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The immuassay handbook parte76

  1. 1. 721© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00059-2 Fertility in males and females is controlled by a homeo- static balance between the hypothalamus, pituitary, and gonads (see Fig. 1). Gonadotropin-releasing hormone (GnRH), formerly known as luteinizing hormone-releasing hormone (LHRH), is secreted from the arcuate nucleus of the hypo- thalamus and passed down neurofibrils to the median emi- nence. Here, it is released into the portal system and taken to the anterior pituitary where it binds to specific receptors on the gonadotroph cells. GnRH stimulates both the syn- thesis and the release of luteinizing hormone (lutropin, LH) and follicle-stimulating hormone (follitropin, FSH). These hormones are also known as gonadotropins. To be effective, GnRH must be secreted in a series of pulses. To initiate normal follicular development in the menstrual cycle and spermatogenesis in the male, the pulse frequency is 90–120min. This is reflected in the pulsed secretion of the gonadotropins, LH in particular. GnRH pulses are modified by the feedback of steroids on the hypothalamus. For instance there is increased amplitude but reduced fre- quency of the GnRH pulses, reflected in LH secretion, in the luteal phase of the menstrual cycle due to higher levels of progesterone. Further light has been shed on the control of GnRH secretion with the discovery of the peptide growth factor kisspeptin (encoded by KISS1) and the kiss1 receptor GPR54. There have been numerous studies to show that the kisspeptin/GPR54 system is a key regulator of puberty and reproduction. Kisspeptin, secreted mainly from the hypothalamus, stimulates the release of GnRH, but it is not yet clear whether kisspeptin itself is secreted in a pul- satile manner. Evidence suggests that steroid feedback on kisspeptin release is the means by which steroids modify GnRH release. However, this does not explain all observa- tions. Many years ago Knobil showed that normal men- strual function could be induced in rhesus monkeys with ablated hypothalami by giving pulsatile GnRH. That is, steroids modify GnRH action on the pituitary in the absence of kisspeptin. LH and FSH are carried in the blood stream to their target organs, the gonads. In the female, the secretion of the gonadotropins is both tonic and cyclic. As in men, the tonic secretion regulates the minute by minute secretion of the gonadal steroids and in turn is modified by the steroidal feedback on the hypothalamus and pituitary by mechanisms described above. The cyclic secretion, present only in women, governs the female menstrual cycle (see Fig. 2). At the beginning of the menstrual cycle, FSH stimu- lates the development of a group of follicles, one of which becomes dominant and matures into the Graafian follicle. These follicles have been developing over the previous two cycles, and it is not until the very end of the second cycle/beginning of the third cycle that FSH is paramount to the development of this cohort. The time taken for follicles to reach preovulatory status is about 85 days. The granulosa cells in the developing follicles secrete anti-Müllerian hormone (AMH). AMH was previously associated only with the sexual differentiation of the male, but it has now been shown that AMH levels at the beginning of the menstrual cycle are proportional to the number of developing follicles. This observation makes AMH a suitable candidate for assessing ovarian reserve. AMH is secreted optimally by the granulosa cells of pre-antral and small antral follicles. Less AMH is secreted by follicles >8mm size and becomes undetect- able when FSH-dependant follicular growth has been initiated. Once the dominant follicle is established, the remaining follicles become atretic. As the follicle matures (for which both LH and FSH are required), it secretes an increasing amount of estra- diol. This steroid has a negative feedback on the hypo- thalamus and pituitary during the early and mid-follicular phase. Thus, during the follicular phase of the menstrual cycle, there is a fall in FSH levels after an initial rise. Another recently isolated nonsteroidal hormone, inhibin, also changes during the menstrual cycle. Inhibin circu- lates as two biologically active forms: inhibin A and inhibin B. Concentrations of inhibin B rise at the time of menses, are maximal during the early and mid-follicular phases of the cycle, fall during the late follicular phase, Infertility Michael J. Wheeler (mjw@the2wheelers.plus.com) C H A P T E R 9.5 FIGURE 1 Control of sex hormone secretion (simplified).
  2. 2. 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 and estradiol. 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 secretion. 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.
  3. 3. 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. Clinical Disorders PRIMARY HYPOGONADISM 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 failure. SECONDARY HYPOGONADISM 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- thalamicdysfunction(hypogonadotropichypogonadism) 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 FUNCTION 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 problem. 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 required. PRIMARY HYPOGONADISM 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
  4. 4. 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. SECONDARY HYPOGONADISM 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 testosterone. 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. Analytes 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%.
  5. 5. 725CHAPTER 9.5 Infertility Function 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 and 120min. 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. Reference Interval All current immunoassay systems use immunometric assays to measure LH (see Table 1). Clinical Application 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. Limitations 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 gonadotropin levels. 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. Assay Technology 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 Mid-cycle 14–96 Luteal phase 1.0–11.0 Postmenopausal 7.7–59.0 Men 1.7–8.6
  6. 6. 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 Common. FOLLICLE-STIMULATING HORMONE (FOLLITROPIN) 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. Reference Interval FSH is measured by immunometric assay, and in most lab- oratories, will be measured along with LH (see Table 2). Clinical Application 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. Limitations 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 Midcycle 4.7–21.5 Luteal phase 1.7–7.7 Postmenopausal 25.8–134.8 Men 1.5–12.4
  7. 7. 727CHAPTER 9.5 Infertility Assay Technology 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 Common. PROLACTIN 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. Reference Interval Table 3. Clinical Application 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. Limitations 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) (Roche)
  8. 8. 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. TRH Test 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. Assay Technology 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 Common. ANTI-MÜLLERIAN HORMONE 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 ovary. Reference Interval 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. Clinical Application 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 Table 5. Assay Technology 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 menopause 20 Pediatrics (intersex disorders) 15 Tumor marker 12 Assessment of puberty 11
  9. 9. 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 a year. INHIBIN 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. Reference Interval 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 39.3±11.0ng/L. Clinical Application 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 choice. 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 infertility. Assay Technology 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 Beckman Coulter. Types of Sample Serum. Frequency of Use Very rare. ESTRADIOL 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 lactation. FIGURE 4 Estradiol.
  10. 10. 730 The Immunoassay Handbook Reference Interval Table 6. Clinical Application 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. Limitations 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 anticonvulsants. 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. Assay Technology 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 Common. PROGESTERONE 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. Reference Interval Table 7. TABLE 6 Reference Intervals for Estradiol Roche (pg/mL) Roche (pmol/L) 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 Roche (ng/mL) Roche (nmol/L) 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
  11. 11. 731CHAPTER 9.5 Infertility Clinical Application 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. Limitations 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. Assay Technology 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 Common. TESTOSTERONE 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.
  12. 12. 732 The Immunoassay Handbook Reference Interval Table 8. Clinical Application The measurement of testosterone for the investigation of hirsutism and virilization in the female is explained in the appropriate chapter. 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- terone levels. 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 hCG test. 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. hCG Test 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). Limitations 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. Assay Technology 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 (Bayer ACS:Centaur) 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 (intramuscularly) Day 2 Blood sample Inject 1500 IU hCG Day 3 Blood sample Inject 1500 IU hCG Day 4 Blood sample
  13. 13. 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 Common. DIHYDROTESTOSTERONE 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. Reference Interval Table 10. Clinical Application 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. Limitations 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 and DHT. 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 Infrequent. 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 (ng/mL) (nmol/L) Women 0.12–0.43 0.4–1.5 Men 0.38–0.72 1.3–2.5
  14. 14. 734 The Immunoassay Handbook Acknowledgment My sincere thanks to UK NEQAS for allowing me to use some of their data. References and Further Reading Balasch, J. Ageing and infertility: an overview. Gynaec. Endocrinol. 26, 855–860 (2010). 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, 1997). 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/ cooper_et_al_hru.pdf 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, 1313–1318 (1986). 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. 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