735© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.
736 The Immunoassay Handbook
used to facilitate egg collection under mild sedation,
replacing the laparoscopic approach, a...
737CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
The drugs used, and their duration and timing of admini...
738 The Immunoassay Handbook
developmental stages are identical to IVF). After 18–21h
each oocyte is examined under the mi...
739CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
provide enough eggs for the embryology laboratory to
740 The Immunoassay Handbook
500pg/mL (1500pmol/L) in a poor responder patient
to >5000pg/mL (>15,000pmol/L) in a high res...
741CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
however they increase before puberty and achieve maxima...
742 The Immunoassay Handbook
AMH Interpretation
The way AMH is reported in an international fertility setting
remains to b...
743CHAPTER 9.6 In Vitro Fertilization and Embryo Transfer (IVF-ET)
The assay is currently in a microplate-based ELISA for-...
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  1. 1. 735© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00060-9 Historical Perspective Research on In Vitro Fertilization (IVF) began as early as the 1930s in the USA when Pincus and Enzmann at Har- vard began their work in the rabbit. However the event marking the beginning of clinical practice throughout the world was the birth of Louise Brown in the UK in 1978. Other IVF births quickly followed: in Australia in 1980, the USA in 1981, and Sweden and France in 1982. Clinical progress in IVF was underpinned by an explo- sion of learned endocrine and physiological studies in the late 1970s and early 1980s. These included the under- standing of human follicular development (reviewed by Baird, 1983) and definition of the critical steps in egg maturation and embryology (reviewed by Edwards and Steptoe, 1983). These studies, together with the develop- ment of ovarian ultrasonography (Hackeloer et al., 1979), played a key role in facilitating the clinical practice of IVF. The ability to measure circulating concentrations of hormones (reproductive steroids and gonadotropins) in primate and human bodily fluids was essential for reveal- ing the mechanisms that control ovarian function. Central to this was the development of (radio)-immunoassay (RIA), a technique that could reliably detect low concentrations of these hormones in the peripheral circulation allowing specific physiological events to be monitored. The biological controller of all the physiological pro- cesses involved in female fertility is gonadotropin-releasing hormone (GnRH) whose decapeptide structure was eluci- dated by Andrew Schally and for which he was awarded his share of the Nobel Prize in 1977. Appropriately, his co-prize winner of that year (Rosalyn Yalow) was responsible for the pioneering development of competitive immunoassays. The ultimate recognition of the importance of advances in assisted reproduction came in 2010, when Bob Edwards was also awarded the Nobel Prize for his principle work, which revealed the sequence and timing of nuclear and cel- lular events leading to maturation of the human egg, which led ultimately to the birth of Louise Brown in 1978. In the early years of IVF there was much debate around ovulation; whether to rely on a woman’s natural cycle, with laparoscopic egg collection, or to induce the development of multiple follicles to obtain more oocytes. The first suc- cessful live birth used a natural cycle but other groups found this difficult to repeat, Trounson and Leeton used clomiphene and human chorionic gonadotropin (hCG) to obtain more oocytes, and Howard and Georgeanna Jones, in the USA, used human menopausal gonadotropin (hMG). Ovarian stimulation produces more oocytes (and so more embryos and more potential pregnancies) and allows better scheduling of egg collection. This became the accepted way forward; however several endocrine and practical issues became apparent. The main endocrine problem is the nature and untimely incidence of luteinizing hormone (LH) surge activity dur- ing the follicular phase of stimulated ovulation and the main practical problem is having to deal with multiple embryo transfers and the issue of excess embryos. In the first instance, the full potential of the stimulated cycles was not achieved and the reasons are clear when the endocrine responses to ovarian stimulation are consid- ered. Multiple follicles, stimulated with hMG or clomi- phene citrate with hMG, secrete estradiol to supranormal levels, attaining normal mid-cycle peak levels well in advance of the normal follicle mid-cycle size and maturity. The high estrogen levels elicit a surge of LH from the pituitary as if it were a normal mid-cycle event, but the event is premature with respect to the stages of follicular development. Exposure of follicles at different degrees of maturity to the signal to luteinize and “ovulate” elicits a variety of responses, due to the broad range of follicles that may be present when the surge occurs. Follicles, in which the mechanisms controlling ovulation have matured, undergo the conventional response of luteiniza- tion and ovulation. Whereas those less able to respond conventionally to the LH surge may be compromised, as high LH activity inhibits the aromatase enzyme, granulosa cell mitosis, and probably many other processes. This phenomenon was recorded in non-ovulatory women as early as 1978 (Gemzell) and became known as “premature luteinization”. The key to resolving this problem was revealed when it was shown that chronic application of a GnRH agonist could reliably block the LH surge and premature ovula- tion for the duration of treatment. Multiple daily applica- tions of a nasal spray of the GnRH agonist, buserelin acetate, effectively blocked all spontaneous LH activity, allowing clinical control of follicular development and ovulation with exogenous gonadotropins (Fleming et al., 1982). The IVF clinic now has both GnRH agonists and antagonists at its disposal for controlling LH activity. However there are important differences in how these two drug types function, with what can be profound dif- ferences in ovarian responses, and these are discussed later. In parallel with the development of endocrine tech- niques to manipulate follicular growth and ovulation, the use of vaginal ultrasound to monitor follicular growth and responses was becoming established (Hackeloer et al., 1979). Improvements in the technique meant it could be In Vitro Fertilization and Embryo Transfer (IVF-ET) Richard Fleming (richard.fleming@gcrm.co.uk) Sherry Faye (safaye@beckman.com) C H A P T E R 9.6
  2. 2. 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 management. 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 TWENTY-FIRST CENTURY 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 discussed below. 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. hCG FSH Injections GnRH Antagonist Day 4/5/6 of FSH Day 2 or 3 of menses ?Pre-treatment to induce menses 2-4 weeks pretreatment GnRH agonist FSH Injections Cycle day 20 - 24 hCG Menses 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).
  3. 3. 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 spray. 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 many centers. 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 agonists. 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. OOCYTE RETRIEVAL 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 of quality. 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 oocyte. 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
  4. 4. 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. Embryo Transfer 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 culture medium. 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 choice. 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 the cycle. In Hormone Replacement Therapy (HRT) cycles, endometrial development is dictated by the start of pro- gesterone treatment. 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 a link. 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 reasons. 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
  5. 5. 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 stimulation first. Analytes 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 Function 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 enzyme. 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 years. Clinical Applications 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.
  6. 6. 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. Limitations 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 advises caution. Assay Technology 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 Characteristics 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. Correlation data 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. Sensitivity 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 programs. Specificity 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 Very common. ANTI-MÜLLERIAN HORMONE (OR MÜLLERIAN-INHIBITING SUBSTANCE) Function 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
  7. 7. 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. Clinical Applications 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., 2002).Itsabilitytopredictthefullrangeofpotentialresponses 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 www.immunoassayhandbook.com).
  8. 8. 742 The Immunoassay Handbook AMH Interpretation The way AMH is reported in an international fertility setting remains to be determined and there are two components to consider: 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. Limitations 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. Assay Technology 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).
  9. 9. 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. Assay Performance 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. Sensitivity 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. Specificity 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 measurable cross-reactivity. Types of Sample Serum or lithium heparin plasma. References and Further Reading Al-Inany, H.G., Abou-Setta, A.M. and Aboulghar, M. Gonadotrophin-releasing hormone antagonists for assisted conception: A Cochrane review. Reprod. Biomed. Online 14, 640–649 (2007). Al-Qahtani, A., Muttukrishna, S., Appasamy, M., Johns, J., Cranfield, M., Visser, J.A., Themmen, A.P. and Groome, N.P. Development of a sensitive enzyme immu- noassay for anti-Müllerian hormone and the evaluation of potential clinical applications in males and females. Clin. Endocrinol. (Oxf.) 63, 267–273 (2005). Baird, D.T. Factors regulating the growth of the preovulatory follicle in the sheep and the human. J. Reprod. Fertil. 69, 343–352 (1983). Bosch, E., Labarta, E., Crespo, J., Simón, C., Remohí, J., Jenkins, J. and Pellicer, A. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum. Reprod. 25, 2092–2100 (2010). Dellenbach, P., Nisand, I., Moreau, L., Feger, B., Plumere, C. and Gerlinger, P. Transvaginal sonographically controlled follicle puncture for oocyte retrieval. Fertil. Steril. 44, 656–662 (1985). Edwards, R.G. and Steptoe, P.C. Current status of in vitro fertilisation and implan- tation of human embryos. Lancet 3(2), 1265–1269 (1983). Fleming, R., Adam, A.H., Barlow, D.H., Black, W.P., Macnaughton, M.C. and Coutts, R.T. A new systematic treatment for infertile women with abnormal hormone profiles. Brit. J. Obstet. Gynaecol. 80, 80–83 (1982). Hackeloer, B.J., Fleming, R., Robinson, H.P., Adam, A.H. and Coutts, J.R.T. Correlation of ultrasonic and endocrinological assessment of follicular develop- ment. Am. J. Obstet. Gynecol. 135, 122–128 (1979). Josso, N., Legeai, L., Forest, M.G., Chaussain, J.L. and Brauner, R. An enzyme linked immunoassay for anti-müllerian hormone: a new tool for the evaluation of testicular function in infants and children. J. Clin. Endocrinol. Metab. 70, 23–27 (1990). Kelsey, T.W., Wright, P., Nelson, S.M., Anderson, R.A. and Wallace, W.H. A validated model of serum anti-Müllerian hormone from conception to meno- pause. PLoS One. 6, e22024 (2011). Kumar, A., Kalra, B., Patel, A., McDavid, L. and Roudebush, W.E. Development of a second generation anti-Müllerian hormone (AMH) ELISA. J. Immunol. Methods 362, 51–59 (2010). La Marca, A., Sighinolfi, G., Radi, D., Argento, C., Baraldi, E., Artenisio, A.C., Stabile, G. and Volpe, A. Anti-Müllerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum. Reprod. Update 16, 113–130 (2010). La Marca, A., Stabile, G., Artenisio, A.C. and Volpe, A. Serum anti-Müllerian hormone throughout the human menstrual cycle. Hum. Reprod. 21, 3103– 3107 (2006). La Marca, A., Broekmans, F.J., Volpe, A., Fauser, B.C. and Macklon, N.S. On behalf of the ESHRE Special Interest Group for Reproductive Endocrinology – AMH Anti-Müllerian hormone (AMH): what do we still need to know? Hum. Reprod. 24, 2264–2275 (2009). Laven, J.S., Mulders, A.G., Visser, J.A., Themmen, A.P., De Jong, F.H. and Fauser, B.C. Anti-Müllerian hormone serum concentrations in normoovula- tory and anovulatory women of reproductive age. J. Clin. Endocrinol. Metab. 89, 318–323(2004). Lee, M.M., Misra, M., Donahoe, P.K. et al. MIS/AMH in the assessment of crypt- orchidism and intersex conditions. Mol. Cell Endocrinol. 211, 91–98 (2003). Nelson, S.M., Yates, R.W. and Fleming, R. Serum anti-Müllerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles– implications for individualization of therapy. Hum. Reprod. 22, 2414–2421 (2007). Nelson, S.M., Yates, R.W., Lyall, H., Jamieson, M., Traynor, I., Gaudoin, M., Mitchell, P., Ambrose, P. and Fleming, R. Anti-Müllerian hormone-based approach to controlled ovarian stimulation for assisted conception. Hum. Reprod. 24, 867–875 (2009). Nelson, S.M., Messow, M.C., Wallace, A.M., Fleming, R. and McConnachie, A. Nomogram for the decline in serum antimüllerian hormone: a population study of 9,601 infertility patients. Fertil. Steril. 95, 736–741 (2011). Palermo, G., Jorris, H., Devroey, P.P. and Van Steirteghem, A.C. Pregnancies after intra cytoplasmic sperm injection of single spematazoon into an oocyte. Lancet 2, 17–18 (1992). Pincus, G. and Enzmann, E.V. Can mammalian oocytes undergo normal develop- ment in vitro? Proc. Natl. Acad. Sci. U.S.A 20, 121–122 (1934). Rey, R.A., Belville, C. and Nihoul-Fékété, C., et al. Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measure- ment. J. Clin. Endocrinol. Metab. 84, 627–631 (1999). Seifer, D.B., MacLaughlin, D.T., Christian, B.P., Feng, B. and Shelden, R.M. Early follicular serum Müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil. Steril. 77, 468–471 (2002). Teixeira, J., Maheswaran, S. and Donahoe, P.K. Mullerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications. Endocrin. Rev. 22, 657–674 (2001). Visser, J.A. and Themmen, A.P. Anti-Müllerian hormone and folliculogenesis. Mol. Cell Endocrinol. 234, 81–86 (2005). Wallace, A.M., Faye, S.A., Fleming, R. and Nelson, S.M. A multicentre evaluation of the new Beckman Coulter anti-Müllerian hormone immunoassay (AMH Gen II). Ann. Clin. Biochem. 48, 370–373 (2011).