results in ambiguity of the eternal genitalia. In males, external genital
development is normal. However, pubertal-type changes occur during early childhood. Seventy-five
percent of classic 21-OH deficiency patients have the salt-wasting varieties. These infants develop
hypernatremia. hyperkalemia, inappropriately high urinary sodium concentrations, low serum and
urinary aldosterone levels, and a resultant high plasma reran activity (PRA) at approximately 7 to 10
days. if untreated. The practitioner usually has a grace period during these first 2 weeks of life to make
the diagnosis before a catastrophic event swill occur. Male infants with salt-wasting CAH are snore
likely to be undiagnosed, and unsuspectingly develop a salt-wasting crisis than are their female
ambiguous counterparts, because the males have a normal phenotype at birth. Infants with salt-wasting
first demonstrate poor feeding and may develop diarrhea and lethargy prior to the full-blown
salt-wasting crisis and death.
No-classic 21-OH deficiency, is almost universally manifested in females only and is not a cause of
intersex disorders. It presents during the peripubertal time and adult menstrual years with signs of
androgen excess, menstrual abnormalities, or both (118). Precocious adrenarche and the peripubertal
development of an atypical polycystic ovarian disease syndrome are the most common presentations.
Non-classic CAH has been reported for 21-OH deficiency, 11β-hydroxylase deficiency, and
3β-hydroxysteroid dehydrogenase deficiency (118). Two forms of non-classic 21-OH deficiency also
exist: symptomatic (adult onset) and asymptomatic (cryptic). Asymptomatic patients with non-classic
CAH appear phenotypically normal (117-120). However, hormonally they are identical to patients with
symptomatic disease: levels of cortisol precursors (i.e., 17-hydroxvprogesterone) and androgens arc
not different for these two groups. Some symptomatic patients occasionally become asymptomatic and
some asymptomatic patients occasionally develop menstrual abnormalities accompanied by signs of
androgen excess. Nonclassic CAH appears to only rarely have an adverse effect on males with these
saint mutations (121,122). Less than a half dozen men with nonclassic disease and oligospermia
corrected by steroid treatment have been reported in the literature (121.122).
Linkage disequilibrium seas found to exist with 21-OH deficiency and the HLA-B and DR antigens
(123,124). Specific HLA-B antigens occur with increased frequency in association with various forms of
21-OH deficiency (e.g., HLA-Bw47 and classic CAH; HLA-B14 and nonclassic CAH). In some families.
20
different forms of 21-OH deficiency-salt-wasting classic, simple virilizing classic, and nonclassic 21-OH
deficiency-have occurred in different members of the same family. Hormonal, HLA, and DNA studies of
these family members demonstrated that patients can harbor a number of different 21-OH mutations
(i.e., one on each chromosome) and that they manifest the least deleterious mutation that they harbor
Sexual Differentiation
(125,126). A patient with a classic salt-wasting mutation on one chromosome and a classic simple
virilizing mutation on the other chromosome would express the simple virilizing form. A patient with a
classic salt-wasting mutation on one chromosome and a nonclassic mutation on the other chromosome
would express the nonclassic mutation. This finding, has serious implications for individuals who may
be at risk of delivering an infant with ambiguity or salt-wasting potential. For example, women with
nonclassic CAH may harbor a classic 21-OH mutation on one chromosome (126.127).
The variability of 21-OH deficiency within the same family prompted investigations into the molecular
nature of these different 21-OH-deficient states. Two genes for 21-OH have been identified and
sequenced (119,127-129). These genes, CYP21 (previously known as 21-hydroxylase B) and CYP21P
(previously known as 21-hydroxvlase A), encode a cytochrome P-450 specific for 21-hydroxylation. The
CYP21 and CYP21P genes lie in tandem with the genes encoding the fourth components of
complement, C4A and C4B, respectively. They are located on chromosome 6 to close association with
the HLA-B and DR loci. While C4A and C-4B genes both encode protein products, only the CYP21 (21hydroxyiase B) gene encodes a specific gene product that is responsible for the 21-hydroxylation step
in steroid biosynthesis. CYP21P (21-hvdroxylase A gene) is a pseudogene (128,129). It harbors three
small mutations in its coding region and other mutations in introns that render it inactive. Mutations
have been found in the CYP21 gene, and render it inactive and cause various forms of 2l-OH
deficiency (119). Approximately 25°/ of these gene mutations are easily identified by Southern blot
techniques rising the 21-OH gene probe. Half of these mutations involve large gene deletions of both
the C-4B and the CYP21 genes (I 19,130). The other half of these easily identifiable gene mutations
involve gene conversions: Any or all of the mutations causing inactivity of the pseudogene are
transferred during chromosomal pairing at meiosis by abnormal crossing over to the active gene,
rendering it also inactive. Seventy-five percent of the mutations identified in patients with classic 21-OH
deficiency are point mutations and not easily identified by routine Southern blot techniques. Diagnosis
for these latter patients has previously involved DNA studies of closely linked HLA-DR gene polymorphisms
(119,131,132). Newer techniques such as denaturing gradient gel electrophoresis are being
utilized to rapidly and more easily identify these point mutations, obviating the need for linkage studies
and potential misdiagnosis (133). Recent studies demonstrated that 95% of mutations are now known
and can be easily identified. Amplification of the CYP21 (active) gene alone by polymerase chain
reaction (PCP,) will allow examination for mutations in it. most of which commonly occur also in the
21
CYP21P (inactive/pseudogene). Southern blotting will identify the large gene deletions or conversions
and oligonucleotide probing of the amplified active gene sequences for common nitrogenous base
changes will find the point mutations (134,135).
Sexual Differentiation
Androgen excess syndromes
In-utero exposure to endogenously produced or exogenously given maternal androgen has been
responsible for varying degrees of masculinization of the external genitalia in 46,XX females with
otherwise normal sexual differentiation. The placenta has the ability to aromatize native androgens.
except DHT, to estrogens and prevent masculinization of a female fetus. In fact, during the end of
pregnancy the fetal adrenal gland makes nearly 200 mg of steroids a day, most of which is DHEA
sulfate (DHEAS). In comparison, at any one time of the menstrual cycle. the regularly menstruating
female never makes more than a total of 511 mg of all the steroids. most of which are estrogens.
Despite the extremely high levels of androgens produced by the normal fetus and high levels of
testosterone produced abnormally by some women during pregnancy, the female fetus does not
masculinize because of placental aromatization.
Several conditions exist in which this "neutralization" of androgens either does not occur or is bypassed
and female fetuses are masculinized. The maternal androgen producing tumor is one such instance in
which androgens may rarely bypass placental aromatization. The likely reason for such masculinization
associated with these tumors is the presence of excessive 5α-reductase activity in the tumor.
Testosterone is converted to DHT, a nonaromatizable androgen, prior to passage across the placenta.
Conceivably, although unlikely, if tumor production rates for testosterone exceed the placenta's ability
to aromatize it to estradiol, then native testosterone could get to the female fetus and cause
masculinizing effects as well. The luteoma of pregnancy is the most common tumor associated with in
utero masculinization of a female fetus and has 5α-reductase activity. This solid tumor appears to be
hCG-dependent and usually disappears after pregnancy. Most cases of genital ambiguity previously
labeled as being idiopathic probably resulted from a luteoma of pregnancy. Very rarely- ovarian and
occasionally adrenal neoplasms have been associated with masculinization of a female fetus. A few
rare testosterone-producing ovarian tumors include arrhenoblastomas, Leydig cell tumors, and
occasionally epithelial tumors that develop within the ovary as a secondary metastasis.
Exogenously given testosterone for such conditions as endometriosis and inadvertent continuation of
this drug during pregnancy rarely masculinize a female fetus. Synthetic hormone derivatives of
22
testosterone such as ethisterone, norethindrone. and danazol (Danocrine) have been more commonly
implicated in masculinization of the female fetus, as they are nonaromatizable.
Sexual Differentiation
Aromatase deficiency was not described until molecular mutations were identified. Aromatase
deficiency recently was found to result from a homozygous recessive inherited aromatase gene
mutation (137-139). Like placental sulfatase deficiency; such pregnant women have inappropriatelylow- levels of estrogens for gestational age in the absence of fetal distress. However, unlike placental
sulfatase deficiency, both the mother and the female fetus are masculinized or androgenized
significantly. The extremely high fetal levels of adrenal DHEAS. which is usually converted to estrogens
by the placenta. remain high and the DHEAS is converted to the more potent androgens, testosterone
and DFIT For male infants with aromatase deficiency. the mothers, but not their sons, are "over
androgenized" at birth. The only phenotypic abnormality described for affected males with estrogen
deficiency is the absence of normal epiphyseal closure and significantly taller suture than would be
predicted by mean parental height estimates (1-10).
Idiopathic genital ambiguity
For a number of 46,XX infants. the cause of genital ambiguity has not been identified. Except for
varying degrees of virilization, these infants are otherwise normal at birth. Endocrine function at puberty
is also normal. While a diagnosis of idiopathic genital ambiguity is given to these infants, it is very likely
that` they were exposed to DHT from a luteoma of pregnancy.
In a number of other 46.XX infants. however, unexplained external genital masculinization .,vas
associated with multiple somatic anomalies. Such anomalies Include the genitourinary, gastrointestinal,
and non-reproductive endocrine systems. These disorders are usually sporadic and may be caused by
unknown teratogens or de novo gene mutations. An autosomal recessive etiology has been implicated
in several consanguineous pedigrees in which two such affected infants were delivered.
IDENTIFICATION AND EVALUATION OF INTERSEX DISORDERS
Few- birth defects require the degree of expertise for early diagnosis and treatment as well as for
concomitant psychosocial counseling as is necessary for those that involve the genitalia. The parents of
these children must deal with routine issues surrounding any birth defect: 1) the delivery of an imperfect
baby: 2) guilt and self blame: and 3) the requirements of "traumatic" testing and potential surgery, for
their newborn. Additionally, these parents are burdened by issues surrounding the sex and future
23
reproduction of their child: 1) the sex-of-rearing decision: 2) subsequent development of gender
identity; and, 3) potential sterility. Sex-of-rearing refers to the label of male or female given to an infant
at birth. Gender identity refers to ones own feelings of sexuality. Children reared with normal
Sexual Differentiation
environmental influences that reinforce sex-of-rearing develop a concordant gender identity. This is true
even for individuals with intersex disorders despite the fact that at birth their karyotype, gonads, or
external genitalia may have been more consistent with the opposite sex-of-rearing. Confusion or
discordant feelings about sex-of-rearing have occurred in intersex patients who have not had
expeditious and long-term medical, surgical, and psychological treatment. These disorders should be
considered medical and surgical emergencies, especially if identified in the newborn, in order to avoid
potentially disastrous outcomes.
Delivery room identification, reaction, and initial evaluation
The delivers- of a child with anything other than normal genitalia calls on the immediate good judgment
of the obstetrician. The physician should understand that ambiguous genitalia potentially caused by an
intersex disorder may include minimal clitoromegalic isolated cryptorchidism (especially if bilateral), and
hypospadias as well as overt ambiguity.
It is important to be able to recognize a potential intersex disorder and to choose the most appropriate
words for discussing this issue with the couple before addressing the sex of
the child.
When the sex of an infant is in question, it is best to withhold the decision on sex-of-rearing. The health
care provider should explain that sexual development of this infant is incomplete and that after a few
studies it will be easy to determine whether the baby is a boy or a girl. After addressing the immediate
usual delivery concerns (e.g., delivery, of the placenta and suturing the episiotomy), the physician
should enter into a more detailed discussion involving causes of ambiguity, concerns, and actions to be
taken. It should be pointed out to the parents that the external genitalia of both male and female infants
appear nearly identical until midgestation. For this reason arrested development in a male infant may
make his genitalia appear underdeveloped; surgery will complete this development. "Overdevelopment"
of an otherwise normal female infant by exposure to male hormones may make her genitalia appear
more masculine. Most of these female infants will appear normal after surgery, and many are fertile.
Spot decisions about sea-of-rearing may be comfortable in the delivery room but are usually risks-.
They often result in the change of sex-of-rearing at least one more time and the potential development
of adverse psychosocial affects for the infant and family. There are two exceptions for which it is usually
24
safe to give the sex-of-rearing immediately. First, an infant with minimal clitoral hypertrophy and
normal-appearing urethral and vaginal orifices can usually be safely given the female sex-of-rearing. It
would be impossible to make a normal functioning penis for this infant. Second. it is usually appropriate
Sexual Differentiation
to give a male set-of-rearing to an infant with a normal-size but hypospadic penis associated with
unilateral cryptorchidism. It is, however, unwise to give spot diagnoses for all other forms of ambiguity.
It would be very- tempting, but very wrong, to give a male sex-of-rearm, to an infant with a
normal-appearing penis and scrotum and bilateral cryptorchidism. This child may be a normal fertile
female with CAH!
Changing sex-of-rearing should be avoided at all cost! It provides the parents and family members with
questions as to whether the final decision for sex-of-rearing is correct. Gender identity and the eventual
emotional well-being of the patient and fancily members largely depend on the comments made during
the first critical moments of life.
Delivery room clues
The delivery of twins concordant or discordant for genital ambiguity is a very rare occurrence bur
provides clues for diagnosis. Concordant ambiguity could occur in identical twins with an enzyme
deficiency and should be suspect if neither of the infants have descended gonads (e.g.. 46,XX CAH) or
if both infants have bilaterally descended testicles (e.g., 46,XY biosynthetic defect for testosterone). By
far the most common cause of ambiguity reported in one or both infants of a set of twins is 45,X/46,XY
gonadal dysgenesis. (111).45,X/46,XY Gonadal dysgenesis has been documented in male-male,
female-female, and male-female identical twins. One or both of these twins usually has one or both
testicles descended and one or both of them may have 45,X/46,XY karyotypes. This disorder is
considered to represent an abnormal attempt at twinning; in the singleton pregnancy. the -46,XY fetus
imperfectly attempts to twin and forms a mosaic ferns instead. In the twin pregnancy, the twinning
process may occur but second attempts at twinning may result in mosaicism.
Evaluation should begin in the delivery room
A review of the maternal history might include questions concerning maternal ingestion of androgenic
compounds (e.g., danazol) and the development during pregnancy of signs or symptoms related to
maternal androgen overproduction (e.g., acne, hirsutism). Family history might reveal similarly affected
25
infants or relatives (e.g.. CAH, AIS). Previous unexplained neonatal deaths of other infants right provide
a clue for the diagnosis of CAH. Maternal blood should be drawn in the delivery room for testosterone,
DHEAS, and estrogen measurements to rule out the presence of an androgen-producing tumor (e.g.,
luteoma, ovarian neoplasm, or adrenal tumor) or placental aromatase deficiency. Similar studies on
cord blood right be helpful.
Sexual Differentiation
Neonatal evaluation
The evaluation of the neonate should be designed to make a diagnosis and determine sex-of-rearing as
expeditiously as possible. The reaction and adjustment of the family depend on timely and informed
counseling.
The diagnostic evaluation should answer the following four questions:
1) Is the ambiguity a part of a syndrome ?
2) Does the infant have CAH ?
3) Is a testis or are testes present ?
4) Can an exact diagnosis be made ?
A number of syndromes described above have been associated with abnormal genital
development. For most of them ambiguity is the result of hormonally induced over
masculinization or under masculinization. For a few syndromes, total disorganization of
external genital development may be accompanied by gross detects of other bode parts such
as the Iimbs or face, and the result of viral-induced or chromosomally related teratogenesis.
Congenital rubella syndrome and the phenotype of unbalanced autosomal translocation,
respectively, are examples of the latter two problems. The infant with an identifiable testis (or
testes) and genital ambiguity does not have life-threatening, salt-wasting 21-OH deficiency;
this syndrome causes ambiguity in affected females, not affected males. Rather, the infant
with as identifiable testis (or testes) may have 45X/46,XY gonadal dysgenesis, one of the
4l6XY syndromes (see Table 16.1). or may be a true hermaphrodite. Finally, understanding
the exact diagnosis will help the clinicians involved to give the most informed counseling about
expectations for the family members.
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Sexual Differentiation
This set of Notes Contributed by Dr. Dorothy Villee PhD (Who gave these lectures for many years)
What is sex?
The Different Kinds of Sex
The most important clinically is the genital sex and the brain sex. Decisions of sex assignment
depend upon the appearance of the external genitalia.
Sex Determination
The processes of morphogenesis and cellular differentiation are dependent upon the activity of
sets of genes normally in complex interacting networks or pathways. However, within these
pathways specific genes occasionally appear to act as switches. One case in which this is
very apparent is in the process of sex determination.
The primary event in sex determination is the differentiation of the gonad. THUS, SEX
DETERMINATION IS THE EQUIVALENT OF TESTIS DETERMINATION IN MAMMALS.
In humans, the Y chromosome acts as a dominant male determinant. If the Y
chromosome is present an embryo will develop as a male independent of the number of X
chromosomes. The activity of the Y chromosome responsible for this has been referred to as
Testis Determining Factor (TDF) in humans.
1. it should be in the minimum portion of the Y chromosome that is shown to be maledetermining
2. it should be conserved on the chromosome
3. there should be a molecular basis for any mutation known to be within the gene
4. it should be present in the somatic portion of the genital ridge, immediately prior to overt
testis differentiation
5. it would be likely to have a structure consistent with a regulatory function
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The most likely candidate for TDF is SRY found in four XX males who had some Y-unique
sequences. It is located within a 35 kb region adjacent to the pseudoautosomal boundary of
the Y-unique portion. It has homology to a number of genes known to bind to DNA in a
nonspecific manner.
In mouse SRY is expressed just prior to testis differentiation (10.5 days) and is switched off by
12.5 days, suggesting that it is not required for maintenance of any differentiated cell type or
Sexual Differentiation
function of the testis. Thus, it is expressed only to trigger the process of overt testis
differentiation. The only site of SRY expression in the embryo is the genital ridge and the
expression is tied to the somatic portion of the gonad and not the germ cell component. Germ
cells are not necessary for testis development.
Transgenic experiments
Fragments of DNA encoding SRY were introduced into early mouse embryos to
determine whether SRY is capable of changing the sex of an XX female embryo. Fourteen
days after the injection the embryos were examined for testis or ovary. Two XX embryos with
developing testes were found. Southern blot analysis showed that the two embryos carried
multiple copies of SRY as a transgene. THIS EXPERIMENT PROVED THAT SRY CAN
INITIATE TESTIS DEVELOPMENT. XX transgenlc male mice look and behave as males
except that their testes are smaller and they are infertile, because XX is incompatible with
spermatogenesis. The rest of the reproductive tract was normally male, indicated that the
Sertoli cells in developing embryos were functionally normal in terms of MIF (Mullerian
inhibiting factor) and that the Leydig cells were also producing testosterone normally.
Gonadal Differentiation
The formation of the gonadal blastema is completed by weeks 5 to 6 of gestation in
human embryos. At this time the primitive ("indifferent") gonad is composed of three distinct
cell types: (1) germ cells (migrated from yolk sac), (2) supporting cells of the coelomic
epithelium of the gonadal ridge that give rise to the Sertoli cells of the testis and the granulosa
cells of the ovary, and (3) stromal ("interstitial") cells derived from the original mesenchyme of
the gonadal ridge.
The first morphological sign of sexual dimorphism in the gonads is the development of
the primordial Sertoli cells and their aggregation into spermatogenic cords in the fetal testis. In
the human this occurs between weeks 6 and 7 of gestational development. In contrast to the
early development of the fetal testis, the fetal ovary shows no characteristic development until
months later in embryogenesis and initially is identified histologically early only by exclusion.
Neither selective destruction of germ cells with drugs nor surgical excision of primordial germ
cells in the anterior germinal crest before they reach the gonadal primordium inhibits gonadal
development Thus, somatic cells can organize into an ovary or a testis irrespective of the
presence or absence of germ cells.
Hormonal Sex
Two substances from the fetal testes are essential for male development:
a nonsteroidal hormone that acts ipsilaterally to cause regression of the mullerian duct
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(Mullerian Inhibiting Factor, MIF) and (2) an androgenic steroid (testosterone) responsible for
virilization of the wolffian duct, urogenital sinus, and urogenital tubercle. Development of the
female urogenital tract occurs in the absence of gonads and does not seem to require
secretions from the fetal ovaries.
Mullerian Inhibiting Factor
MIF is a large (140,000 daltons) dimeric glycoprotein formed by the Sertoli cells of
Sexual Differentiation
the fetal and newborn testis. It acts locally to Suppress mullerian duct development.
Persistence of the Mullerian ducts is usually accompanied by failure of the testes it descend.
Mullerian-inhibiting activity is Tower in biopsied testicular cells from newborn boys with
cryptorchidism than from normal newborns. Therefore, MIF may play a role in the descent of
the testes, particularly in the transabdominal migration.
Androgens
Testosterone formation in the testis begins shortly after the onset of differentiation
of the spermatogenic cords and is concomitant with the histological differentiation of the Leydig
cells. In the rabbit the onset of testosterone synthesis and the resulting differentiation of the
male urogenital tract are independent of gonadotropin control. It is uncertain whether a similar
situation exists in embryonic sexual differentiation in humans. LH receptors are present in
human fetal testes as early as the twelfth week of gestation and human fetal testes respond to
human chorionic gonadotropin stimulation by exhibiting increased testosterone synthesis at
this time. It is not known what happens between weeks 8 and 11, when the major portion of
male phenotypic differentiation takes place. Testosterone synthesis is gonadotropin
dependent during the latter two-thirds of gestation.
Genital Sex
Internal genitalia
Prior to the eighth week of human development, the urogenital tract is
identical in the two sexes. The internal accessory organs of reproduction develop from a dual
duct system (wolffian and mullerian) that forms within the mesonephric kidney early in
embryogenesis (Figure .2). Within the substance of the mesonephros, tubules connect
primitive capillary networks with a longitudinal mesonephric (wolffian duct). The wolffian duct
extends caudally to the primitive urogenital sinus. At about six weeks the development of the
paramesonephric (mullerian) ducts begins in embryos of both sexes as an evagination in the
coelomic epithelium, just lateral to the wolffian ducts. Mullerian duct development cannot take
place in the absence of the wolffian duct.
The first sign of male differentiation of the urogenital tract is degeneration of the mullerian
ducts adjacent to the testes, a process that begins just after formation of the spermatogenic
cords in the testes. Eventually, the mullerian ducts of the male undergo almost complete
regression. The transformation of the wolffian ducts into the male reproductive tract begins
subsequent to the onset of mullerian duct regression. The portion of the wolffian duct adjacent
to the testis becomes convoluted to form the epididymis; the central portion of the duct
becomes the vas deferens.
The differentiation of the Wolffian duct is dependent upon local high concentrations of
testosterone. The regression of the mullerian ducts is dependent upon local high
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concentrations of MIF.
External genitalia
Prior to week six of gestation, the anlagen of the external genitalia are
indistinguishable in the two sexes. The genital eminence is a rounded mass between the
umbilicus and the tail and is composed of a genital tubercle flanked by prominent genital
swellings. The opening of the urogenital sinus between the genital swellings (the urethral
Sexual Differentiation
groove) is surrounded by genital folds. At week 7 of gestation in the human, the genital
tubercle begins to elongate; a shallow, circular depression defines the glans of the tubercle. At
this stage of development, there are no differences between the external genitalia of male and
female embryos.
The external genitalia of the male begin to develop shortly after the onset of
virilization of the wolffian ducts and urogenital sinus. The genital tuberde elongates, and the
urethral folds begin to fuse over the urethral groove from posterior to anterior. The two
urogenital swellings move posterior to the genital tubercle and eventually fuse to form the
scrotum. The elongated urogenital cleft becomes closed to form the penile urethra.
Male differentiation of the external genitalia depend upon the androgen
dihydrotestosterone, which is formed from testosterone by
5 alpha reduction. dihydrotestosterone binds to the androgen receptor with greater affinity
than does testosterone and the dihydrotestosterone-receptor complex is more readily
transformed to the DNA-binding state than is the testosterone-receptor complex. The net
consequence is that dihydrotestosterone formation generally amplifies the androgenic signal
Brain Sex
Animal experiments have shown that androgens and estrogens can affect cyclicity to the
hypothalamus and sexual behavior if administered during the critical period of brain sexual
differentiation.
In humans patients who are 46XX and have adrenocortical hyperplasia with excessive
production of adrenal androgens demonstrate an increased incidence of tomboy behavior.
There is a higher incidence of homosexuality in such patients compared with normal females.
Homosexual men have been shown to have size differences in sexually dimorphic areas
of the brain compared to non-homosexual men.
Such data suggests that sexual differentiation of the brain is dependent upon hormonal
influences and that anatomical differences in these sexually dimorphic areas of the brain
maybe responsible for sexual identity and behavior.
Present evidence suggests that testosterone must be converted to estradiol in brain cells
for these changes to occur. In the human brain sexual differentiation is thought to occur in the
latter half of pregnancy.
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Sexual Differentiation
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