722 The Immunoassay Handbook
and have a brief secondary rise just after ovulation. Cur-
rent evidence suggests that inhibin B modiﬁes the pitu-
itary sensitivity to GnRH rather than having an effect at
the hypothalamus. At about day 12, estradiol feedback
changes. The mechanism is still unclear, but the steroidal
feedback becomes positive causing a sharp surge of LH
and FSH. This in turn stimulates ovulation, that is, rup-
ture of the Graaﬁan follicle and release of the ovum into
the fallopian tube.
The remaining granulosa and thecal cells of the follicle
develop into the corpus luteum. LH is required for the
maintenance of this structure, which secretes progesterone
Therefore, during the luteal phase, there is a smaller
rise in estradiol and a large increase in progesterone
concentrations. Concentrations of inhibin B remain at
low levels during the luteal phase. Inhibin A concentra-
tions, which are low during the early follicular phase,
begin to increase in the late follicular phase and reach
peak levels at the mid-luteal phase. Levels reﬂect growth
of the corpus luteum from which this hormone is
secreted. Changes in inhibin A concentrations closely
follow changes in progesterone concentration. If no fer-
tilization of the ovum takes place, there is then a fall in
progesterone levels. This, in turn, leads to necrotic
changes in the endometrium with loss of the bulk of the
tissue accompanied by the menstrual bleed. Inhibin A
concentrations also fall at the end of the luteal phase and
are associated with an increase in FSH concentrations
suggesting that inhibin A suppresses FSH secretion dur-
ing the luteal phase.
Eventually, the supply of oocytes declines so that in the
late ﬁfth and early sixth decade of life, the woman enters
the menopausal transition or perimenopause. AMH levels
have been shown to gradually decrease with age and
become undetectable in the menopause. There have been
several large studies to both stage the period from the
beginning of the perimenopause to the postmenopause
and to ﬁnd early markers of ovarian decline that herald the
beginning of the menopause. Most of these studies have
been recently reported, and references to them may be
found in the review by Butler and Santoro. Generally,
these studies divide the perimenopause or menopausal
transition, the time at which menstrual cycles start to become
irregular until the ﬁnal menstrual period, into two stages.
The early stage is associated with cycles of variable length
being more than 7 days greater than the normal cycle.
The second stage, is marked by periods of amenorrhea of
more than 60 days and more than 2 missed periods. At the
end of this stage, there is a period of amenorrhea of 12
months after which the women is postmenopausal. Early
transition is associated with variable sex hormone levels
with initially higher estradiol levels, higher FSH levels,
and lower inhibin B levels. In the late transition, FSH con-
tinues to increase with falls in estradiol, inhibin A, and
inhibin B. Sex hormone-binding globulin (SHBG) was
also found to decrease during the menopausal transition
with a corresponding increase in free testosterone. An
Australian study found that inhibin B began to decrease
before the transition indicating loss of follicular function
or size although they could not identify a single marker
for an individual woman for approaching perimenopause.
Obesity is associated with less ovarian reserve and lower
FSH and LH levels although obese women do not have an
early age of entering the postmenopausal period. Evidence
suggests that obesity affects both the ovarian function and
the hypothalamic/pituitary function independently. In
addition, the menopausal transition has been associated
with increasing hot ﬂushes, depression, and short-term
memory function. Eventually, menstruation and ovarian
function cease completely, and the woman enters the
menopause. Gonadotropin concentrations are initially
very high, thereafter declining slowly with age; estrogen
levels are very low. Some women experience an early
menopause due to premature ovarian failure. AMH may
have a role in determining whether anovulation is a result
of premature ovarian failure or some other cause.
In the male, as in the female, the same mechanism for
the control of LH and FSH secretion exists. LH and
FSH concentrations are low before puberty although
there is a signiﬁcant rise of testosterone in the ﬁrst 2
months of neonatal life. At puberty, surges of LH, FSH,
and testosterone occur during the night. As puberty pro-
ceeds, there is an overall increase in gonadotropin and
testosterone secretion with the gradual loss of circadian
LH stimulates the Leydig or interstitial cells of the testis
to secrete the male sex hormone, testosterone. The main
site of action of FSH is in the Sertoli cells in the seminifer-
ous tubules. These cells secrete the peptides inhibin B and
androgen-binding protein (no inhibin A is detectable in
the serum of men). Androgen-binding protein is involved
in the transport of the androgens, testosterone, and
5α-dihydrotestosterone (DHT), to the developing sperm
cells, whose development they support. Inhibin B secre-
tion is stimulated by FSH, but there appears to be an addi-
tional FSH-independent component to its secretion.
Testosterone and inhibin B are carried in the bloodstream
back to the hypothalamus and pituitary where they exert a
negative feedback. Several studies have conﬁrmed that
inhibin B has a negative feedback on the secretion of FSH,
whereas estradiol, produced by the aromatization of tes-
tosterone, feeds back negatively on LH secretion.
FIGURE 2 Serum hormone changes during the menstrual cycle.
723CHAPTER 9.5 Infertility
A male climacteric has been proposed in the past, but
now studies suggest that testosterone concentrations do
not fall until about the age of 60 years. Free testosterone
(nonprotein bound) falls earlier than this due to a rise in
SHBG, which starts at approximately 50 years of age. There
is a corresponding gradual increase in gonadotropin levels.
IN THE FEMALE
Just under half of the women who present with infertility
have ovarian dysfunction. An organized investigative
approach is likely to lead to a faster diagnosis with a lower
cost and less inconvenience to the patient. Breckwoldt
et al. (1993) suggested that a detailed medical history and a
meticulous physical examination, with special attention to
the target organs for sex steroids, frequently provided
better information than a battery of uncoordinated labora-
tory tests. A number of algorithms have been proposed
which help in a logical approach to female infertility. The
classiﬁcation proposed in 1976, by the WHO Scientiﬁc
Group on Agents Stimulating Gonadal Function in the
Human, has been widely used, and other algorithms are
largely based on this classiﬁcation.
Gonadal dysgenesis is associated with the failure of
the gonads to develop properly. They are present, if at all,
as streaks of tissue. Two classes of patient may be conve-
niently recognized. Firstly, those in whom development is
associated with an abnormality of the sex chromosome,
namely Turner’s syndrome and its variants. In the typical
condition, the karyotype is 45XO and is associated with
short stature, sexual infantilism, and several somatic abnor-
malities. All the patients are female. A partial abnormality
of the second sex chromosome is associated with fewer
abnormalities. Gonadotropins are elevated as expected in
postpubertal patients. The second class of patients has a
normal or near normal karyotype, 46XX or 46XY, but the
gonads are absent. Usually, the somatic abnormalities of
Turner’s syndrome are absent, and patients are of normal
or tall height. Affected men have variable sexual develop-
ment, presumably dependent upon the time at which the
gonads degenerated. If this occurred before 6 weeks of
fetal life, the patient will have a complete female pheno-
type. If later, there will be variable development of the
genital ducts and ambiguous genitalia. Gonadotropin con-
centrations will be increased in all adult patients.
Gonadotropin levels are also raised in women who have
entered the menopause. A very high FSH concentration
is usually diagnostic. (An exception would be if a very rare
gonadotropin-secreting tumor was present.) Laparoscopy
studies in some women, who appeared to have entered an
early menopause, showed them to have normal ovaries.
These studies suggested that the ovaries were insensitive
to the elevated gonadotropins (resistant ovary syn-
drome). This syndrome may be associated with primary
amenorrhea (failure of menstruation to commence at
puberty) or secondary amenorrhea (absence of men-
struation postpuberty). Again, levels of gonadotropins in
the blood are increased, LH more signiﬁcantly than FSH.
Some women have premature ovarian failure, and in the
early stages, LH and FSH may not be consistently
increased. It has been shown that AMH is undetectable in
premature ovarian failure but normal in functional hypo-
thalamic amenorrhea. Therefore, the measurement of
AMH may help the clinician conﬁrm premature ovarian
IN THE FEMALE
Secondary hypogonadism may result from dysfunction of
the pituitary, hypothalamus, or higher brain centers;
gonadotropin levels may be within or below the normal
follicular range. For instance, tumors of the hypothalamus
(craniopharyngioma) and pituitary (prolactinomas,
chromophobe adenomas) may be associated with nor-
mal or low LH and FSH concentrations, whereas hypo-
is associated with low levels. Low gonadotropin concen-
trations are found in cases of anorexia nervosa, and after
irradiation to the pituitary area, which can lead to either
hypothalamic or pituitary dysfunction.
Oligomenorrhea and amenorrhea associated with hir-
sutism are discussed in the HIRSUTISM AND VIRILIZATION IN
THE FEMALE chapter.
INFERTILITY AND NORMAL MENSTRUAL
Cyclic activity of the hypothalamus, pituitary, and ovary is
required for cyclic hormonal changes. However, it is still
possible for some endocrine deﬁciency to be present.
Some women have a high incidence of anovulatory cycles
or poor development of the corpus luteum with inade-
quate secretion of progesterone. Obesity is known to
contribute to anovulation and infertility. Careful monitor-
ing of basal body temperature (BBT) and the measure-
ment of luteal progesterone are helpful in determining the
If there is cyclic hormonal secretion with amenorrhea,
degeneration of the endometrium or adhesions within the
uterus are indicated. Once associated with tuberculosis,
this is now rare.
Examination of the male partner is indicated if there is
normal endocrine function and menstruation in the
woman. In some cases where both partners appear endo-
crinologically normal, and hostility of the secretions of the
female tract has been excluded, sex counseling may be
IN THE MALE
LH and FSH concentrations are increased because of
reduced feedback by testosterone and inhibin. Testoster-
one may be very low or only slightly reduced.
Males with Klinefelter’s syndrome have an extra
X chromosome. The classical form is XXY although cases
with further additional X chromosomes have occurred.
Most cases are diagnosed after the age of 17 years. A study by
724 The Immunoassay Handbook
the Klinefelter’s Syndrome Association (www.ksa-uk.
co.uk) found that 16% of patients were diagnosed under
the age of 10 years, 11% between 11 and 17 years, 29%
between 18 and 28 years and 39% between 29 and 55 years.
Affected men typically have small, ﬁrm testes and a tall
stature with eunuchoid appearance and gynecomastia.
Social maladjustment and low IQ are also reported. Tes-
tosterone levels range from very low to just within the
normal reference range. Gonadotropin concentrations are
elevated. The syndrome has an incidence of about 0.2%,
and diagnosis is conﬁrmed by chromosome analysis.
Patients with male Turner’s syndrome have features
similar to women with Turner’s syndrome but their chro-
mosome pattern is normal. Affected individuals have small,
soft testes, low testosterone, and increased gonadotropin
levels. In anorchia (absence of the testes), patients remain
prepubertal. In the Sertoli cell-only syndrome, the tes-
tes are almost normal in size although the germ cells are
completely absent. The seminiferous tubules are diagnos-
tically not hyalinized as in the other conditions above.
Because Leydig cell function is normal, LH and testoster-
one levels are normal. The azospermia is associated with
increased FSH concentrations.
Viral orchitis is the most common type of acquired tes-
ticular failure. About 30% of men who develop orchitis
after puberty experience testicular atrophy. Other causes
of acquired testicular failure are trauma, radiation, and
drugs. Damage by radiation is related to duration and
dose. Spironolactone, ketoconazole, and ethanol lower
testosterone levels by inhibiting its synthesis.
Primary testicular dysfunction is also associated with a
number of systemic diseases such as renal failure and cirrhosis
of the liver. It is difﬁcult to determine whether the effect on
the testes is due to the disease or concomitant malnutrition.
Androgen resistance (pseudohermaphrodism: Reif-
enstein’s syndrome, testicular feminization) is charac-
terized by a complete or incomplete female phenotype.
Unlike primary hypogonadism, testosterone concentra-
tion is normal or even elevated in the presence of increased
LH and FSH values.
IN THE MALE
As in the female, hypogonadism can result from dysfunc-
tion of the pituitary, hypothalamus, or higher brain centers.
Isolated gonadotropin deﬁciency may occur as in Kall-
man’s syndrome or be part of a more generalized pitu-
itary failure. In the former condition, individuals do not
have a normal pubertal development and typically have
anosmia (absence of a sense of smell). This and other forms
of hypogonadotropic hypogonadism (e.g., Prader–Willi
and Bardet–Biedl syndromes), are a result of a hypotha-
lamic defect. This is indicated by the efﬁcacy of repeated
administrations of GnRH therapy, which can eventually
stimulate LH and FSH secretion by the pituitary.
Hyperprolactinemia due to a pituitary adenoma causes
infertility and azospermia. Men usually present with head-
aches and visual problems, caused by a large pituitary
tumor, rather than with infertility. Prolactin seems to
interfere with the normal synthesis of LH, FSH, and
Men with anorexia nervosa and with psychogenic
infertility show gonadotropin secretion similar to women.
Psychiatric treatment, rather than hormone replacement,
is required and if successful can restore normal hormone
secretion. Frequently psychogenic infertility is accompanied
by impotence, but in most cases, gonadotropin and testos-
terone concentrations remain within the normal range.
With the availability of more convenient modes of testos-
terone treatment, there has been greater interest in the loss
of sexual function in the older male, a condition now
referred to as Late Onset Hypogonadism. This has led to
much discussion as to which men should be treated. It is
agreed by the professional bodies that a man with a testos-
terone concentration <7nmol/L (202ng/dL) has hypogo-
nadism. The difﬁculty lies in the range from 7nmol/L to the
lower limit of normal due to the variability in a) testosterone
methods, b) the testosterone secretion, and c) the quoted
reference ranges by laboratories (partly methodological and
partly due to ethnicity of the reference population). It is
suggested that for testosterone levels in the equivocal
region, a free testosterone or bioavailable testosterone level
should be obtained to clarify the diagnosis. The professional
bodies also agree that only aging men with low testosterone
concentrations should be given replacement therapy.
IMPAIRED SPERM TRANSPORT
AND SPERM VIABILITY
The WHO current guidelines (Cooper et al., 2010) deﬁne an
adequate semen specimen as having a volume ≥1.5mL, a
sperm count ≥15million/mL, total motility ≥40%, total
sperm number ≥39million/ejaculate, vitality ≥58%, and Kru-
ger strict morphology ≥4%. These are evidence-based values
differing from previous WHO guidelines. The grading of
motility has also changed from four to three categories: pro-
gressive motility, nonprogressive motility, and immotile.
Infertility in the male may result from an obstruction in
the reproductive tract, either unilateral or bilateral. Hor-
mone concentrations are normal, and diagnosis is made
from seminal analysis, vasograms, and, if necessary, testicu-
lar biopsy. Poor sperm viability is another cause of infertility.
There may be oligospermia, a greater than normal percent-
age of abnormal forms, poor motility, or the presence of
antisperm antibodies. An increase in FSH concentrations is
usually the only hormone abnormality although the levels of
this hormone are frequently normal. Treatment with testos-
terone or gonadotropins is frequently unsatisfactory.
LUTEINIZING HORMONE (LUTROPIN)
LH is structurally similar to FSH, thyroid stimulating
hormone/thyrotropin (TSH), and human chorionic gonad-
otropin (hCG). They are all glycoproteins consisting of
two subunits. The α subunit is similar in all four hor-
mones, but the β subunit is unique to each of them. The
latter subunit confers biological activity to the hormone
by governing receptor-binding speciﬁcity. The LH mol-
ecule is about 30kDa with 89 amino acids in the α chain
and 129 amino acids in the β chain. The carbohydrate
content is between 15 and 30%.
725CHAPTER 9.5 Infertility
LH is both stored and secreted by the anterior pituitary. In
men, it acts on the Leydig cells of the testes, stimulating
the synthesis and secretion of testosterone. In women, LH
is involved in the development of the follicle, ovulation at
midcycle, and maintenance of the corpus luteum. It is
cleared by the liver and has a biphasic half-life of about 40
Synthesis and secretion of LH are controlled by the
stimulation of the gonadotroph cells of the anterior pitu-
itary by GnRH from the hypothalamus. GnRH is secreted
as pulses with a frequency of about 90min in men and in
the follicular phase of women. This is reﬂected in the epi-
sodic secretion of LH. The amount of GnRH and LH
secreted is, in turn, controlled by the negative feedback of
the sex steroids released from the gonads. Thus, high ste-
roid levels suppress LH secretion and low levels increase
it. However, in the middle of the menstrual cycle, estradiol
exerts a positive feedback, stimulating LH secretion.
All current immunoassay systems use immunometric
assays to measure LH (see Table 1).
LH levels are increased in patients who have suffered pri-
mary gonadal failure. Conversely, where there is hypotha-
lamic dysfunction (hypogonadotropic hypogonadism,
anorexia nervosa), the concentration of LH is low. It may
also be low in any cause of hypopituitarism; for example, it
may be low or normal when a pituitary tumor is present.
Therefore, if the concentration of LH is low, other pitu-
itary hormones should be investigated. LH secretion is very
variable in the perimenopausal period, and FSH should be
measured instead. LH levels are normal in many cases of
infertility, and other hormone assays are required. The
pituitary reserve of LH can be examined with a GnRH test.
This may be particularly helpful after pituitary surgery or
in the investigation of hypogonadotropic hypogonadism.
G The technical limitations are the same as those
described for TSH (see THYROID).
G LH is suppressed by estrogen, but in women taking oral
contraceptives, the concentration of LH may be nor-
mal or low.
G Excessive dieting and weight loss may lead to low
G It is not uncommon for amenorrhea to be investigated
in a woman who presents with amenorrhea but is, in
fact, pregnant. Most current immunometric assays
have little or no cross-reaction with hCG, and both LH
and FSH concentrations will be very low or undetect-
able. Vivekanandran and Andrew (2002) have reported
that the DPC Immulite method has sufﬁcient cross-
reaction with hCG to give a falsely detectable LH con-
centration. The UK NEQAS scheme has shown that a
hCG concentration of 16kIU/L results in an LH result
of between 7 and 9IU/L in the Bayer Immuno 1 and
the DPC Immulite LH assays. In pregnancy, estradiol,
testosterone, SHBG, and prolactin concentrations will
be elevated. In these circumstances, the hCG concen-
tration should be measured in the same serum sample.
G Some pairs of monoclonal antibodies in immunometric
assays fail to recognize certain epitopes of LH that
seem to be biologically active. This is discussed more
fully in ASSAY TECHNOLOGY below.
G The presence of heterophilic antibodies in serum may
lead to erroneous results, which may be higher or lower
than the true value. This problem may be encountered
in any immunometric assay using two monoclonal anti-
bodies. Ismail et al. examined the TSH, LH, and FSH
results of 5310 patients. They reported that 0.53% of
results were analytically incorrect due to interference.
G LH is released in a pulsed manner. The value in a single
blood specimen may not be representative of the 24-h
secretory mean level.
GnRH Stimulation Test
GnRH is a decapeptide secreted by the hypothalamus. It has
been synthesized and is available commercially. An intrave-
nous injection of 100µg GnRH is given, and blood is taken
at 0, 20, and 60min. A normal response shows an increase in
LH values of 5- to 10-fold at 20min over the basal level. At
60min, LH levels usually decrease again but may still
increase slightly in a small proportion of patients (see Fig. 3).
An exaggerated response is found in patients with pri-
mary hypogonadism and is also typical of the polycystic
ovary syndrome (see HIRSUTISM AND VIRILIZATION IN THE
FEMALE). However, these observations are rarely of help in
diagnosis. Both hypothalamic and pituitary dysfunction
may lead to little or no response to a GnRH test, and
therefore, a lack of response does not indicate pituitary
dysfunction per se. When hypothalamic dysfunction is
present, repeated injections or pulsed administration of
GnRH will lead to increased secretion of the gonadotro-
pins. A GnRH test is also helpful in establishing whether
gonadotroph function is normal after pituitary surgery.
All participants in the UK NEQAS for LH and FSH use
non-isotopic immunometric assays run on fully automated,
random-access analyzers. The immunometric technique has
the beneﬁts of faster assays, a wider working range and fre-
quently greater sensitivity. The Roche analyzer has the great-
est number of users in UK NEQAS. The between laboratory
agreement for all methods in 2010 is approximately 15%.
The between laboratory agreement for users of a single
method is <10%. Companies have strived to remove
TABLE 1 Reference Intervals for LH
Roche (IU/L, IS 80/552)
Women Follicular phase 2.4–12.6
Luteal phase 1.0–11.0
726 The Immunoassay Handbook
interferences from their assays, but one should be aware of
these interferences when viewing abnormal results in partic-
ular. Some individuals have anti-mouse antibodies in their
serum which can interfere with antibody binding in these
assays. Also when analyte concentrations are very high in
patient sera, suppression of tracer binding may occur, leading
to falsely low results (the “high-dose hook” effect). A knowl-
edge of the limitations of each assay is therefore very impor-
tant when choosing which one to use.
Two monoclonal antibodies are usually used in immu-
nometric assays. The epitopes of LH measured in a method
depend on the pair of monoclonal antibodies used. In some
patient sera, signiﬁcant differences in LH results have
been found between kits. Some women produce a genetic
variant of LH, which is biologically active but not recog-
nized by some monoclonal antibodies. Undetectable LH
values are recorded in these patients, who have normal
cyclical activity of their steroids.
Some immunometric assays have a polyclonal capture
antibody and a monoclonal-labeled antibody for signal gen-
eration, an arrangement which overcomes most of the prob-
lems associated with a dual monoclonal antibody system.
In routine clinical laboratories, totally automated random-
access analyzers are now used to perform this assay. Such
assays have a sensitivity of about 0.1IU/L and may have a
total imprecision of <3% over the normal reference range.
Types of Sample
Normally serum or plasma. However, some kits are only
suitable for serum.
Frequency of Use
As with LH, FSH has two subunits, α and β. The β subunit
differs between the two hormones and bestows their inde-
pendent biological activities. FSH is cleared more slowly
from the circulation than LH and has a biphasic half-life of
3.9 and 70h. Its control by the hypothalamus is similar to
that already described for LH but, because of its longer
half-life, episodic secretion is less obvious. Stimulation of
the pituitary with exogenous GnRH, either by a bolus
injection or by pulsed infusion, stimulates FSH synthesis
and secretion. FSH acts on the granulosa cell of the ovary,
stimulating steroidogenesis. It is involved in the develop-
ment of the next cohort of follicles at the beginning of
each menstrual cycle. It does not seem to be essential for
either ovulation or the maintenance of the corpus luteum.
Secretion of FSH from the hypothalamus and pituitary is
controlled by the negative feedback of estradiol, testoster-
one, and inhibin.
In men, FSH acts on the Sertoli cells of the testis, stimu-
lating the synthesis of inhibin and androgen-binding pro-
tein. Thus, it indirectly supports spermatogenesis. The
negative feedback of testosterone on the pituitary and
hypothalamus controls FSH in the same way as LH. How-
ever, inhibin speciﬁcally inhibits FSH secretion. In cases
of azospermia, inhibin concentrations are low, and FSH
levels are speciﬁcally increased.
FSH is measured by immunometric assay, and in most lab-
oratories, will be measured along with LH (see Table 2).
FSH is diagnostically the best hormone for conﬁrming
primary hypogonadism in women. In general, changes in
FSH and LH concentrations are concurrent in women.
However, in men with a spermatogenic defect FSH con-
centrations can be increased when LH and testosterone
concentrations are normal. Increased LH levels indicate
dysfunction of the Leydig cells.
Technical limitations are the same as described for TSH
(see THYROID) and LH (above). The problem encountered
with the measurement of LH epitopes by immunometric
assays has not been reported for FSH methods at present.
The effects of heterophilic antibodies on an immunomet-
ric assay system are the same as for LH.
FIGURE 3 Examples of LH responses in LHRH stimulation tests. LH
concentrations after injection of 100 µg LHRH in normal subjects,
patients with hypopituitarism and patients with primary hypogonadism.
TABLE 2 Reference Intervals for FSH
Roche (IU/L, IS 94/632)
Women Follicular phase 3.5–12.5
Luteal phase 1.7–7.7
727CHAPTER 9.5 Infertility
Methods for LH and FSH have been developed in parallel
since these hormones are usually measured at the same
time in most laboratories. Assays have a similar sensitivity
and imprecision to the LH assays. Between laboratory
agreement is <10% for participants in UK NEQAS.
Types of Sample
Serum or plasma.
Frequency of Use
Prolactin is a protein hormone secreted from the lacto-
trophs of the anterior pituitary. In most people, the major-
ity of prolactin circulates as a monomer consisting of a
single 23kDa polypeptide chain of about 200 amino acids.
Other variants also circulate. Big prolactin has a molecular
mass of about 50kDa and makes up most of the remaining
prolactin. The remainder is big big prolactin or macropro-
lactin. If these variants are present in high amounts, they
can cause problems in the measurement of prolactin as will
be discussed later. The main action of prolactin is on the
mammary gland where it is involved in the growth of the
gland and in the induction and maintenance of milk pro-
duction. There is evidence that it may be involved in ste-
roidogenesis in the gonad, acting synergistically with LH.
Certainly, very high levels of prolactin seem to inhibit ste-
roidogenesis as well as having a local effect on the pitu-
itary, inhibiting LH and FSH production.
Prolactin shows a noticeable circadian rhythm, being
elevated during sleep. It rises rapidly after conception and
continues to rise until the third trimester. After delivery,
levels in the suckling mother rapidly fall but may be main-
tained for several months at concentrations just above the
reference range. Suckling itself stimulates the release of
Prolactin measurement is used for the diagnosis of hyper-
prolactinemia and for monitoring the effectiveness of sub-
sequent treatment. Microadenomas can be treated with
the dopamine agonists, such as bromocriptine, carbergo-
line, and pergolide. Bromocriptine therapy usually reduces
the size of even large prolactin-secreting pituitary tumors,
and therefore, pituitary surgery is only rarely required
nowadays. Where surgery is deemed necessary dopamine
agonist treatment may be given ﬁrst to shrink the size of
the tumor making subsequent surgery easier.
G Because prolactin is raised during sleep, particularly just
before waking, it has been recommended that blood for
prolactin measurement should be taken 2h after sleep.
G Moderate increases of up to twice the upper limit of the
normal range can occur in stressed patients and after mild
exercise (e.g., climbing several ﬂights of stairs). Therefore,
it is important that patients are well rested before blood is
taken. Multiple venepuncture may also increase prolactin
levels. In particularly anxious patients, especially when a
slightly raised prolactin level has been previously reported,
some clinicians make use of a butterﬂy needle. After inser-
tion of the needle, the patient is left to rest for about
20min before collecting blood. Others prefer to take
blood every 10min for 30min. A fall in prolactin is
observed if the raised prolactin level was due to stress.
G Prolactin is elevated in about 30% of acromegalics, in
hypothyroidism (in concert with TSH), and in polycys-
tic ovarian disease. A moderate increase is reported in
epileptic patients immediately following a seizure.
G Rarely after pituitary surgery, very high concentrations
of prolactin may be found, with a steady fall in levels
over the following months. Presumably, this represents
residual pituitary tissue, containing lactotrophs that are
no longer under hypothalamic control. This residual
tissue gradually regresses.
G Prolactin secretion is increased by drugs such as reser-
pine, methyldopa, morphine, metaclopromide, and the
psychotropic drugs. These impair the action of dopa-
mine from the hypothalamus, which inhibits prolactin
secretion. A careful drug history and clinical examina-
tion is therefore required before hyperprolactinemia
due to a prolactinoma can be diagnosed.
G Prolactin is also increased in hyperthroidism due to
stimulation by the increased levels of thyrotropin-
releasing hormone (TRH).
G In some patients, monomeric prolactin circulates bound
to a 150kDa IgG molecule. This form is called macro-
prolactin, is not thought to be biologically active and
can lead to falsely high results. It has been reported that
up to 26% of cases of hyperprolactinemia are due to the
presence of macroprolactin. The degree to which assays
detect macroprolactin varies. Most laboratories now
screen for the presence of macroprolactin, in specimens
with an elevated prolactin concentration, using a simple
PEG precipitation method (see Fahie-Wilson et al.,
1997; Leslie et al., 2001). One must ensure that the assay
system being used is unaffected by the presence of PEG.
Occasionally, where results do not appear to ﬁt the clin-
ical picture, estimation of macroprolactin should be car-
ried out by gel ﬁltration chromatography.
Some workers have advocated the use of a TRH test to distin-
guish hyperprolactinemia due to a prolactinoma from other
causes. A normal response shows a marked increase of prolac-
tin at 20min with prolactin levels falling at 60min. Patients
TABLE 3 Reference Intervals for Prolactin
Women 6.0–29.9ng/mL 127–637mIU/L
(third IRP 84/500)
Men 4.6–21.4ng/mL 98–456mIU/L
(third IRP 84/500)
728 The Immunoassay Handbook
with a prolactinoma have a ﬂat response. However, many
have found the test to be unreliable, and it is little used today.
An intravenous, bolus injection of 200µg is given. Blood is
taken at 0, 20, and 60min. A normal response is an increase
of 100% over the basal concentration or a threefold
increase of the basal concentration (see Carmine and
Lobo, 1997). Patients with a prolactinoma frequently show
a suppressed or ﬂat response.
As with LH and FSH prolactin is measured by immuno-
metric assay. Despite the presence of dimeric as well as
monomeric prolactin, there is a good agreement between
assays. Routine measurement of prolactin is carried out in
routine clinical laboratories on fully automated immunoas-
say analyzers. These assays have sensitivities down to about
10mIU/L, and a total imprecision of <3% over the refer-
ence range. The between laboratory agreement between
all methods for participants in UK NEQAS is about 20%.
This high ﬁgure is a result of the variation between meth-
ods since the between laboratory agreement for a single
method is <10%.
Types of Sample
Serum or plasma.
Frequency of Use
AMH is a dimeric glycoprotein of the transforming
growth factor β family of growth and differentiation fac-
tors. It is located on chromosome 19p13.3. Until recently,
the role of AMH was associated with the sexual differen-
tiation of the male. It has now been shown to be secreted
by the granulosa cells of the developing follicles. Its
actions in the adult ovary are mainly autocrine and para-
crine via a transmembrane serine/threonine kinase type
II receptor. It is secreted by granulosa cells <8mm in size,
and serum levels of AMH have been shown to be propor-
tional to the number of developing follicles in the
At the time of writing, Beckman Coulter is the only com-
pany supplying a kit for the measurement of AMH. The
expected values this company quotes for the AMH Gen II
ELISA method are shown in Table 4.
AMH measurement in the UK appears to be mainly for
investigating ovarian reserve. This may be in the investi-
gation of premature menopause, pre-IVF treatment, or
infertility. AMH has also been shown to be increased in
patients with polycystic ovarian syndrome and can help
with a diagnosis where reliable ultrasound is not avail-
able. AMH might also be helpful in predicting normaliza-
tion of menstrual function with weight loss in these
patients. Equally, AMH could be helpful in assessing
ovarian function and normalization of menstrual cycles in
anorectic patients. Because of the large difference in
AMH levels between young boys and young girls, some
clinicians are using AMH measurement to assess intersex
disorders. Recently, the UK National External Quality
Assessment Scheme carried out a survey of the clinical
settings for AMH measurement. The results are given in
Currently Beckman Coulter is the only company with a kit
for AMH, the Gen II ELISA. This is an enzymatically
ampliﬁed two-site immunoassay. The wells of a microti-
tration plate are coated with anti-AMH antibody.
TABLE 4 Expected Values for Anti-Müllerian Hormone
Samples Median Age (y) Median Conc (ng/mL) 2.5–97.5 Percentile (ng/mL)
Random males (n=136) 38 5.7 1.3–14.8
Random females (n=95) 30 2.4 ND–12.6
Males fertility clinic (n=100) 37 5.3 0.8–14.6
Females third of cycle (n=106) – 1.5 ND–10.6
Postmenopausal females (n=45) 71 ND ND
Boys (n=36) 4.8 56.3 3.8–159.8
Girls (n=36) 5.0 1.3 ND–8.9
(Beckman Coulter AMH Gen II ELISA)
TABLE 5 Clinical Setting for the Measurement of AMH
Clinical Setting No. of Labs
Investigation of infertility 40
Assisted conception assessment 35
Polycystic ovary syndrome 27
Prediction/assessment of the
Pediatrics (intersex disorders) 15
Tumor marker 12
Assessment of puberty 11
729CHAPTER 9.5 Infertility
Types of Sample
Serum and lithium heparin plasma.
Frequency of Use
The workload of laboratories varies considerably. The sur-
vey carried out by UK NEQAS showed that the lowest
workload was 150 tests per year and the highest 50,000
The inhibins are glycoproteins that are heterodimers.
They comprise a common alpha subunit and one of two
beta subunits. In women, inhibin B is produced by the
developing follicles, while inhibin A is produced by the
corpus luteum. In men, only inhibin B is secreted from
the Sertoli cells of the testes.
Using the most commonly reported assay developed by
Groome, concentrations of inhibin B in normal men are
reported as 187±28ng/L.
Inhibin A and B concentrations change markedly dur-
ing the menstrual cycle. Peak levels of inhibin B occur in
the early follicular phase when concentrations are reported
as 86.8±13.8ng/L, whereas inhibin A concentrations
peak in the mid-luteal phase to 59.5±15ng/L. The surge
of inhibin A at midcycle reaches concentrations of
Inhibins have been investigated as a marker in several
areas of gynecology and obstetrics. It has been shown
that the measurement of inhibin A helps in assessing the
risk of Down’s syndrome in pregnancy, and with AFP,
βhCG, and estriol, has led to the quadruple test for
Down’s syndrome screening (see Wald et al., 2003). A
recent study (Wald et al., 2003) indicated that this is the
best test for women who present in the second trimester
of pregnancy. For women who present in the ﬁrst trimes-
ter of pregnancy, the integrated test, which combines
markers from the ﬁrst and second trimesters, is the test of
Inhibin measurement is also used in the assessment of
IVF treatment, and its use is also being investigated in the
investigation of the perimenopause, premature ovarian
failure, and as a tumor marker. No clinical use has been
established for the inhibins in the investigation of
The ﬁrst assay to be developed for the measurement of
inhibin was a radioimmunoassay and is referred to as the
Monash assay. Although it was thought that this assay
measured dimeric inhibin, it was later found to cross-
react with the circulating α-subunit and other nonbio-
logically active forms of inhibin. Speciﬁc ELISAs for
inhibin A and B, developed by Professor Nigel Groome
at the Oxford Brooke University, UK, have been used in
a large number of studies. Other groups are now devel-
oping their own in-house assays, and commercial assays
are available, using Professor Groome’s reagents, from
Types of Sample
Frequency of Use
The major estrogens in man are estradiol, estrone, and estriol,
of which estradiol is biologically the most active (see Fig. 4).
The estrogens are characterized by a phenolic A ring. A
small amount of estradiol, which may constitute as much
as 30% of the total estrogen production in some men, is
produced by the testes. The ovaries are the main source of
estrogen in the nonpregnant woman. During the follicular
phase of the menstrual cycle, there is a steady increase in
the concentration of estradiol, which reﬂects the follicular
growth in the ovary. Over the ﬁrst 10 or 11 days of the
cycle, estradiol has a negative feedback on the pituitary.
This is reﬂected by a small fall in FSH concentration over
this period. At about day 12, the feedback of estradiol
becomes positive and stimulates the mid-cycle surge of
LH and FSH at day 14. There is a fall in estradiol levels at
mid-cycle, but the concentrations increase again in the
luteal phase, peaking at mid-cycle.
In postmenopausal women, most of the estrogen pro-
duced is from the peripheral conversion of androstenedi-
one to estrone (15–60µg per day). About 95% of the
androstenedione production in the postmenopausal woman
is from the adrenal glands. Androstenedione is converted
to estrone in peripheral body fat. Therefore, the more body
fat there is, the higher the estrone concentration.
Estradiol circulates in the blood bound to SHBG and
albumin. Only 1–3% of the total circulating estradiol
remains unbound. It is a matter of great controversy at the
moment whether any or all of the bound steroid is available
to tissues (see REFERENCES AND FURTHER READING).
Estradiol is the main reproductive hormone in women
and is involved in the development and maintenance of the
reproductive organs. It is responsible for the development
of the reproductive tract in the fetus and the female habi-
tus at puberty. In the adult, it is involved in the maturation
and maintenance of the uterus during the menstrual cycle
and the control of female reproductive behavior. It is also
essential for the development of the mammary gland and
FIGURE 4 Estradiol.
730 The Immunoassay Handbook
Although estradiol estimation is one of the most frequently
requested hormone tests, its usefulness in the investigation
of infertility in women is generally considered to be lim-
ited (see FURTHER READING). A progestogen challenge is
more helpful in establishing estrogenization of the uterus.
A single intramuscular injection of 100mg progesterone
results in uterine bleeding within the next 7 days in women
who have sufﬁcient estrogen to produce endometrial pro-
liferation. It also demonstrates that the ovary is responding
to LH and FSH secretion from the pituitary and that the
endometrium is responsive to estrogen and progesterone.
Estradiol is not helpful in establishing whether a woman
has entered the menopause. The measurement of FSH is
the most useful test in this case.
There is a large variation in the estradiol concentra-
tion both within and between women receiving hormone
replacement therapy. This is especially so in women who
are taking tablets or who have had implants. Levels are
more constant when women have patches containing
estradiol. However, menopausal symptoms frequently
do not correlate with the estradiol concentration, so
again the measurement of total estradiol is unhelpful.
G There is a large overlap of the reference intervals during
the menstrual cycle and for the menopause. This can
make the interpretation of estradiol results difﬁcult.
G Drugs can affect the ﬁnal estradiol result either by
cross-reacting in the assays, e.g., danazol metabolites or
by altering SHBG concentrations, e.g., androgens and
G Estradiol results in women on oral contraceptives are
unreliable due to the variable cross-reaction of syn-
thetic and horse estrogens, in these preparations,
with the estradiol antibodies used in different assays.
For the purist, the only accurate way to measure steroids is
by solvent extraction and column chromatography followed
by radioimmunoassay. Chromatography may be required to
remove estrone, which can have a high cross-reaction with
antiserum to estradiol. However for routine purposes, most
laboratories now use commercial non-extraction methods.
Although the majority of laboratories use automated
non-isotopic methods, some participants of UK NEQAS
use kit methods, both radioactive and nonradioactive.
A working range of about 50–2000pmol/L is required for
the investigation of infertility, whereas for IVF purposes, a
range of 150–15,000pmol/L is needed. No single assay can
adequately cover the complete range of 50–15,000pmol/L.
Estradiol assays are available on all the current automated
immunoassay analyzers, but they generally have a functional
sensitivity of about 150pmol/L. Recently launched assays
are showing slightly improved functional sensitivity. Recent
reports show poor precision and bias of many of these assays
especially at low concentrations. Between laboratory agree-
ment is 20–30% for concentrations <150pmol/L falling to
about 15% at concentrations >400pmol/L.
Types of Sample
Serum or plasma.
Frequency of Use
Progesterone is produced from pregnenolone in all steroid-
producing cells. It can then be further synthesized to
17α-hydroxyprogesterone or androstenedione. Large
amounts (up to 30mg per day) are produced by the corpus
luteum and by the placenta (between 250 and 500mg per
day in late pregnancy) (see Fig. 5).
The main site of action of progesterone is on the uterus
where, during the luteal phase, it increases the vascular
bed, the tortuosity of the glands, and glandular secretion,
and reduces myometrial activity. In this way, it prepares
the uterus for implantation and supports the developing
fetus. During pregnancy, progesterone is required for the
maintenance of the placenta.
TABLE 6 Reference Intervals for Estradiol
Women Follicular phase 12.5–166 46–607
Mid-cycle 85.5–498 315–1828
Luteal phase 43.8–211 161–774
Postmenopausal 5.0–54.7 18.4–201
FIGURE 5 Progesterone.
TABLE 7 Reference Intervals for Progesterone
Women Follicular phase 0.2–1.5 0.6–4.7
Mid-luteal phase 0.8–3.0 2.4–9.4
Luteal phase 1.7–27 5.3–86
Postmenopausal 0.1–0.8 0.7–4.3
731CHAPTER 9.5 Infertility
The measurement of progesterone concentrations is used
to show that ovulation has occurred, and the corpus luteum
is functioning normally. A single sample may be inconclu-
sive and three samples of blood may be taken around the
mid-luteal phase to determine whether secretion of pro-
gesterone is adequate.
Progesterone measurement is also used in some centers
during IVF therapy. Concentrations are monitored just
before ovulation is due, when rising levels indicate that
ovulation has or is about to occur.
A single blood specimen taken during the luteal phase may
be inadequate as an indicator of normal luteal function. In
a cycle of normal length, three specimens, on days 19, 21,
and 23, are more informative.
BBT rises when progesterone levels begin to increase at
mid-cycle. However, BBT is quite variable between cycles
and can be difﬁcult to interpret. A rise in BBT will usually
indicate that ovulation has occurred but does not suggest
that corpus luteum function or progesterone concentra-
tions are normal.
Very few laboratories use methods with solvent extraction
before immunoassay; most use commercial immunoassays.
Progesterone assays are available on all automated systems.
Between laboratory agreement between participants in UK
NEQAS is >20% at concentrations <10nmol/L. This is a
result of method biases since between laboratory agreement
for a single method is <10% even at low concentrations.
Types of Sample
Serum or plasma.
Frequency of Use
During fetal life, at approximately 12 weeks, there is a rise
in testosterone concentration in the male fetus due to
stimulus of the Leydig cells in the developing testes by
hCG. Testosterone falls to low levels in the third trimester
of pregnancy, but there is another increase in the male
neonate after about 3 weeks of life, reaching a maximum at
about 2 months. Concentrations may be almost into the
adult normal range at peak secretion. After about 6 months,
the concentration falls to less than 1.0nmol/L (0.3ng/mL)
and remains at low levels until puberty (Fig. 7).
Testosterone is the main male sex hormone and is secreted
by the Leydig or interstitial cells of the testes. Small amounts
of testosterone are secreted by the ovary and the adrenal, but
about 50% of the testosterone production in women is
derived from androstenedione by peripheral conversion.
Concentrations are less than 1nmol/L (0.3ng/mL) pre-
pubertally. There is a gradual rise to adult levels during
puberty in the male. Initially, an increase in concentration
occurs at night, but as puberty progresses, daytime levels
also increase. In the adult, secretion of testosterone is epi-
sodic. There is a small circadian rhythm, which, until
recently, has been regarded as clinically insigniﬁcant. We
have shown that if testosterone is measured in the serum of
normal men during the afternoon, concentrations may be
below the reference interval. In such cases, normal men
may be wrongly diagnosed as hypogonadal (see Fig. 6).
Testosterone in the male has a negative feedback on the
hypothalamus and pituitary. Most of the testosterone cir-
culates in blood bound to SHBG and albumin. About 2%
of the total plasma testosterone in men is nonprotein
bound, about 1% in women. It has been suggested that
salivary testosterone represents this “free” fraction.
Testosterone has a variety of actions in the body. In
both sexes, it stimulates secondary sexual hair growth,
alters the concentration of several enzymes of the kidney,
stimulates erythropoiesis, and increases libido, competi-
tiveness, and aggression. In men, it is responsible for the
change in voice at puberty and promotes growth and
development of the sex glands and organs.
FIGURE 6 Testosterone concentrations in men at 9 a.m. and 4 p.m.
FIGURE 7 Testosterone.
732 The Immunoassay Handbook
The measurement of testosterone for the investigation of
hirsutism and virilization in the female is explained in the
Testosterone may be measured in boys with delayed
puberty. Increases in the secretion of the gonadotropins
and testosterone, particularly during sleep, indicate that
puberty is progressing. Random blood samples may be
taken at 3- to 6-month intervals to monitor testosterone
levels; an increase in concentration may precede deﬁnite
clinical evidence of puberty.
Infertile men may have normal testosterone concentra-
tions. In these cases, seminology tests should be done. Low
libido may be related to low testosterone concentrations,
but impotence is more commonly associated with neuro-
pathic, vascular or psychogenic causes with normal testos-
Low testosterone concentrations may be due to pri-
mary or secondary hypogonadism. Measurement of the
gonadotropins aids the diagnosis; increased gonadotro-
pins indicate primary hypogonadism. The capacity of the
testes to secrete testosterone can be determined from a
Treatment of infertility in men is often unsuccessful
although pulsed GnRH therapy has been found effective
in patients with hypogonadotropic hypogonadism, and in
some cases, the GnRH may be supplemented with FSH to
achieve complete spermatogenesis. Injections of hCG are
also used to stimulate testosterone production and again
FSH may be used as a supplement to therapy to achieve
spermatogenesis. In primary hypogonadism, one of several
injectable analogs can be used to maintain libido and
androgenization. In these cases, there is usually no treat-
ment for infertility.
There are a variety of regimens in use. The one given here
is our standard protocol to investigate the ability of the
adult testes to secrete testosterone. Blood specimens taken
on day 1 and day 4 are usually adequate for clinical
diagnosis (see Table 9 and Fig. 8).
G Total testosterone measurement is greatly inﬂuenced
by the level of SHBG, which is increased by estrogens
and anticonvulsants, in cirrhosis of the liver and some
cases of hypothyroidism. In these situations, the con-
centration of free testosterone may be low despite a
normal total testosterone level. Anticonvulsant therapy
is associated with primary hypogonadism with elevated
LH and FSH concentrations, but this is masked by the
increased SHBG concentrations. Low testosterone
values are found in patients with a variety of systemic
diseases. Many centers measure serum SHBG concen-
trations in addition to total testosterone to provide an
indication of free testosterone levels (see HIRSUTISM
AND VIRILIZATION IN THE FEMALE).
G Testosterone is released in a pulsed manner. The value
in a single blood specimen may not be representative of
the 24-h secretory mean level. There is a marked circa-
dian rhythm in men, and blood samples should be taken
preferably between 9 and 10 am.
Direct testosterone assays are now available on most auto-
mated immunoassay analyzers. The performance of the
methods varies widely as does the agreement. Some meth-
ods appear to suffer from either interference or cross-
reaction from unidentiﬁed substances in serum that leads
to spuriously high results. It has not been possible to asso-
ciate these results with any particular clinical condition. As
TABLE 8 Reference Intervals for Testosterone
Women 0.06–0.82ng/mL 0.22–2.9nmol/L
Men 2.8–8.0ng/mL 9.9–27.8nmol/L
FIGURE 8 Percentage change in testosterone concentration after
1500IU hCG after 1, 2 and 3 days in normals (dashed lines) and men
with primary hypogonadism (solid lines).
TABLE 9 Regimen for hCG test
Day 1 Blood sample Inject 1500 IU hCG
Day 2 Blood sample Inject 1500 IU hCG
Day 3 Blood sample Inject 1500 IU hCG
Day 4 Blood sample
733CHAPTER 9.5 Infertility
well as interference, these methods also have poor sensi-
tivity, the functional sensitivity being usually no more
than 1.5nmol/L. Some laboratories are now using tandem
mass spectrometry to measure low concentrations of tes-
tosterone in female serum. However, imprecision of these
methods is often >20%. Even at concentrations in the
male normal range, some methods show high imprecision.
Methods that employ solvent extraction before immuno-
assay have greater sensitivity. These methods can have a
functional sensitivity of 0.1nmol/L and can be further
modiﬁed so that the low levels of testosterone in saliva and
hair can be measured. In 2007, The Endocrine Society
published a position paper on the measurement of testos-
terone. They do not recommend the use of direct com-
mercial assays for total testosterone for the investigation
of levels found in women and children.
Free testosterone radioimmunoassay kits are marketed
by DPC and DSL. Good correlation between results
from the DPC kit and total testosterone, free testoster-
one, and the free androgen index has been reported.
However, it is also reported that the results from this kit
are about half those measured by equilibrium dialysis and
steady-state gel ﬁltration, and it is suggested that the kit
does not measure free testosterone but simply a fraction
of testosterone (see Rosner, 2001). The Endocrine Soci-
ety suggests that one step assays for FT measurement
should be avoided.
Types of Sample
Serum and plasma. Some centers measure testosterone
levels in saliva for research purposes.
Frequency of Use
Testosterone is converted in many tissues to DHT by the
enzyme 5α-reductase. In most bioassays, DHT is more
active than testosterone (Fig. 9).
DHT is required for the normal development of the
prostate, and the urogenital sinus that forms the penis and
scrotum. DHT is also produced in skin, brain, lung, sali-
vary glands, and heart muscle.
The measurement of DHT is used in the investigation of
neonates with ambiguous genitalia, which could result
from a deﬁciency of the enzyme 5α-reductase. Normal
men have a testosterone:DHT ratio of 10:1. This is greatly
increased in 5α-reductase deﬁciency.
Adult men with this deﬁciency present with poor mas-
culinization and a microphallus. Where diagnosis is uncer-
tain, a hCG test may uncover the deﬁciency. Normally,
there is a proportional increase in testosterone and DHT
following stimulation of the testes with hCG, but in
5α-reductase deﬁciency, the increase in the secretion of
DHT is greatly reduced.
The concentration of DHT is very low both in men and in
women, and a sensitive assay is required. In addition, anti-
sera raised against DHT have a signiﬁcant cross-reaction
with testosterone which has a concentration up to 10 times
that of DHT. Thus, testosterone must be removed from
specimens before DHT is measured. This can be achieved
by column chromatography, high-performance liquid
chromatography or the oxidation of testosterone with
potassium permanganate. The latter method was adopted
by Nycomed Amersham (now GE Healthcare) to produce
a research kit for the measurement of testosterone
The above methods use solvent extraction and are tech-
nically difﬁcult. Therefore, the measurement of DHT is
not suited to the routine laboratory.
Types of Sample
Serum or plasma.
Frequency of Use
Test Strategy for
Infertility in Women
Many different strategies are used for investigating infer-
tility. As an example, a simpliﬁed scheme for the investiga-
tion of infertility in women is shown in Fig. 10.
FIGURE 9 5α-Dihydrotestosterone.
TABLE 10 Reference Intervals for DHT
RIA After Extraction and HPLC
Women 0.12–0.43 0.4–1.5
Men 0.38–0.72 1.3–2.5
734 The Immunoassay Handbook
My sincere thanks to UK NEQAS for allowing me to use
some of their data.
References and Further
Balasch, J. Ageing and infertility: an overview. Gynaec. Endocrinol. 26, 855–860
Breckwoldt, M., Zahradrick, H.P. and Neulin, J. Classification and diagnosis of
ovarian insufficiency. In: Infertility: Male and Female, (eds Insler, V. and
Lunenfeld, B.), 229–251 (Churchill Livingstone, Edinburgh, 1993).
Broekmans, F.J., Visser, J.A., Laven, J.S.E., Broer, S.L., Themmen, A.P.N. and
Fauser, B.C. Anti-mullerian hormone and ovarian dysfunction. Trends
Endocrin. Met. 19, 340–347 (2008).
Butler, L. and Santoro, N. The reproductive endocrinology of the menopausal
transition. Steroids 76, 627–635 (2011).
Carmina, A. and Lobo, R.A. Dynamic tests for hormone evaluation. In: Infertility,
Contraception and Reproductive Endocrinology, (eds Lobo, R.A., Mishell, D.R.,
Paulsen, R.J. and Shoupe, D.), 471–483 (Blackwell Science Ltd, Massachusetts,
Cooper, T.G., Noonan, E., von Eckardstein, S. et al. World Health Organization
reference values for human semen characteristics. Hum. Reprod. Update 16,
231–45 (2010). http://www.who.int/reproductivehealth/topics/infertility/
Fahie-Wilson, M.N. and Soule, S.G. Macroprolactin: contribution to hyperprolac-
tinemia in a district general hospital and evaluation of a screening test based on
precipitation with polyethylene glycol. Ann. Clin. Biochem. 34, 252–258 (1997).
Greene, S., Zachmann, M., Manella, B., Hesse, V., Hoepffner, W., Willgerodt, H.
and Prader, A. Comparison of two tests to recognize or exclude 5α-reductase
deficiency in prepubertal childhood. Acta Endocrinol. 114, 113–117 (1987).
Groome, N.P., Illingworth, P.J., O’Brien, M., Pai, R., Rodger, F.E., Mather, J.P.
and McNeilly, A.S. Measurement of dimeric inhibin B throughout the human
menstrual cycle. J. Clin. Endocrinol. Metab. 81, 1401–1405 (1996).
Hampl, R., Snajderova, M. and Mardesic, T. Antimullerian hormone (AMH) not
only a marker for prediction of ovarian reserve. Physiol. Res. 60, 217–223 (2011).
Huhtaniemi, L. and Forti, G. Male late-onset hypogonadism: pathogenesis, diag-
nosis and treatment. Nat. Rev. Urol. 8, 335–344 (2011).
Hwang, K., Walters, R.C. and Lipshultz, L.I. Contemporary concepts in the evalu-
ation and management of male infertility. Nat. Rev. Urol. 8, 86–94 (2011).
Illingworth, P.J., Groome, N.P., Byrd, W., Rainey, W.E., McNeilly, A.S., Mather, J.P.
and Bremner, W.J. Inhibin-B: a likely candidate for the physiologically impor-
tant form of inhibin in men. J. Clin. Endocrinol. Metab. 81, 1321–1325 (1996).
Imperato-McGinley, J., Gautier, T., Pichardo, M. and Shackleton, C. The diagno-
sis of 5α-reductase deficiency in infancy. J. Clin. Endocrinol. Metab. 63,
Ismail, A.A.A., Walker, P.L., Barth, J.H., Lewandowski, K.C., Jones, R. and Burr,
W.A. Wrong biochemistry results: two case reports and observational study in
5310 patients on potentially misleading thyroid-stimulating hormone and
gonadotrophin immunoassay results. Clin. Chem. 48, 2023–2029 (2002).
Leslie, H., Courtney, C.H., Bell, P.M., Hadden, D.R., McCance, D.R., Ellis, P.K.,
Sheridan, B. and Atkinson, A.B. Laboratory and clinical experience in 55
patients with macroprolactinemia identified by a simple polyethylene glycol
precipitation method. J. Clin. Endocrinol. Metab. 86, 2743–2746 (2001).
Luisi, S., Florio, P., Reis, F.M. and Petraglia, F. Inhibins in female and male repro-
ductive physiology: role of gametogenesis, conception, implantation and early
pregnancy. Hum. Reprod. Update 11, 123–135 (2005).
Matzuk, M.M. and Lamb, D.J. The biology of infertility: research advances and
clinical challenges. Nat. Med. 14, 1197–1213 (2008).
Meczekalski, B., Podfigurna-Stopa and Genazzani, A.R. Why kisspeptin is such
important for reproduction. Gynaec. Endocrinol. 27, 8–13 (2011).
Odame, I., Donaldson, M.C.D., Wallace, A.M., Cochran, W. and Smith, P.J. Early
diagnosis and management of 5α-reductase deficiency. Arch. Dis. Child. 67,
Plant, T.M. Hypothalamic control of the pituitary-gonadal axis in higher
primates: key advances over the last two decades. J. Neuroendocrinol. 20,
Rosner, W. An extraordinary inaccurate assay for free testosterone is still with us.
Clin. Endocrinol. Metab. (Letter) 86, 2903 (2001).
Rosner, W., Auchas, R.J., Azziz, R., Sluss, P.M. and Raff, H. Position statement:
utility, limitations and pitfalls in measuring testosterone: an Endocrine Society
position statement. J. Clin. Endocrinol. Metab. 92, 405–413 (2007).
Smith, T.P., Kavanagh, L., Healy, M.-L. and McKenna, T.J. Technology insight:
measuring prolactin in clinical samples. Nat. Clin. Pract. 3, 279–289 (2007).
Stenvers, K.L. and Finlay, J.K. Inhibins: from reproductive hormones to tumor
suppressors. Trends Endocrin. Met. 21, 174–180 (2009).
Suresh, P.S., Rajan, T. and Tsutsumi, R. New targets for old hormones: inhibins
clinical role revisited. Endocrinol. J. 58, 223–235 (2011).
Vivekanandran, S. and Andrew, C.E. Cross-reaction of human chorionic gonado-
trophin in Immulite 2000 luteinizing hormone assay. Clin. Chem. 39, 318–319
Wald, N. Antenatal screening for Down’s syndrome with the quadruple test: 5 year
results from a screening programme. Lancet 361, 835–836 (2003).
Wald, N.J., Rodeck, C., Hackshaw, A.K., Walters, J., Chitty, L. and Mackinson,
A.M. First and second trimester antenatal screening for Down’s syndrome: the
results of the serum, urine and ultrasound study (SURUSS). J. Med. Screen. 10,
Wang, C., Nieschlag, E., Swerloff, R., Behre, H.M., Hellstrom, W.J., Gooren, L.J.,
Kaufman, J.M., Legros, J.-J., Lunenfeld, B., Morales, A., Morley, A., Schulman,
C., Thompson, I.M., Weidner, W. and Wu, F.C.W. Investigation, treatment
and monitoring of late-onset hypogonadism in males. Int. J. Androl. 32, 1–10
Wheeler, M.J. and Barnes, S.C. Measurement of testosterone in the diagnosis of
hypogonadism in the ageing male. Clin. Endocrinol. 69, 515–525 (2008).
Yen, S.S.C., and Jaffe, R.B. (eds), Reproductive Endocrinology: Physiology,
Pathophysiology and Clinical Management (Saunders, Philadelphia, 2004).
FIGURE 10 Simpliﬁed scheme for investigation of female infertility. Prl, prolactin; T, testosterone; FT, ‘free’ testosterone; OHP,
17α-hydroxyprogesterone; SHBG, sex hormone-binding globulin.