736 The Immunoassay Handbook
used to facilitate egg collection under mild sedation,
replacing the laparoscopic approach, and improving egg
yields (Dellenbach et al., 1985). The end result of these
reﬁnements has been a steadily improving success rate
per cycle as well as more convenient and safer clinical
When IVF was ﬁrst introduced it was considered a
potential beneﬁt for tubal and idiopathic infertility in the
female, and also for mild male infertility. The develop-
ment of intracytoplasmic sperm injection (ICSI) by Pal-
ermo et al. in 1992, led to widespread use of IVF techniques
in more profound male infertility.
IVF and ICSI are now commonplace procedures in all
developed countries, thanks to these scientiﬁc develop-
ments, in combination with permissive laws for the prac-
tice of reproductive medicine, reimbursement policies and
the acceptance and conﬁdence of the public. So much so
that in Europe they account for between 2 and 7% of all
births and in the USA just over 1%.
IVF PROGRAMMES INTO THE
Even though the majority of the practical steps in clinical
IVF processes have been elucidated, the measurement of
hormones is intrinsic to all good treatment programmes.
The two main reasons for this are the range of ovarian
responses to the drugs that stimulate follicular growth
(Follicle Stimulating Hormone (FSH)), and the need to
ensure that the LH surge is suppressed in controlled
ovarian stimulation (COS).
A number of FSH drugs are now available to stimulate
follicular growth and there is also a choice of GnRH ago-
nists or antagonists to suppress LH activity. Both
components inﬂuence follicular recruitment, and the
eventual response to COS depends upon a number of fac-
tors: the choice of drugs, their dosage and the biological
status of the individual woman (including body mass index
(BMI), smoking status and ovarian reserve). These are
The IVF Programme
In general, IVF programmes follow a familiar sequence of
events to attain their goal of placing a limited number of
embryos at an appropriate stage of development in the
uterus of the woman. The sequence is shown in Fig. 1.
These steps are discussed in detail below together with
the appropriate timing of hormone investigations.
CONTROLLED OVARIAN STIMULATION
There are two main protocols (long and short) for control-
ling the LH environment during stimulation of the ovaries
with exogenous FSH, as shown in Fig. 2:
1. The long protocol uses a GnRH agonist, and relies
on the down-regulation of the GnRH receptor in
the pituitary to suppress the pituitary-ovarian axis.
This requires prolonged treatment, usually 10–14
days before starting ovarian stimulation.
2. The short protocol uses a GnRH antagonist, which
has an immediate effect via competitive inhibition
of the GnRH receptor, and treatment is only
required for a short period; when there is a risk of
premature luteinization, i.e., after a few days of
induced follicular growth.
FIGURE 1 The sequence of steps in an IVF programme.
Day 2 or 3
to induce menses
2-4 weeks pretreatment GnRH agonist
20 - 24
FIGURE 2 The ﬂow of the two main stimulation protocols. The vertical arrows indicate points of ovarian activity assessment. (The color version of
this ﬁgure may be viewed at www.immunoassayhandbook.com).
737CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
The drugs used, and their duration and timing of adminis-
tration in relation to the menstrual cycle are shown in Fig. 2.
Despite the apparently improved simplicity of the GnRH
antagonist methods, the different decision making required,
and published clinical results, in terms of pregnancy rates
and live births, do not support the use of GnRH-antagonist
protocols for all women, and the longer conventional
GnRH agonist control remains the most popular method
deployed world-wide (Al-Inany et al., 2007).
The checkpoints and hormone evaluations required in
these two types of COS are different, and are described
more fully below.
GnRH Agonist-Controlled COS
A patient usually starts the GnRH-agonist (pre-) treatment
in the mid-luteal phase (day 20–25) of the cycle prior to
stimulation with FSH. Prolonged pre-treatment with GnRH
agonists is to fully establish ‘down-regulation’ of GnRH
receptors and thus circulating FSH and LH levels. The
down-regulation effect takes 1–3 weeks of continued treat-
ment, while in the ﬁrst few days of treatment, FSH and LH
concentrations actually increase (the ‘ﬂare’ effect). GnRH
agonists can be administered in three ways: depo formula-
tions, which last for approximately one month, daily subcu-
taneous injections, or by multiple applications of a nasal
Pituitary ‘down-regulation’ is established when subse-
quent menstruation occurs and is characterized by a low
estradiol concentration. This is an important decision
point requiring an assessment of the circulating estradiol,
because in exceptional cases, the estrogen can be elevated,
which may indicate cyst formation, following the ‘ﬂare’
effect. If this occurs the stimulation phase should not be
initiated. Furthermore, if there is ambiguity regarding the
nature of the menstruation, or endometrial thickness, an
evaluation of circulating hCG should be made, as this may
be due to an early spontaneous pregnancy.
The phase of ovarian stimulation is usually 10–14
consecutive days of FSH injections, and the degree of
follicular development is monitored by ultrasound
scans, although many centers conﬁrm observations by
evaluating the concentration of estradiol in the circulation.
The ﬁrst assessment point (ultrasound scan and estradiol
measurement) is after approximately a week of injections,
when some FSH dose modiﬁcations can be instituted, and
the second is between 10 and 14 days of injections. This
second assessment point is used to determine the timing of
the ovulation trigger (an injection of hCG), and is based on
follicular size and number. The values of estradiol during
the last week of stimulation are generally elevated, well
above ‘normal cycle’ concentrations. The degree of eleva-
tion depends upon the response to FSH injections, and is
proportional to the number and size of the induced folli-
cles. If values are more than 10 times normal cycle peak
concentrations, this indicates a risk of subsequent ovarian
hyperstimulation syndrome (OHSS).
GnRH Antagonist-Controlled COS
GnRH antagonists prevent premature luteinization with-
out the pre-treatment required with GnRH agonists. They
are also associated with a lower incidence of excessive
responses and OHSS is signiﬁcantly lower in programs
controlled with the GnRH-antagonist. However, there is a
slightly lower egg yield, which is deemed a disadvantage in
In cycles controlled with the GnRH antagonists, the
start of FSH injections is directed by the patient’s men-
struation. In some cases (women with oligomenorrhea)
menses may need to be induced with oral contraceptive
steroids. Many clinics use induction of menses routinely to
‘program’ cycles and control the number of egg collections
in any one week. As there is no down-regulation, responses
tend to be more rapid than in a cycle controlled by GnRH
It is commonplace to assess estrogen concentrations at
the start of stimulation, and at the ﬁrst and subsequent
assessment points, along with ultrasound measurements of
follicular development. As with GnRH-agonist controlled
cycles the estradiol and ultrasound results are used to
determine the timing of the triggering hCG injection. If
there is ambiguity regarding the nature of the menstrua-
tion, an evaluation of circulating hCG should be performed
to detect a spontaneous pregnancy. If the response to FSH
is excessive, the trigger mechanism (hCG injection) can be
replaced by using a single injection GnRH agonist, which
elicits a short LH surge—sufﬁcient to mature the eggs, but
the degree of subsequent luteinization is diminished, and
the risk of OHSS is almost eliminated.
When the ovarian follicles have reached a size consistent
with maturity, an injection of hCG is administered as the
trigger for all subsequent events. The trigger hCG acts as
an analog of LH and elicits maturation of the egg and
luteinization of the follicle, turning it into a corpus luteum,
which secretes large amounts of progesterone. It is the
time of this trigger that dictates when the eggs are col-
lected, when they are fertilized, when the embryo divides,
and when they are observed for morphological assessment
The egg collection is carried between 34 and 40h after
the hCG injection, just prior to when the follicle would
rupture and ovulate. At this stage the eggs should be
mature (Metaphase II stage), and are ready for fertilization
by IVF or ICSI.
Oocyte retrieval is performed transvaginally under
ultrasound guidance. Every ovarian follicle is aspirated
using a guided needle although the eggs from the more
mature follicles tend to perform better in subsequent
embryo development. The follicular ﬂuid is passed from
the collection to the laboratory as quickly as possible
and examined under a microscope to search for the
FERTILIZATION AND EMBRYO CULTURE
The sperm and egg are incubated together in a “fertiliza-
tion dish” in droplets of specialized media. Approxi-
mately 200,000 sperm are placed per oocyte or 500,000
per 3–4 oocytes and incubated for 18–21h. Usually fresh
ejaculated sperm are used, but in certain situations, such
as low sperm count or motility, a single sperm may be
directly injected into an egg using ICSI (subsequent
738 The Immunoassay Handbook
developmental stages are identical to IVF). After 18–21h
each oocyte is examined under the microscope to deter-
mine the presence of two pro-nuclei, which indicate nor-
mal fertilization. At this stage, all fertilized ‘embryos’ are
transferred to a growth medium with low glucose con-
tent, designed to support further embryo development in
the incubator for the next 24–48 h. If there is timely divi-
sion of the cells, then embryo development is deemed
promising and further incubation to the blastocyst stage
can be undertaken. Each developmental stage normally
leads to some attrition of the cohort of embryos, but this
is counter-balanced by increased validity of the selection
processes. Correspondingly, blastocysts (day 5 of cul-
ture) have a signiﬁcantly higher implantation rate than
embryos at the earlier stages.
in the Fresh IVF Cycle
Embryo transfer can take place on embryo development
day 2 (four cell stage), 3 (eight cell stage), or 5 (blastocyst
stage). The outcomes tend to improve, the longer the
transfer is delayed, because of the beneﬁts of improved
qualitative embryo selection.
Transfer is performed with a semi-rigid, plastic cathe-
ter, which is passed though the cervix into the uterine cav-
ity and, once in position, the embryologist ﬁlls the inner
catheter with one or more embryos in a small volume of
The number of embryos transferred to the uterus
depends on the clinic guidelines (sometimes dictated by
national legislation). There is a strong shift toward extend-
ing the use of single embryo transfers in most countries,
aiming to reduce the incidence of multiple pregnancies.
Remaining embryos can be stored at all stages using
either the traditional controlled ‘slow freeze’ methodol-
ogy, or more recently and more successfully, rapid cool-
ing vitriﬁcation. The attrition rate of this latter method
shows less cellular damage, and in many cases, undimin-
ished implantation rates. The traditional slow freeze
methods showed reduced implantation rates, so it is
likely that vitriﬁcation will evolve into the method of
EMBRYO TRANSFER IN THE FROZEN
EMBRYO TRANSFER CYCLE
Frozen embryo transfer can be conducted in a normal
cycle, or in one created using hormone replacement ther-
apy superimposed upon suppression of endogenous func-
tion. In both cases, the embryos should be thawed and
transferred according to the stage of their development
and at the appropriate stage of endometrial development.
In the normal cycle, endometrial development is dic-
tated by the endocrine changes elicited by the LH surge
and the rise of progesterone. The LH surge and onset of
luteinization can be detected by measuring blood hormone
concentrations of estradiol, LH, and progesterone
around mid-cycle. It is also common practice to use uri-
nary ovulation prediction kits to determine this stage of
In Hormone Replacement Therapy (HRT) cycles,
endometrial development is dictated by the start of pro-
DETECTION OF PREGNANCY
Many centers now use quantitative serum hCG measure-
ments to detect pregnancy. This test is usually carried out
15–17 days after the hCG injection that initiated egg matu-
ration prior to oocyte collection. A quantitative evaluation
can provide some guidance as to the eventual outcome of
the pregnancy. This is desirable as a quarter of all positive
tests are followed by pregnancy failure.
SAFETY AND ETHICAL CONSIDERATIONS
The major complications of IVF result from ovarian stim-
ulation and the availability of multiple embryos. Obstetric
complications are directly related to the practice of trans-
ferring multiple embryos and include pregnancy loss, pre-
maturity, and neonatal morbidity, with the potential for
long-term damage to the child. Strict legal limits on the
numbers of embryos transferred have been imposed by
some countries to reduce these risks, and there is an
increasing trend toward elective single embryo transfer
(SET) in many European countries. This is an issue of
applying appropriate professional standards.
Of similar importance, although less well reported, is
the problem of OHSS, which is an acutely presenting and
potentially fatal sequela of clinical practice.
The issue of birth defects as a result of ovarian stimula-
tion and embryo culture is a controversial topic in IVF,
with studies yielding conﬂicting evidence; some showing
an increase in birth defects whilst others do not support
In the early years, IVF research and clinical practice was
performed in a different and sometimes hostile environ-
ment. The moral and religious discussions that these tech-
niques raised clearly demonstrated that the new assisted
reproductive technologies (ART) required an ethical
framework in which to operate. In many countries legisla-
tion provides the framework and government agencies,
e.g., the UK’s Human Fertility and Embryology Authority
(HFEA), Australia’s Infertility Treatment Authority (ITA)
have oversight, monitor adherence, grant licenses, store
records etc. Ethical issues include laboratory errors result-
ing in the transfer of wrong embryos, pregnancy after the
menopause, screening (in or out) particular genetic traits
using pre-implantation genetics and sex selection for social
OVARIAN RESERVE TESTING AND
PREDICTION OF RESPONSE TO COS
The most important factor inﬂuencing response to COS is
a woman’s ovarian reserve (or number of primordial folli-
cles). The woman with a high ovarian reserve often
responds to exogenous FSH with excessive degrees of fol-
licular recruitment and growth. This woman is at high risk
of OHSS, especially if she achieves a pregnancy. At the
other extreme, the woman with a low ovarian reserve
struggles to elicit the growth of sufﬁcient follicles to
739CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
provide enough eggs for the embryology laboratory to
work with in an efﬁcient manner. These extremes of
response are independent of the woman’s age, although
the high responding woman tends to be younger and the
poor responder older.
Measurement of circulating FSH concentrations, or
estimating ovarian volume and antral follicle counts with
vaginal ultrasound scans, performed in the early follicular
phase, showed promise in predicting these response
extremes. However, it has recently been shown that circu-
lating Anti-Müllerian hormone (AMH, or Müllerian-
inhibiting substance, MIS), has the greatest ability to
predict ovarian responses to exogenous FSH injections.
Furthermore, AMH is stable across the menstrual cycle,
and can therefore be measured at any time in the cycle.
The debate surrounding this is best covered by a review
article addressing each of the main issues by La Marca
et al. (2010).
Predicting the response to COS is essential. It has been
shown in numerous analyses of conventional programs of
IVF that women producing less than ﬁve eggs demonstrate
a lower pregnancy rate than their age-matched cohort with
5 or more eggs. It is important that a woman knows the
risks of achieving such a suboptimal response so she can be
counseled/advised in order to reduce the emotional and
ﬁnancial burden associated with a lack of response to exog-
enous FSH. However, it is possibly more important for the
clinic to be able to identify women at risk of an excessive
response as these women are at risk of potentially fatal
OHSS or at least risk-canceled cycles.
It is now established that a simple AMH evaluation can
predict these responses with high precision (reviewed by
La Marca et al., 2010). The clinic can therefore adopt COS
protocols and doses of FSH that are suitable for the indi-
vidual—whether predicted to be poor, moderate, or high
responder—without having to subject the patient to a full
It is clear from the account above that a number of analytes
are required to be measured under different circumstances,
and these include AMH, hCG, LH, progesterone, and
estradiol. The principal and most routine assays would
now include AMH (once prior to treatment), estradiol
during treatment, and hCG at the end of treatment.
During treatment, vaginal ultrasound is the single most
important tool for monitoring responses to treatment, but
it is clariﬁcation of the apparently exceptional circum-
stance that dictates a requirement for estradiol measure-
ments on a number of occasions.
Estradiol-17β (known as E2) is arguably the most impor-
tant product of the human ovary. It is produced by the
maturing follicle under the drive of both FSH and LH
under the 2-cell, 2-gonadotropin mechanism for estradiol
biosynthesis. The primary precursor is cholesterol (with
27 carbon atoms), which is metabolized by side-chain
cleavage to the C21 product pregnenolone, which is freely
transformed to progesterone in the granulosa cells. There
is no further route of metabolism of these products within
the granulosa cells, but the theca cells, under the drive of
LH, metabolize pregnenolone to the C19 androgens,
mainly androstenedione. The androgens then return to
the granulosa cells to be converted to estradiol and estrone
back in the granulosa cell compartment by the aromatase
During the normal cycle the role of estradiol is to induce
the proliferation of the endometrium, which is then con-
verted to a secretory structure ready to support the implan-
tation by the increased concentrations of progesterone
secreted by the corpus luteum after ovulation.
The structure of estradiol is shown in Fig. 3, and its dis-
tinguishing phenolic A ring with two hydroxyl groups in
positions 3 and 17 mean that it has been relatively easy to
produce antibodies for immunoassay purposes for many
During the normal cycle, there is a direct correlation
between circulating E2 and the size of the maturing pre-
ovulatory follicle. However, during the induction of mul-
tiple follicular development, each of the multiple growing
follicles secretes estrogen, and the concentration in the
peripheral circulation therefore rapidly progresses to
supranormal concentrations, and it tends to represent the
number and advancement of follicles that are growing. It is
this phenomenon that makes serum estradiol the most
important laboratory hormonal assays for routine IVF.
Serum estradiol is measured at the initiation of the
induction of follicular growth to indicate that the patient is
either down-regulated (agonist-controlled COS) or at
least showing representative values for the early menstrual
cycle (antagonist-controlled COS). These early values
should be low (usually lower than 50pg/mL [150pmol/L]).
The stimulation phase then follows, and is usually re-
assessed after about a week of stimulation. By this stage the
circulating E2 concentrations should be in the supranor-
mal range. In women with a high ovarian reserve the
degree of elevation above normal will be greater than the
woman with a low ovarian reserve.
Stimulation is continued until the diameter of the lead
follicles achieves 17–20mm. At this stage the circulating
concentrations can achieve a wide range of values from
FIGURE 3 The structure of Estradiol-17β. Estradiol is produced in
large amounts by maturing follicles and assays are required to assess
concentrations from the lows of menopausal levels (ca 10pg/mL)
through to high levels which exceed 5000pg/mL in patients at risk
of excessive responses.
740 The Immunoassay Handbook
500pg/mL (1500pmol/L) in a poor responder patient
to >5000pg/mL (>15,000pmol/L) in a high response.
The trigger hCG (usually 5000 to 10,000IU) is admin-
istered at this stage at a precise time—starting the subse-
quent steps of oocyte maturation prior to fertilization and
precisely timing the clinical steps of egg collection and
insemination or ICSI.
If during the stimulation phase, the serum E2 concentra-
tions show unexpected changes, the other analytes (LH
and progesterone) may be assessed to determine whether
premature luteinization has taken place. When this is
identiﬁed, it usually results in cycle cancellation.
Excessive values of E2
When the estrogen is grossly elevated such as >5000pg/
mL (>15,000pmol/L) the risk of OHSS is greatly increased,
and clinical decisions of how to handle the next steps of the
treatment cycle are required. This can involve cycle can-
cellation, discontinuation of FSH injections and waiting a
number of days until the E2 level has declined to lower
levels (‘coasting’), or cryopreservation of all embryos, prior
to transfer in a subsequent normal menstrual cycle.
There is some difﬁculty predicting the amount of estra-
diol produced by each follicle, due to the different states
(sizes) of development. Furthermore, the amount of
estrogen produced per follicle depends as much on the
circulating LH activity as on size and FSH drive, and
therefore the nature of the FSH product administered
plays an important role in the estrogen proﬁle. Patients
treated with recombinant FSH alone, generally show
lower estradiol concentrations than those treated with a
product containing LH activity—despite producing at
least as many eggs.
In general, it is adequate to start induction of ovulation
with very low levels of estradiol. However, at the other end
of the spectrum, there is no absolute elevated value that
would indicate that cycles should be cancelled—it merely
Many analyzer menus include competitive immunoassays
that perform well in the circumstances required, covering
appropriate concentration ranges with low level sensitivity
and reliable dilution characteristics for excessive values.
Dilution is required as competitive immunoassays have a
limited range (See INFERTILITY chapter).
Desirable Assay Performance
Serum (or plasma) estradiol is an analyte with exceptional
performance requirements in IVF, because most of the
clinical decisions made for the induction of ovulation, such
as whether to proceed or stop the cycle, are based on levels
of this hormone in response to the gonadotropins admin-
istered. Therefore the range required is extensive, from
perimenopausal to many times supranormal, and sample
dilutions are not exceptional.
Isotope dilution-gas chromatograph/mass spectrometry
(ID-GC/MS) is considered the reference method for
estradiol measurement, but only automated random access
immunoassay analyzers offer the ease of use and turn-
around times required for the fertility clinic.
A wide range of patient samples should be compared with a
well-established method, in order to check for satisfactory
correlation. The level of agreement between different
methods for serum estradiol is variable, and because of the
considerable bias differences between methods, a clinical
follow-up of a patient should always be performed using
the same, ﬁt-for-purpose and well-validated assay. It should
also be noted that there is usually a non-linear recovery and
concentration dependent bias when compared against
ID-GC/MS and the problems of comparability and vari-
ability are more severe at low E2 concentrations. Nonethe-
less external quality assessment schemes generally show
performance characteristics that are adequate for most
main assay systems and platforms.
The minimum detectable concentration of estradiol
should be around 10pg/mL (35pmol/L) for use in the fer-
tility clinic. Most assays are able to detect this low concen-
tration of estradiol, which is adequate for use in IVF
Assay methods for E2 generally show good speciﬁcity with
the family of estrogen molecules, and the proﬁles of E2
generally exceed those of estrone and estriol (except dur-
ing pregnancy). Method literature should be checked for
sample interferences (lipemia, icterus, and hemolysis).
Types of Sample
Serum or plasma.
Frequency of Use
ANTI-MÜLLERIAN HORMONE (OR
AMH or MIS, is a 140kDa dimeric glycoprotein hormone
composed of two 72kDa monomers linked by disulﬁde
bridges and is a member of the transforming growth factor
β family. Secretion of AMH by the Sertoli cells of the testes
commences during embryogenesis and continues through-
out life. Its primary role is to cause regression of the Mülle-
rian duct in the male fetus, allowing testosterone to lead
normal development of the male reproductive tract (Wolff-
ian ducts). Levels are high at birth in boys and then rise dur-
ing infancy before gradually declining at and after puberty
(Teixeira et al., 2001). In females, AMH is produced by the
ovarian granulosa cells and circulating concentrations are
generally an order of magnitude lower than for males
741CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
however they increase before puberty and achieve maximal
concentrations in early adulthood. Thereafter, a steady and
unrelenting decline is seen until AMH levels become unde-
tectable before or at the menopause (Kelsey et al., 2011).
Maximum expression of AMH occurs in pre-antral and
small antral follicles up to 5mm in size (Laven et al., 2004).
However expression is lost during the FSH-dependent
phase of follicular growth. This suggests that basal levels of
AMH reﬂect the total developing follicular cohort, or the
‘functional ovarian reserve’, which in turn represents the
non-growing primordial pool of follicles or total ovarian
reserve (Fig. 4).
Visser and Themmen (2005) suggest that the pattern of
expression indicates that AMH has an important role in
regulating the number of follicles that are recruited from
the primordial pool.
Increased understanding of the role of AMH in pathophysi-
ology and recent availability of commercial immunoassays
for its measurement have led to its increased use as a diag-
nostic tool in reproductive medicine. The main roles include
investigations into disorders of sexual differentiation (Lee
et al., 2003; Rey et al., 1999), polycystic ovarian disease (La
Marca et al., 2006), female fertility (La Marca et al., 2009)
and for the assessment of all women undergoing assisted
reproduction technologies (below). AMH is also used as a
tumor marker in granulosa cell tumors.
The dramatic increase in AMH requesting over the last
few years is predominantly related to its use as an aid to clini-
cal decision making in COS. The hormone’s potential for
predicting ovarian responses to exogenous FSH injections
was ﬁrst described by David Seifer in 2002 (Seifer, et al.,
to stimulation was determined prospectively by Nelson et al.
(2007) and conﬁrmed as superior to all other indicators by
meta-analysis (La Marca et al., 2010). Nelson et al. (2009)
subsequently demonstrated how AMH could be used to indi-
cate the most appropriate method of stimulation, maximizing
safety in high responding women, and allowing aggressive
treatment in women with a reduced ovarian reserve.
AMH should be measured before determining the mode
of COS to be used and the dose of FSH. Its importance lies
in the marker’s ability to predict the degree to which a patient
is likely to respond to the exogenous FSH injections. This
knowledge can be used by the clinic to dictate the advice it
gives to patients, and how best to stimulate that patient safely,
but aiming to obtain a healthy cohort of follicles and eggs.
Furthermore, as AMH levels ﬂuctuate very little throughout
the menstrual cycle, it can be measured at any time making it
more convenient for the patient and clinic than other mark-
ers of ovarian response such as FSH and Inhibin B, which
need to be measured in the follicular phase on day 3–5.
Figure 5 explains how AMH values can be used to deter-
mine the strategic approach to COS in women undergoing
IVF, optimizing safety and outcomes.
FIGURE 4 Role of AMH in follicular development (the gray area represents the granulosa cell layer, the red (dark gray) area, the oocyte and the
white area the follicular ﬂuid). (The color version of this ﬁgure may be viewed at www.immunoassayhandbook.com). From: Broekmans et al. Trend
Endocrinol. Metabol. 19: 340–347, 2008.
FIGURE 5 An example of how AMH can be deployed to indicate COS
treatment strategy. (The color version of this ﬁgure may be viewed at
742 The Immunoassay Handbook
The way AMH is reported in an international fertility setting
remains to be determined and there are two components to
1. age-speciﬁc reference ranges
2. predicted response to FSH injections
At the Glasgow Centre for Reproductive Medicine
(GCRM) a reference range for 25- to 50-year-old women
is reported, which is based on two publications by Nelson
et al. in 2011 and is shown in Fig. 6.
The original data set was based on a population study of
9601 infertile patients and showed that the decline of
AMH with age was optimally modeled by a quadratic
equation. Subsequent external validation in 15,834 US
women conﬁrmed this relationship. The original analysis
was performed using the Diagnostic Systems Limited
(DSL) AMH assay which gives values approximately 40%
lower than the AMH Gen II assay currently in use. The
above ﬁgure uses a conversion factor derived from a multi-
center evaluation of AMH Gen II versus the original DSL
AMH assay (Wallace et al., 2011).
Many patients and clinicians like to know, not only what
the AMH value is, but how this relates to women of a simi-
lar age. As such, large datasets are required but very few
are available other than those described above. Their
potential utility of this evaluation is to inform the individ-
ual of the length of their potential fertility. If AMH is low
for a given age, then there are important implications for
family planning. Furthermore, as multiple eggs are
required for success at assisted conception, AMH can pro-
vide guidance regarding when to access fertility treatment.
It can be seen that functional ovarian reserve (AMH)
undergoes an inexorable decline after age 25 years.
There are numerous further questions to be answered
regarding AMH and its potential roles. These include longi-
tudinal nomograms and analyses in different ethnic groups.
Figures 5 and 6 show how AMH values are used at
GCRM to determine the strategic approach to COS in
women undergoing IVF. Other centers have established
their own differential approaches to COS, broadly based
on the same or similar observations. The original informa-
tion derives from a prospective study of almost 600
patients, which showed that directing the stimulation
strategy based on AMH alone signiﬁcantly reduced the
risk of OHSS, treatment burden and cycle cancellation,
and increased clinical pregnancy rates. Although the study
has limitations, including a non-randomized design, the
cut-off points derived from the study have been proven in
routine clinical use since 2009.
There is no international standard for AMH and therefore
different assays can report markedly different values on the
same patient specimen. Furthermore historically there
have been several “in house” and commercially available
assays whose performance has changed over the assay life-
time. Therefore there is a clear warning that when review-
ing the literature care must be exercised to ascertain which
assay is deployed and to understand that reference inter-
vals and threshold values obtained with one assay are not
interchangeable with another or even, possibly with the
same assay over time.
Certain assays (see below) show variability in AMH
concentrations when study samples have been frozen and
thawed so care must also be taken when interpreting
published results using these assays.
The literature also uses both mass and molar units, for
which the conversion equation is 1ng/mL=7.14pmol/L.
The ﬁrst reported AMH immunoassays were developed by
Hudson et al. and Josso et al. in 1990. The Hudson assay
used a pair of monoclonal antibodies raised against human
recombinant AMH, both directed to epitopes in the pro
region but were shown to give variable AMH results due to
storage and freeze-thaw instability. The variability in the
AMH concentrations observed may have been due to pro-
cessing of the AMH protein in vivo. Assays using one or
both antibodies directed against the pro region are likely
to exhibit this instability and careful attention to sample
collection and storage may be required if reliable results
are to be obtained from these assays. The assay developed
by Josso et al. (1990) does not suffer from this issue but was
designed to investigate gonadal function in children
(males), and so was less sensitive.
Two commercial assays for AMH, one from Immuno-
tech (IOT), Marseille, France, based on the Josso assay and
one from Diagnostic Systems Limited (DSL, now part of
Beckman Coulter Inc.), Texas, USA, were brought to the
market in 1990 and 2004 respectively and have enjoyed
widespread application. Despite the antibodies being raised
to different epitope combinations of the molecule, the two
assays showed closely parallel results, although they give
different values and have slight differences in sensitivity.
Both IOT and DSL were acquired by Beckman Coulter,
and in order to harmonize AMH values, the DSL assay has
been replaced by AMH Gen II, standardized to the IOT
assay. The AMH Gen II assay uses a pair of monoclonal
antibodies directed to epitopes in the mature region of
AMH (Al-Qahtani et al., 2005) and correspondingly the
AMH measured by this assay is less affected by proteolysis.
In addition, the Gen II assay measures AMH in human,
monkey, bovine, and other mammalian species.
FIGURE 6 The age-related decline in AMH in women between ages
25 years and 45 years with indicated discriminators of response. AMH is
reported here as assayed by the Gen II assay. (The color version of this
ﬁgure may be viewed at www.immunoassayhandbook.com).
743CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
The assay is currently in a microplate-based ELISA for-
mat, although plans to automate the AMH Gen II assay on
the Beckman Coulter Access II immunoassay platform are
now well advanced.
The DSL and IOT assays have been extensively used to
provide the evidence base for the use of the AMH assay to
assess ovarian responsiveness to exogenous FSH stimula-
tion and both have been adopted into routine clinical prac-
tice. Recent publications show good correlation and
agreement between the AMH Gen II and IOT assays
(Kumar et al., 2010) and similar precision and excellent
correlation between AMH Gen II and the DSL assay
although assay values are approximately 40% higher using
the AMH Gen II ELISA (Wallace et al., 2011). Assay
results in separated serum are stable over time, and through
freeze-thaw cycles (Kumar et al., 2010), although changes
with time in whole blood require full elucidation.
For the AMH Gen II assay the limit of detection or lowest
concentration of AMH in a sample that can be detected
with a 95% probability is 0.08ng/mL (0.57pmol/L). The
limit of quantitation, estimated minimum dose achieved at
20% total imprecision, is 0.16ng/mL (1.14pmol/L), from
Kumar et al., 2010.
The antibody speciﬁcity for human, monkey, mouse, rat,
bovine and horse samples has been published (Al-Qahtani
et al., 2005). Structurally related proteins and some of the
other members of the transforming growth factor beta
(TGF-β) super family, Inhibin A (10,000pg/mL), activin A
(10,000pg/mL), follicle stimulating hormone (450mIU/
mL), and luteinizing hormone (100µIU/mL) do not show
Types of Sample
Serum or lithium heparin plasma.
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