746 The Immunoassay Handbook
peripubertal daughters of PCOS women indicating that
ovarian abnormalities are present as early as infancy.
Testosterone concentrations are frequently above the
reference interval in patients with PCOS, androstenedione
concentrations occasionally so. Free testosterone concen-
tration may be increased proportionately more than the
total testosterone concentration because of the lowered
sex hormone-binding globulin (SHBG) concentration.
The concentration of SHBG has been shown to be
inversely correlated with weight, but this seems to be in
turn related to insulin resistance in these patients. A large
number of studies have now shown that insulin appears to
diminish SHBG concentration and hence increases free
testosterone concentration. In most patients, the ovaries
are the major source of the increased androgen secretion.
Up to 10% of patients with PCOS may have increased
dehydroepiandrosterone sulfate (DHEAS) concentration
in the serum indicating increased androgen secretion from
the adrenal. Several groups have investigated adrenal func-
tion in PCOS patients, and subtle defects in adrenal ste-
roidogenesis have been reported in 12–40% of these
patients although no increase in adrenocorticotropic hor-
mone (ACTH) secretion has been found.
Luteinizing hormone (LH) concentration in PCOS may
be increased above or at the top of the reference interval
for the follicular phase. The follicle-stimulating hormone
(FSH) concentration is usually normal. The ratio of
LH:FSH has been used to indicate the presence of PCOS,
and a ratio of greater than three was said to be diagnostic.
Although this was a helpful diagnostic tool when radioim-
munoassays were used to measure the gonadotropins, cur-
rent immunometric methods for LH give lower values and
this ratio is no longer valid. Although some groups have
tried to reestablish a diagnostic ratio, generally, it has been
found to be unhelpful, and the most useful indicator of
polycystic ovaries is a LH concentration greater than
10IU/L. Patients may present with hirsutism, or infertility
due usually to oligomenorrhea or amenorrhea. The use of
anti-androgens, for example, cyproterone acetate or spi-
ronolactone, is the most effective treatment for hirsutism.
A number of different procedures have been carried out to
achieve conception. These include clomiphene citrate,
conventional gonadotropin therapy, pulsatile GnRH,
GnRH agonist with gonadotropin therapy, and diathermy.
Although for most of these treatments, a conception rate
of 50–80% has been reported, spontaneous abortion has
been reported to be as high as 40% in some studies. Those
obese women with PCOS who are able to lose weight will
often resume normal menstrual function.
Insulin resistance is present in about 40% of patients
with PCOS. It occurs both in obese and non-obese PCOS
patients although obesity further exacerbates insulin resis-
tance. Hyperinsulinemia is thought to play a key role in
PCOS. Reported actions include increase of hyperandro-
genemia, decrease in SHBG concentration, increase in
LH secretion from the pituitary, and increased estrogen
production by the ovaries. The key role is demonstrated by
the fact that suppression of androgen concentration does
not lead to normal insulin sensitivity (see FURTHER READING
for reviews). Harbourne et al. (2003) reported that treat-
ment with the anti-hyperglycemic drug, metformin,
reduced androgen slightly but showed a greater reduction
in the Ferriman–Gallwey score when compared with Dia-
nette. Patients perceived greater reduction of hirsutism
with metformin treatment. In support of these ﬁndings,
most studies examining the effect of metformin treatment
of PCOS patients ﬁnd a modest reduction in circulating
Polycystic ovaries are frequently present in other clini-
cal disorders that are associated with increased secretion of
androgens from the adrenal (congenital adrenal hyperpla-
sia [CAH], see below and Cushing’s syndrome, see ADRENAL
CORTEX). Because about 50% of patients with PCOS are
hirsute and obese, it may be difﬁcult to decide whether the
patient has Cushing’s syndrome or PCOS. Appropriate
tests can quickly exclude or conﬁrm Cushing’s syndrome.
Some women have hirsutism without the other features of
PCOS being present. In some cases, the hirsutism may be
due to genetic or racial factors. For instance, it is well rec-
ognized that women of Mediterranean origin are more
hirsute, and dark-haired women may be more conscious of
visible hair on the upper lip in particular. Social pressures
can also have an important role to play. In these women,
androgen concentration is normal.
However, there is a separate group of women who have a
marked increase of hair on the face, which may be present
on other areas of the body. In the strict deﬁnition of the
term, androgen concentration is within the reference inter-
val. An index of the free testosterone concentration may
show this parameter to be increased, but no pathology is
obvious with no ultrasound evidence of polycystic ovaries.
A number of androgen indicators have been used to disclose
an abnormality of androgen synthesis or secretion, and
these are discussed further under the appropriate analyte.
Some investigators have seen this condition as one end of
the spectrum of the polycystic ovary syndrome and would
so classify these patients. In 2008, the Endocrine Society
published guidelines for the evaluation and diagnosis of hir-
sutism. Using the Ferriman–Gallwey score, hirsutism was
deﬁned as a score of ≥8. They did not advocate measuring
androgens in cases of mild hirsutism (score 8–15) because it
was unlikely that a medical disorder would be revealed that
would change management or outcome. In these cases, ini-
tial action would be a trial of cosmetic, dermatologic, or
oral contraceptives. This was also the approach in women
with a score >15 but with a normal morning testosterone.
These ﬁrst two conditions would fall into the category of
idiopathic hirsutism especially when menstrual cycles are
normal. Other investigations would be carried out when the
testosterone was elevated. What is unclear is what weight is
put on free testosterone. These days many, if not most,
laboratories would measure testosterone and SHBG in the
ﬁrst instance and calculate the free testosterone. Therefore,
some clinicians may put more weight on the free testoster-
one result rather than the total testosterone concentration.
Certainly, one colleague indicated that his treatment of hir-
sutism was likely to be more aggressive if the free testoster-
one was elevated, i.e. using anti-androgen therapy rather
than creams or oral contraceptives. Hirsutism is associated
with hyperprolactinemia, acromegaly, hyperthyroidism,
Cushing’s syndrome, and late-onset CAH (LOCAH). In
747CHAPTER 9.7 Hirsutism and Virilization in the Female
the initial endocrine workup of the patient, it is common to
include the measurement of prolactin, 17-hydroxyproges-
terone, and thyrotropin (thyroid-stimulating hormone).
Other hormones such as growth hormone and cortisol
would only be requested if there was a high suspicion of
acromegaly or Cushing’s disease. Treatment of idiopathic
hirsutism is usually inﬂuenced by the distress it causes the
patient. The Endocrine Society advocates the use of creams,
physical removal of hair, or oral contraceptives as the ﬁrst
line of treatment for idiopathic hirsutism. More aggressive
treatment includes insulin lowering drugs, dexamethasone,
anti-androgens (spironolactone, cyproterone acetate), and
gonadotropin-releasing hormone analogs, not all advocated
by the Endocrine Society. Cyproterone acetate is not avail-
able in the United States.
OF THE OVARY
Androgen-secreting tumors of the ovary are very rare.
Several different types of tumor can occur, depending on
the main type of cell present. The testosterone concentra-
tion is typically greater than 1.7ng/mL (6nmol/L). Andro-
stenedione may also be elevated in concentration. Patients
may present with hirsutism or with evidence of virilization.
Hirsutism of sudden onset and of increasing severity
should alert the clinician to the possibility of an androgen-
CONGENITAL ADRENAL HYPERPLASIA
CAH is due to an enzyme deﬁciency in steroid biosynthe-
sis. The consequent fall in cortisol secretion leads to
increased secretion of ACTH. This, in turn, increases the
stimulus of this hormone to the adrenal and hence causes
hyperplasia of the adrenal cortical cells. A number of
enzyme deﬁciencies have been identiﬁed, but about 95%
of the cases are due to a deﬁciency of the 21-hydroxylase
enzyme (see Fig. 1). The reported incidence of classical
CAH is 1 in 5000 to 1 in 15,000 with variation between
ethnic and racial backgrounds.
In this particular condition, there is increased secretion
of 17α-hydroxyprogesterone (17-OHP), the steroid
immediately before the enzyme deﬁciency. At the same
time, more 17-OHP is converted to the androgens, and the
increased concentration of these steroids causes virilization
of the female fetus. Patients are treated with exogenous
glucocorticoids, but, as up to 75% of the individuals also
suffer salt loss, exogenous mineralocorticoid must be given.
Diagnosis of CAH is made by measuring the steroid
immediately before the deﬁcient enzyme. Therefore,
nearly all cases may be diagnosed by measuring 17-OHP
in the serum or saliva of the neonate.
Effective treatment will minimize further virilization
and allow puberty to proceed at a more normal rate. There-
fore, it is most important to control androgen levels.
Although testosterone has been used to monitor treatment,
androstenedione may be viewed as a more appropriate
androgen. When androstenedione is suppressed well into
the reference interval, the concentration of 17-OHP may
be as much as four times the upper limit of its reference
interval. If 17-OHP levels are within the reference interval,
it is likely that the patient is being overtreated, and growth
will be retarded as well as other side effects of glucocorti-
coid excess being manifest. Therefore, it is inappropriate to
monitor treatment with 17-OHP measurement alone.
To date, 127 mutations have been reported in the
cyp21a2 gene. These range from complete loss of enzyme
function to partial enzyme activity. The classical form of
CAH has been described as an autosomal recessive gene,
closely linked to the human leukocyte antigen-B (HLA-B)
locus. It is now suggested that the pathophysiology is more
complicated than suggested by an autosomal recessive dis-
order as variations at other loci may inﬂuence steroid
metabolism and steroid responsiveness. HLA typing can be
carried out in families with an affected individual to assess
the carrier status of relatives. Individuals who are heterozy-
gous for the gene mutation also show abnormalities of ste-
roid synthesis. 17-OHP may be slightly above or just
within the reference interval but stimulation of steroido-
genesis with ACTH results in a greater increase of 17-OHP
secretion than seen in normal subjects. This abnormality
may be further exposed by calculating the 17-OHP:cortisol
ratio. Patients usually have no physical abnormality.
One group of patients shows no sign of abnormal steroid
secretion until after puberty. These patients are said to have
FIGURE 1 Steroid pathways.
748 The Immunoassay Handbook
LOCAH. Patients have hirsutism, oligomenorrhea, and
infertility, and since they also have polycystic ovaries (as do
most patients with CAH), they may be wrongly diagnosed
as having PCOS if appropriate biochemical tests are not
carried out. 17-OHP concentrations are above the refer-
ence interval for the follicular phase of the menstrual cycle
and give an exaggerated response to ACTH stimulation.
Patients may be treated with exogenous glucocorticoids
although anti-androgens may also be required for effective
treatment of hirsutism. Addition of anti-androgens may
allow lower doses of glucocorticoid therapy and may even
allow replacement of cortisone with prednisolone. The bal-
ance is between suppressing androgen secretion and main-
taining adrenal function. As androgens decrease with age it
may allow decrease in steroid dosage with increasing age.
Most other cases of CAH are due to a deﬁciency of the
11β-hydroxylase enzyme. There is increased production of
11-deoxycortisol, the steroid produced immediately before
the enzyme block (see CONGENITAL ADRENAL HYPERPLASIA),
and diagnosis is made by measurement of this steroid in
serum. There is also increased secretion of 11-deoxycorti-
costerone and the androgens. Patients develop hyperten-
Other enzyme deﬁciencies have been described, but these
are very rare.
See also ADRENAL CORTEX.
Cushing’s syndrome results from an overproduction of
cortisol. It may be the result of a pituitary adenoma (Cush-
ing’s disease), an adrenal adenoma, adrenal carcinoma, or
an ectopic source of ACTH. The disease is nine times more
common in women. Increased steroidogenesis leads to
increased secretion of androgens. The resulting hirsutism is
generally mild in Cushing’s disease, but as adrenal tumors
secrete greater quantities of androgens, severe hirsutism,
clitoromegaly, and deepening of the voice may occur.
Rarely, cortisol and androgen secretion is greatly increased
when an adrenal carcinoma is present, but in the case of
adrenal adenomas, DHEAS is usually below the reference
interval. Treatment is appropriate for the abnormality and
includes pituitary surgery, adrenalectomy, or removal of an
ACTH-secreting tumor. Further treatment by pituitary
irradiation or chemotherapy may be required. Careful
assessment of pituitary and adrenal function is required after
surgery and exogenous glucocorticoid given if necessary.
LUTEINIZING HORMONE AND FOLLICLE-
See also INFERTILITY.
The following section describes the measurement of
these hormones in the diagnosis of androgen disorders.
The secretion of LH and FSH is suppressed by very high
levels of androgen. Thus, LH and FSH concentration is
usually low normal or below the reference interval in
LOCAH and Cushing’s syndrome. Although the concen-
tration of FSH is normal in patients with PCOS, LH con-
centration is frequently above the reference range and may
be up to twice the upper limit of the reference interval. A
LH concentration greater than 10IU/L with a normal
FSH concentration is suggestive of polycystic ovaries for a
serum sample taken day 2–5 of the menstrual cycle or in an
G The measurement of LH and FSH is not helpful in the
diagnosis of Cushing’s syndrome, CAH, or androgen-
G Although the mean ratio of LH:FSH is increased in
PCOS, there is a large overlap with normal subjects, and
the ratio is unhelpful in diagnosis. It has been questioned
whether LH and FSH assays have a role in the diagnosis
of PCOS when good ultrasound facilities are available.
Frequency of Use
ANTI-MÜLLERIAN HORMONE (AMH)
See also INFERTILITY.
This following section discusses the clinical use of AMH
measurement in the investigation of PCOS.
Although it is uncertain whether AMH shows cyclical secre-
tion or not during the menstrual cycle, intra- and inter-
cycle ﬂuctuations may be considered to be low enough to
permit random blood samples for AMH measurement. All
studies have found more than a twofold difference in mean
AMH concentration in PCOS patients compared with nor-
mal menstruating women. The increase in AMH appears to
be related to the severity of the disease. The AMH concen-
tration is higher in PCOS with insulin resistance compared
with patients with normal insulin sensitivity. AMH is also
higher in amenorrheic PCOS women compared to PCOS
patients with oligomenorrhea. Since clinical speciﬁcity and
sensitivity are reported as 92% and 67%, respectively, it has
been suggested that AMH may be useful in the diagnosis of
PCOS where ultrasound is not available or unclear.
It has been shown that in overweight PCOS patients
who show menstrual improvement with weight loss, there
is a signiﬁcant fall in AMH concentration. Therefore,
AMH measurement may be helpful is assessing therapy in
PCOS patients. It is also reported that AMH measurement
may be helpful in predicting outcome to clomiphene
citrate therapy as well as evaluating efﬁcacy with insulin
sensitizers such as metformin.
See also INFERTILITY.
The following section describes the use of testosterone
measurement in the investigation of hirsutism and
749CHAPTER 9.7 Hirsutism and Virilization in the Female
A testosterone measurement is usually requested in every
case of hirsutism. The primary use of this parameter is to
diagnose an androgen-secreting tumor when values are
typically greater than 1.7ng/mL (6nmol/L). The clinician
should be alerted to the possibility of a tumor when hirsut-
ism is of sudden onset with increasing severity.
The NIH 1990 criteria and the Rotterdam 2003 crite-
ria require testosterone to be raised in PCOS. This is also
true using the Endocrine Society guidelines of 2008. Dis-
crimination between patients with PCOS, idiopathic hir-
sutism, and normal women is enhanced by including the
measurement of SHBG. From these two parameters, an
indication of the free testosterone level may be achieved.
Testosterone may be normal, but if the SHBG is low, the
concentration of free testosterone may be abnormally
high. It is questionable whether knowing the result of any
of these three parameters will change the clinician’s man-
agement of the patient with isolated mild hirsutism, and
the Endocrine Society does not advocate the measure-
ment of androgens in such patients. However, when
there is a mixed ethnic population, an indication of the
circulating free testosterone concentration may help to
distinguish racial or genetic causes of hirsutism from an
abnormal pathology. In addition, they may be helpful in
conﬁrming suppression or compliance during treatment
with suppressive therapy.
Testosterone concentrations are higher in patients
with CAH, LOCAH, and Cushing’s syndrome, but the
measurement of this hormone has no place in the diagno-
sis of these diseases. However, the measurement of testos-
terone has been used to monitor the treatment of patients
G Total testosterone measurement is greatly inﬂuenced
by SHBG levels. The SHBG concentration is increased
by estrogens and anticonvulsants and in cirrhosis of the
liver and some cases of hypothyroidism. In hirsutism,
SHBG is often low, resulting in an elevated concentra-
tion of nonprotein-bound testosterone. This may be a
result of obesity or accompanying insulin resistance in
these patients. In these situations, measurement of total
testosterone alone can be particularly misleading. Free
testosterone concentration may be estimated by using
an SHBG measurement to derive a free androgen index.
G The measurement of testosterone gives no indication
of the source of increased androgen secretion in
women. The testosterone concentration does not pre-
dict the efﬁcacy of any treatment instituted to treat hir-
sutism, apart from when an androgen-secreting tumor
G As about half the circulating testosterone in women is
derived from the peripheral conversion of the weaker
androgens secreted by both the adrenals and ovaries,
testosterone is likely to be less sensitive than andro-
stenedione in monitoring the treatment of CAH.
G It has been reported that unidentiﬁed steroid metabo-
lites, which cross-react in direct assays, may occasionally
be secreted in large amounts. A falsely high testosterone
concentration will be achieved, and any results from a
direct assay that are unexpectedly above 1.7ng/mL
(6nmol/L) should be conﬁrmed by an extraction assay.
In the UK, this assay is available through the Supra-
Regional Assay Service.
G Testosterone exhibits diurnal variation, being highest
in the early morning and falling by 25–30% to a mini-
mum in the early evening. Normal males can have a
testosterone concentration in the late afternoon at the
bottom or just below the 9am reference range. Testos-
terone concentrations close to the lower limit of the
reference range, in samples taken in the afternoon,
should always be checked with a 9am sample.
G Testosterone levels may be elevated owing to alcohol
abuse, stress, or hard physical exercise of short duration.
It has been shown that extended exercise such as mara-
thon running lowers the testosterone concentration.
See INFERTILITY for more details.
The lack of sensitivity and poor precision of direct
methods at female concentration levels have led to two
approaches in the measurement of testosterone in women
and in the investigation of hirsutism. One approach has
been in place for a number of years and that is the use of
extraction methods. Such methods are more sensitive and
speciﬁc and enable the investigator to have more conﬁ-
dence in normal and elevated levels. The other approach is
more recent and that is the use of tandem mass spectrom-
etry (MS). There are 19 participants using tandem MS in
the UK NEQAS. Not surprisingly, these methods show
less variability than the automated methods. Nevertheless,
most routine laboratories use automated methods, and
many would send clinical cases that were difﬁcult to inter-
pret to a specialist lab using tandem MS for a more deﬁni-
The UK NEQAS has recently shown that the presence
of norethisterone interferes in the Roche automated
method for testosterone. This occurs at concentrations
that might be expected in women being given the drug for
heavy periods or taking oral contraceptives. Some interfer-
ence may even occur in the Siemens Centaur® assay.
Frequency of Use
SEX HORMONE-BINDING GLOBULIN
SHBG is a β-globulin, has a molecular mass of about
52kDa, and is secreted by the liver. Secretion of this pro-
tein is stimulated by estrogens and suppressed by andro-
gens. This results in a sex difference in plasma
concentration. In the circulation, SHBG binds several ste-
roids. It has a high avidity for testosterone and DHT
(about 1.5×109 mol/L) but a lower avidity for estradiol
(5.0×108 mol/L). Nevertheless, even the binding with
estradiol is considerably higher than the binding of these
steroids to albumin (3.7–6.4×104 mol/L).
An inverse correlation between SHBG and obesity and
SHBG and insulin resistance has been demonstrated, situ-
ations which are found in patients with PCOS.
750 The Immunoassay Handbook
Measurement of SHBG and testosterone levels enables
the concentration of biologically active testosterone to be
There is still much debate about the precise role of
SHBG. It has been suggested that SHBG dampens any
large ﬂuctuations in steroid concentration, thus main-
taining a fairly constant level of unbound steroid avail-
able to the tissues. Considering the marked episodic
secretion of testosterone, this would seem a reasonable
hypothesis. However, other experiments suggest that, if
this is true, it is an oversimpliﬁcation of the function of
SHBG. Of greatest interest are the recent studies that
have shown the presence of cell membrane receptors for
SHBG, as well as evidence for internalization of the pro-
tein. Hence, the whole role of SHBG at the cellular level
is yet to be elucidated. Two recent reviews (see FURTHER
READING) deal with SHBG receptor binding, and its inter-
nalization in some tissues where it can effect androgen
and estrogen binding.
(Orion SHBG IRMA)
The measurement of SHBG is used in the investigation of
hirsutism. When combined with a testosterone test, the
free testosterone index (or free androgen index) can be cal-
culated (see below). This is particularly useful when the
total testosterone concentration is normal but the SHBG
is low, resulting in an elevated concentration of nonpro-
tein-bound testosterone. Undetectable SHBG has been
reported but is very rare.
During treatment of hirsutism with the combined treat-
ment of anti-androgen and estrogen, the SHBG concen-
tration is usually above, often twice, the upper limit of the
reference interval. Therefore, the measurement of SHBG
concentration can be used to conﬁrm or refute compliance
in patients who are not responding to therapy.
1. The concentration of SHBG is increased by estro-
gens (oral contraceptives, pregnancy) and decreased
by androgens. It is also altered in a number of clini-
cal conditions and by several drugs, e.g. anti-epilep-
tics and barbiturates.
2. SHBG concentration is increased in hepatic cirrho-
sis, thyrotoxicosis, testicular feminization and hypo-
gonadism in the male.
3. SHBG concentration is decreased in myxedema,
Cushing’s syndrome, CAH, and acromegaly.
4. Exogenous T3 increases the secretion of SHBG, as
do most of the anticonvulsant drugs. Dexametha-
sone is reported to cause a small increase in SHBG
Early methods were based on the binding of tritiated testos-
terone to SHBG after endogenous steroids had been
removed from the serum with charcoal. Tritiated DHT
soon replaced tritiated testosterone because its higher bind-
ing afﬁnity for SHBG meant that removal of endogenous
steroids was not required. Another procedure used Con-
canavalin A-Sepharose 4B to bind SHBG before addition of
tritiated DHT, whereas Iqbal and Johnson used a column
of Cibacron-blue Sepharose 4B layered on top of LH-20.
Although manual assays are still available all participants
in the UK NEQAS use commercial assays with the Sie-
mens Immulite® 2000 and the Roche methods being the
International Reference Preparations are available for
the calibration of assays. The ﬁrst preparation was made
available in 1998, but recently, this has been superseded by
the 2nd International Reference Preparation, code 08/266.
It is not always clear what companies have used for the
calibration of their assays, but this information should be
available on request.
Type of Sample
Frequency of Use
Only about 1% of the testosterone in the blood of women
remains unbound to protein; in men, about 2% remain
unbound. This “free” fraction has been traditionally regarded
as the biologically active portion. However, over recent years,
this view has been challenged. Some workers suggest that
both the free and albumin-bound fractions should be deter-
mined as the biologically important portions, whereas, more
controversially, the SHBG-bound part has been suggested
as the fraction available to tissues, see FURTHER READING.
Nevertheless, there is little doubt that the nonprotein-bound
steroid correlates well with the clinical state.
Free testosterone concentration is often measured rou-
tinely in the investigation of hirsutism. Because the direct
measurement of free testosterone is complex and the com-
mercial kits unreliable, an indication of the free testoster-
one concentration can be achieved by two methods. Firstly
from the ratio of testosterone and SHBG concentrations –
the free testosterone or free androgen index. Secondly, a
closer approximation to the free testosterone concentra-
tion can be calculated (derived free testosterone) from the
testosterone, SHBG, and albumin concentrations, and a
variety of calculations have been published using some or
all of these parameters (see ASSAY TECHNOLOGY).
Women: 2.88–14.4pg/mL (10–50pmol/L)
Men: 38.6–243pg/mL (134–844pmol/L)
(Determined by micro-steady-state gel ﬁltration in the
751CHAPTER 9.7 Hirsutism and Virilization in the Female
An estimate of the free testosterone concentration may
inﬂuence the treatment of hirsutism in women who have a
normal total testosterone concentration. Treatment may
be more aggressive in women where an underlying andro-
gen abnormality is revealed.
In general terms, the levels of free and total testosterone
are positively correlated with the degree of hirsutism and
virilization in women. Nevertheless, there is considerable
interindividual variation; some women have increased
androgen concentrations but little or no hirsutism, whereas
others have normal androgen results but quite marked
Clinicians disagree about the importance of a free tes-
tosterone measurement. Some clinicians are guided in
their treatment by the result, although most argue that
they treat the hirsutism, and whether the androgen con-
centration is normal or slightly elevated is irrelevant.
The percentage of free testosterone in serum can be deter-
mined by equilibrium dialysis, steady-state gel ﬁltration,
ultracentrifugation, and micro-ﬁltration (see FURTHER
READING). These methods are lengthy, tedious, and require
good technical skill and experience. They are unsuited to
routine clinical use.
An estimate of the free testosterone concentration may
be made from the total testosterone and SHBG results.
There is a good correlation between the percent free tes-
tosterone and the SHBG concentration (see Fig. 2).
An equation describing this correlation can be used to
calculate the percent free testosterone from the SHBG
result. The free testosterone concentration can then be
calculated from the total testosterone result. Some labo-
ratories calculate a free testosterone index or free andro-
gen index from the ratio of the total testosterone and
SHBG results. Laboratories should establish their own
reference intervals because of differences between
It has been reported that albumin-bound testosterone
diffuses readily from the circulation into tissues. It has
been proposed, therefore, that the free testosterone plus
the albumin-bound testosterone (the non-SHBG-bound
testosterone) represent the fraction of testosterone
available to target cells. The measurement of this frac-
tion can be carried out using a simple ammonium sulfate
precipitation of the SHBG-bound testosterone before
A number of mathematical models have been devised to
calculate the non-SHBG-bound testosterone. Their com-
plexity depends on the number of different parameters
(e.g. SHBG, albumin, testosterone, cortisol-binding glob-
ulin) that are included in the calculation.
A radioimmunoassay kit is available from Siemens. Studies
that have examined the reliability of this kit have shown
good correlations with total testosterone, free testoster-
one, and the androgen index, although values about half
those obtained by equilibrium dialysis have been reported.
It has been suggested that rather than measuring the free
testosterone concentration, the method measures a con-
stant proportion of the total testosterone (see FURTHER
READING). Rosner (2001) has been particularly critical of
Vermeulen et al. (1999) evaluated the methods for esti-
mating free and non-SHBG-bound testosterone, com-
paring results with equilibrium dialysis measurement of
the free testosterone. They concluded that neither the
free testosterone using DPC’s kit (now Siemens) nor the
free androgen index was a reliable parameter of free tes-
tosterone concentration. The free testosterone calcu-
lated from the testosterone and SHBG measurement was
rapid, simple, and a reliable index of bioavailable testos-
terone. It was comparable to the results from equilibrium
dialysis and suitable for clinical use, except in pregnancy.
Calculated nonspeciﬁcally - bound testosterone reliably
reﬂected the measured non-SHBG-bound testosterone
concentration. The website of the International Society
for the Study of the Aging Male provides calculation of
free testosterone from the SHBG and testosterone val-
ues. It assumes a normal albumin level, but this may be
changed if the albumin concentration is available. The
calculation is not valid for very abnormal albumin
Type of Sample
Frequency of Use
Androstenedione is secreted by the testis, ovary, and adre-
nal. It is a weak androgen that is converted in peripheral
tissue to the more potent testosterone. Therefore,
increased secretion of androstenedione gives rise to hirsut-
ism through this conversion (see Fig. 3).
FIGURE 2 Percentage of free testosterone against log SHBG. The
correlation between the percentage of free testosterone, measured by
steady-state gel ﬁltration, and the log SHBG concentration measured by
752 The Immunoassay Handbook
Women: 30–330ng/dL (1.0–11.5nmol/L)
Men: 60–310ng/dL (2.1–10.8nmol/L)
(Quoted for the Siemens Immulite 2000 assay)
G Most requests for the determination of androstenedi-
one concentration are for the investigation of hirsut-
ism. However, there is disagreement among clinicians
about the usefulness of this measurement. Our own
investigations suggest that only 3% of hirsute women
with PCOS have an increased androstenedione con-
centration alone (see Fig. 4).
G Apart from salt loss, the major problem of CAH is vir-
ilization. Treatment of CAH should aim at maintain-
ing androgen concentration at physiological levels in
order to minimize further virilization and to allow
puberty to develop more normally. Although the
measurement of 17-OHP, androstenedione and total
testosterone have all been used to monitor the treat-
ment of patients with CAH, androstenedione seems
the most appropriate. Its main source in CAH is from
the adrenal, whereas testosterone is from peripheral
conversion of androstenedione. It is well established
that when androstenedione concentration is sup-
pressed to physiological levels, 17-OHP remains at
concentrations up to four times the upper limit of its
reference interval in men and in women for the fol-
licular phase. Thus, suppression of 17-OHP into the
normal range leads to overtreatment with exogenous
G A deﬁciency of the 17β-hydroxysteroid dehydrogenase
enzyme results in reduced testosterone synthesis by the
testis. This is a rare condition associated with incom-
plete masculinization in the male. Nearly, all affected
neonates have been assigned female status. Theoreti-
cally, androstenedione secretion by the testes will be
increased but practically, a human chorionic gonado-
tropin (hCG) test (see INFERTILITY) is required to uncover
the defect. An increased ratio of androstenedione to tes-
tosterone is diagnostic.
G An androstenedione result provides little additional
information in the investigation of hirsutism. Andro-
stenedione is secreted by both the ovary and the
adrenal, and the measurement of this steroid does
not establish the source of increased androgen
G Levels in the neonate and the child are <0.9ng/mL
(<0.3nmol/L), which is below the sensitivity of many
assays. In addition, it is difﬁcult to establish reference
intervals for neonates, and few laboratories have expe-
rience at accurately diagnosing 17β-hydroxysteroid
dehydrogenase deﬁciency. Such investigations should
be referred to an expert center.
Over 90% of the participants in the UK NEQAS use com-
mercial kits with about 50% using the Siemens Immulite
2000 method. Spiking experiments by the UK NEQAS
and comparison with tandem MS methods indicate that
commercial kits overestimate androstenedione by between
10% and 20%. There are signiﬁcant differences between
methods so laboratories should establish a reference inter-
val appropriate for the method they are using.
Types of Sample
Plasma or serum.
Frequency of Use
Essentially all of the DHEAS in the circulation is derived
from direct secretion by the adrenal glands. Because it is
sulfated, it has a long half-life, and hence lacks a circadian
rhythm (see Fig. 5).
The reference interval changes with age, peaking just after
puberty, and gradually falling after the third decade of life
(see Fig. 6 and Table 1).
FIGURE 3 Androstenedione.
FIGURE 4 A Venn diagram showing the percentage of patients with
polycystic ovarian syndrome who had one or more abnormal androgen
results. In only 3.1% of patients was androstenedione the only abnormal
result, whereas 20.6% had a raised testosterone level. No hormonal
abnormality, 14.2%; not every test done, 4.7%.
753CHAPTER 9.7 Hirsutism and Virilization in the Female
About 10% of patients with PCOS have a concentration of
DHEAS above the reference interval although some studies
have reported up to 42% of patients. Because this suggests
that the adrenal is the major source of increased androgen
secretion, exogenous glucocorticoid may be used initially to
treat such patients. Response to this treatment is variable.
Patients with Cushing’s syndrome due to an adrenal
adenoma frequently have a DHEAS concentration below
the reference interval.
G Although DHEAS concentration may be increased in
hirsute patients, this does not exclude increased andro-
gen secretion by the ovary. In fact, studies have shown
that it is very rare to have increased androgen secretion
from the adrenal alone.
G A normal DHEAS concentration in a patient with
Cushing’s syndrome does not exclude an adrenal
Because the concentration of DHEAS is 1000-fold greater
than the other androgens, specimens are usually diluted by
at least 100-fold before assay. Therefore, assays do not suf-
fer from serum effects. Iodinated DHEAS is available, and
several simple non-extraction assays have been developed
commercially. However, most routine clinical laboratories
use automated immunoassay methods, the majority the
Siemens Immulite 2000 platform. There are an increasing
number of laboratories using tandem MS, currently, seven
participants in the UK NEQAS.
It has been reported that some assays appear to cross-
react with as yet unidentiﬁed metabolites, leading to spuri-
ously high results. No speciﬁc clinical condition has been
associated with these metabolites.
Type of Sample
Frequency of Use
Gonadal and adrenal tissues are able to synthesize
17α-hydroxyprogesterone (17-OHP) from progesterone
(see Fig. 7). 17-OHP is readily converted to 11-deoxycor-
tisol in the adrenal and to androstenedione in the gonads
and adrenal. Levels of the steroid rise in the late follicular
phase and peak at the same time as estradiol. A second
increase occurs in the luteal phase that is similar to proges-
terone. Thus both the follicle and the corpus luteum
appear to secrete 17-OHP during the menstrual cycle.
Follicular phase: 0.55–1.84ng/mL (1.7–5.7nmol/L)
Luteal phase: 0.55–6.31ng/mL (1.7–19.6nmol/L)
The measurement of serum 17-OHP is used for the diag-
nosis of CAH caused by a deﬁciency of the 21-hydroxylase
enzyme. Affected infants have a concentration greater than
9.7ng/mL (30nmol/L). Heterozygote individuals also
have increased 17-OHP concentrations compared with
FIGURE 5 Dehydroepiandrosterone sulfate (DHEAS).
FIGURE 6 Diagrammatic representation of the changes in DHEAS
levels throughtout life.
FIGURE 7 17α-Hydroxyprogesterone.
TABLE 1 Reference Intervals for DHEAS
Approximate ranges for post-puberty
15–30 years 0.7–4.5µg/mL (1.7–11.5µmol/L)
30–40 years 1.2–4.2µg/mL (3.1–10.8µmol/L)
40–50 years 0.8–4.0µg/mL (2.0–10.2µmol/L)
50–60 years 0.3–2.7µg/mL (0.8–6.9µmol/L)
>60 years 0.2–1.8µg/mL (0.4–4.7µmol/L)
The graph and ranges are based on data from the author’s laboratory.
The conversion factor used (2.56) is based on the use of the sodium salt, which is used
as a standard.
754 The Immunoassay Handbook
the normal population. Identiﬁcation of heterozygotes is
important in genetic counseling.
Patients with LOCAH usually have 17-OHP concen-
trations above the reference interval for the follicular
phase of the menstrual cycle. A synacthen test will identify
those patients whose results are equivocal. In affected
patients, there is an exaggerated increase in the secretion
of 17-OHP (more than three times the basal level).
G 17-OHP is mildly elevated in those cases of CAH that
are due to an 11β-hydroxylase deﬁciency. This enzyme
abnormality should always be considered when hyper-
tension is present, and the concentration of 17-OHP is
only moderately increased.
G Interpretation of results is complicated in the ﬁrst 24h
of life because of maternal steroids still present in the
G 17-OHP should not be used to monitor the effective-
ness of treatment for CAH. If 17-OHP is suppressed
into the normal range, it is likely that the patient is
being overtreated. Androstenedione is the most
appropriate analyte for monitoring treatment for
“In-house” extraction assays are common. Siemens manu-
factures a direct kit that has a good correlation with the
extraction assay for 17-OHP concentration in the adult.
However, when using this kit, serum from neonates and
children less than six months old should be extracted
before the measurement of 17-OHP concentration due to
the cross-reaction with other steroids present in the
serum. Six of 53 participants use tandem MS in the UK
NEQAS. There are no methods developed for automated
Cross-reaction with other steroids can confuse the
interpretation of results when using immunoassay meth-
ods. In the UK, a GC–MS is available through the Supra-
Regional Assay Laboratory at the University College
The investigation of CAH is usually made in neonates
and prepubertal children where the collection of blood in
sufﬁcient quantity is difﬁcult. Methods have been devel-
oped to measure 17-OHP in saliva (see FURTHER READING)
and from blood spots.
Types of Sample
Serum, plasma, saliva, or blood spot.
Frequency of Use
DHT is formed in peripheral tissue from testosterone by
the enzyme 5α-reductase. Its biological activity, relative to
testosterone, depends on the bioassay and animal species
used. However, it is generally regarded as the more potent
androgen with a higher binding afﬁnity for SHBG. Testos-
terone is converted to DHT in the testes, skin, brain, sali-
vary glands, lung, heart, and pectoral muscle.
Women: 377–725pg/mL (1.3–2.5nmol/L)
Men: 116–435pg/mL (0.4–1.5nmol/L)
(Derived by an radioimmunoassay (RIA) method after
high performance liquid chromatography (HPLC) separa-
tion of DHT from the other androgens.)
Some studies suggested that the measurement of DHT
might be helpful in the investigation of hirsutism. How-
ever, because DHT is produced in many tissues, it is not a
sensitive indicator of DHT production in the skin.
The concentration of DHT is low in both sexes, and it
is difﬁcult for most assays to achieve the required sensi-
tivity. This is because it is difﬁcult to avoid losses during
the separation of DHT from testosterone and other
Antisera raised against DHT conjugates have a high cross-
reaction with testosterone. Because attempts to produce a
speciﬁc monoclonal antibody have been unsuccessful, tes-
tosterone must be removed from patient sera before DHT
can be measured.
DHT has usually been separated from testosterone by a
chromatographic procedure after an initial solvent extrac-
tion. These methods have the advantage of separating out
all the androgens, which can then be measured by RIA.
However, these procedures are usually limited to research
A slightly simpler method uses potassium permanganate
to oxidize the testosterone. Several companies now sell
commercial kits. These include DSL (Beckman Coulter);
Research Diagnostics Inc., New Jersey, USA; and IBL-
Hamburg, Germany. The ELISA from IBL-Hamburg is a
simple method with no oxidation or extraction steps. To
achieve good precision and reliability in the extraction
methods, a high degree of experience and skill is required.
Types of Sample
Serum or plasma.
Frequency of Use
ANDROSTANEDIOLS AND THEIR
DHT is rapidly converted to a variety of other compounds
that include androsterone and the androstanediols,
5α-androstane-3α, 17β-diol (3α-diol) and 5α-androstane-3β,
17β-diol (3β-diol), by a number of tissues. These include the
755CHAPTER 9.7 Hirsutism and Virilization in the Female
accessory sex glands, skin, brain, salivary glands, and heart
muscle. These steroids are further metabolized to their gluc-
uronides and sulfates. All these steroids have been investi-
gated as markers of hirsutism. The most abundant of these
steroid metabolites is androsterone sulfate. However,
Zwicker and Rittmaster (1993) showed that this steroid was
not suitable as a marker of hirsutism. It correlated poorly
with other androgens, and concentrations were not elevated
in hirsute women. It was shown that the steroid is derived
almost entirely from the adrenal glands. Little or no 3β-diol
is formed in the human and so studies have focused on
3α-diol and its glucuronide (3α-diol G). The latter has been
measured in both urine and serum although recent studies
have measured 3α-diol G in serum. Many of these studies
have reported that 3α-diol and 3α-diol G have a role in the
investigation of hirsutism.
Adult Males 3.4–22.0ng/mL
ARUP Laboratories RIA
Studies have shown that DHT concentrations are infre-
quently increased in idiopathic hirsutism. The 3α-diol was
investigated as a better indicator of increased androgen
activity in peripheral tissue as it is the end metabolite of
androgen metabolism in the skin. Findings have been vari-
able. Whereas one group of investigators reported
increased 3α-diol concentrations in 93% of their patients
with idiopathic hirsutism, other workers have found nor-
Interest switched to 3α-diol glucuronide (3α-diol G)
when it was reported that this metabolite is predomi-
nantly formed in sexual tissue and skin. Early studies
showed that 3α-diol G concentration was greatly
increased in hirsute women, whereas testosterone was
only modestly increased. It was later shown that method-
ological deﬁciencies in the early method caused errone-
ously elevated results. More recent studies have shown
that 3α-diol G is only modestly increased in hirsutism. In
a detailed review Rittmaster (1991) examined studies
that had looked at the origin and clinical use of 3α-diol
and 3α-diol G. He concluded that the primary source of
precursors for androgen conjugates appeared to be the
adrenal glands and that the liver may be the major source
of glucuronide conjugation. Therefore, he concluded
that the clinical utility of 3α-diol G in the investigation
of hirsutism was limited and androgen-dependent hir-
sutism is best assessed by examining the distribution of
body hair. Nevertheless there continue to be many stud-
ies of PCOS and hirsutism where 3α-diol and 3α-diol G
have been measured. Chen and et al. (2002) discuss these
studies and their varied outcomes in a more recent
Results from different studies have been inconsistent, cast-
ing doubt on the usefulness of these assays. They probably
add little to the information achieved from the measure-
ment of other androgens. Azziz et al. (2000) concluded that
the routine measurement of serum 3α-diol G is not recom-
mended in the evaluation of idiopathic hirsutism or in other
hirsute patients. The assays are not routinely available.
Commercial assays are available from Beckman Coulter
and from ARUP Laboratories, Salt Lake City, UT 84108-
Type of Sample
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