9.3.2 Endocrine evaluation

The main constituent of endocrine laboratory diagnosis of testicular dysfunction is the determination of the gonadotropins, luteinizing hormone and follicle-stimulating hormone (FSH) secreted from the pituitary gland, of testosterone secreted from the Leydig cells, and of inhibin-B secreted from the Sertoli cells. Where hypothalamic or pituitary disorders are suspected as causes of testicular dysfunction, a gonadotropin-releasing hormone (GnRH) stimulation test can be performed for further differentiation. A human chorionic gonadotropin (hCG) stimulation test is done for evaluation of the endocrine reserve capacity of the testis. Additional hormone measurements are performed for special diagnostic questions, e.g. of oestradiol in cases of gynaecomastia, or hCG and oestradiol upon suspicion of a testicular tumour. Various steroid hormones, including dihydrotestosterone, androgen receptors, or androgen metabolizing enzymes (e.g. 5α-reductase) in the target organs are analysed in patients with disturbances of sexual differentiation.

The evaluation of serum levels of luteinizing hormone and FSH in combination with testosterone provides information for specifying the cause of hypogonadism, which is important for adequate therapy. High gonadotropin levels in serum, in combination with low testosterone levels, indicate hypogonadism of testicular origin (primary hypogonadism); low gonadotropin levels point to a central cause (secondary hypogonadism).

In interpreting basal luteinizing hormone values one must consider the physiological pulsatility of pituitary secretion, with ensuing oscillations of serum levels. A normal man shows approximately 8–20 luteinizing hormone pulses per day. Patients with primary hypogonadism have increased average serum concentrations as well as elevated luteinizing hormone pulse frequency. When hypothalamic GnRH secretion fails, only sporadic luteinizing hormone pulses, if any, can be measured. High luteinizing hormone levels in combination with high testosterone serum concentrations indicate androgen resistance.

FSH displays only minor oscillations in serum levels, and therefore a single measurement is representative. To a certain extent, FSH serum concentrations reflect spermatogenesis (1). High FSH levels in the presence of a small, firm testis (less than 6 ml) and azoospermia are indicators for Klinefelter’s syndrome; low FSH levels indicate a hypothalamic or pituitary deficiency (Fig. 9.3.2.1). If testicular volume exceeds 6 ml and azoospermia or severe oligozoospermia is simultaneously present, elevated FSH indicates primary impairment of spermatogenesis. Within wide margins, the extent of FSH elevation is correlated with the number of seminiferous tubules lacking germ cells (Sertoli cell-only tubules) (Fig. 9.3.2.2) (2). Normal FSH values in combination with azoospermia, normal testicular volume and low values of epididymal markers in the ejaculate raise the suspicion of bilateral obstruction or aplasia of the seminal ducts (Fig. 9.3.2.2).

For the determination of gonadotropin levels in serum, competitive assays such as radioimmunoassays (RIA) or the more sensitive noncompetitive immunoassays such as immunoradiometric assays (IRMA), immunofluorometric assays (IFMA), or enzyme-linked immunosorbent assays (ELISA), are performed. In addition, in vitro bioassays for luteinizing hormone and FSH have been developed. In most cases, the bioactivity and immunoactivity of gonadotropins are well correlated, and in vitro bioassays are unnecessary for routine clinical diagnostics (3).

Mutations of the gonadotropin genes are rare. Inactivating mutations of the luteinizing hormone β subunit lead to infertility and lack of spontaneous puberty. Inactivating mutations of the FSH β subunit gene lead to azoospermia and infertility (4). The rare mutations of gonadotropin receptor genes are classified into activating (gain-of-function) and inactivating (loss-of-function) mutations. Activating luteinizing hormone receptor mutations cause pubertas praecox; inactivating mutations cause Leydig cell hypoplasia and hypogonadism. Inactivating FSH receptor mutations result in variable suppression of spermatogenesis; the only activating FSH receptor mutation described so far maintained spermatogenesis in a hypophysectomized patient (4, 5).

Serum concentrations of GnRH in the general circulation are too low to be measurable by existing immunoassays.

The GnRH test is performed to measure the gonadotropin reserve capacity of the pituitary, and is indicated particularly in the event of low to normal luteinizing hormone and FSH values, which cannot always be differentiated from pathologically low basal values. The rise of luteinizing hormone should be at least threefold 30–45 min after an injection of 100 μg GnRH, and the increase in FSH should be 1.5 times over basal levels. However, the results should be judged by an experienced clinician.

In some patients with GnRH deficiency, the gonadotrophs respond to a GnRH stimulus in a physiological fashion only after a certain period of ‘GnRH priming’. Differentiation between a hypothalamic and a pituitary disorder as the cause of absent gonadotropins, or their blunted increase after GnRH administration, can be achieved using the so called GnRH pump test. For a period of up to 7 days, 5 μg GnRH is given subcutaneously every 90–120 min with a portable minipump. Normalization of the gonadotropin response to a GnRH bolus after 36 h or 7 days indicates a hypothalamic source of the testicular dysfunction. In contrast, a primary pituitary problem must be suspected if the gonadotrophs remain functionally resistant to a GnRH bolus. A GnRH test after 36 h pulsatile GnRH application can diff-erentiate constitutional delayed puberty (which displays a normalized GnRH test after 36 h of pulsatile GnRH application) from Isolated Hypogonadotropic Hypogonadism (IHH) or Kallmann’s syndrome (where a normalized GnRH test results only after 7 days of pulsatile GnRH application) (6, 7). Magnetic resonance imaging (MRI) should be performed for further differentiation. When basal gonadotropin levels are high, which points to a primary testicular disorder, no additional information can be gained by a GnRH test.

Recently, mutations of various genes involved in the control of GnRH secretion, of the GnRH gene, and of the GnRH receptor gene have been identified as causes for Isolated Hypogonadotropic Hypogonadism (IHH) or Kallmann’s syndrome (Chapter 9.2.2) (4). Upon suspicion of these disorders, molecular genetic diagnostics and counselling should be offered to patients (4).

The determination of prolactin in men does not play as pivotal a role as it does in women. Fertility disorders of unclear origin, erectile dysfunction and loss of libido, gynaecomastia, galactorrhoea, and/or other symptoms that indicate a pituitary disorder, or suspicion of pituitary tumour, should prompt prolactin serum measurements via noncompetitive immunoassays. Prolactin is the hormone most commonly secreted by pituitary adenomas. In interpreting the results, it should be remembered that numerous drugs, particularly psychotropic drugs, and stress increase prolactin secretion.

In stress-induced hyperprolactinaemia the basal prolactin levels generally do not exceed twice the upper normal limit. High values (> 2000 mU/l) are typical of a macroprolactinoma; however, serum levels can be variable (4, 8). In general, endocrine tests such as the thyroid releasing hormone (TRH) stimulation test are not suited to and are not longer recommended for the differential diagnosis of hyperprolactinaemia (9).

Testosterone in serum is the most important laboratory value for confirming clinical suspicion of hypogonadism and for monitoring testosterone substitution therapy. In interpreting testosterone values, diurnal variations should be considered; these result in morning serum concentrations that are approximately 40% higher than evening values (10).

Short, intense physical exercise can increase serum testosterone levels, whereas extended, exhausting physical exercise, and high-performance sports, can lead to their decrease. Nearly all chronic diseases, and particularly those of the liver, kidneys, and the cardiovascular system, lead to a decrease in testosterone (Chapter 9.4.8), as does stress, anaesthesia, drugs and certain medications (e.g. ketoconazole).

Low levels of testosterone, and especially of free testosterone, are found more often in elderly men (11). This decrease may be partially caused by various diseases or conditions, including obesity, or by a combination of different diseases (multimorbidity), but it is also seen in healthy elderly men (11). To date, no age-specific normal ranges for testosterone have been established. However, the combination of clinical symptoms of hypogonadism with low serum concentrations of testosterone is regarded as late-onset hypogonadism (LOH), and is an indication for testosterone substitution therapy (12).

There is increasing evidence that there are specific thresholds for the signs and symptoms of hypogonadism in young as well as aging men (13, 14). This might explain the different threshold values for the diagnosis of hypogonadism in different countries (15).

In addition, it has been demonstrated that CAG-repeat polymorphism of the androgen receptor gene modulates the bioactivity of testosterone at the cellular level (16). Although measurement of the CAG repeats of the androgen receptor gene is not included in current recommendations for the diagnosis, monitoring and treatment of hypogonadism, molecular diagnostics might be warranted in the future, for individualized diagnosis and treatment of hypo-gonadal men (12, 16, 17).

Considering these factors, a normal testosterone concentration in serum in the adult male lies between 12 and 40 nmol/1 during the first half of the day; values lower than 8 nmol/l are certainly pathological, values between 8 and 12 require additional testing (12). Boys before puberty and castrated men have serum levels lower than 4 nmol/l.

In most laboratories, serum concentrations of testosterone are determined by radioimmunoassay, enzyme immunoassay, fluoro-immunoassay, or chemiluminescence immunoassay. However, these methods are low in precision and accuracy for the measurement of low testosterone serum levels (18). Tests based on mass spectrometry are more accurate and precise for low testosterone concentrations, and are increasingly recognized as the methods of choice (18, 19). For practical reasons, the established immunoassays are sufficient for clinical diagnosis of hypogonadism in adults, if respective reference values have been established for each laboratory (12).

The stability of testosterone is high, even after repeated freezing and thawing. Normally, a single blood sample is sufficient for the assessment of testosterone serum levels; repeated measurements on the same day or serum pooling is not necessary (20).

In blood, testosterone is bound to a protein, specifically, sex hormone binding globulin (SHBG). Only approximately 2% of testosterone is unbound and available as free testosterone for biological effects. The free testosterone concentration obtained by equilibrium dialysis, and the fraction of serum testosterone not precipitated by ammonium sulphate (non-SHBG-testosterone, bioavailable testosterone), represent reliable indices of biologically readily available testosterone. However, these measurements are too time-consuming for routine clinical practice. For practical purposes, the free and the so-called bioavailable testosterone can be calculated from total testosterone and SHBG (21).

Since total testosterone is well correlated with free testosterone, the calculation of free testosterone is necessary only in certain cases. As an example, hyperthyroidism and antiepileptic drugs cause an increase in SHBG levels and thereby increase testosterone concentration in the serum, without a parallel increase of the biologically active free testosterone levels. Low testosterone levels are found in extreme obesity; however, if this occurs in combination with low SHBG values, then the free testosterone fraction might remain normal.

Testosterone can also be measured in the saliva. Experimental studies demonstrated that salivary testosterone concentrations are correlated with free testosterone in serum (22). However, determination of salivary testosterone is not recommended for routine diagnostics, since the methodology has not been standardized and ranges for adult men are not available in most reference laboratories (12).

The endocrine reserve capacity of the testis can be tested by stimulation with hCG, which has activity predominantly similar to luteinizing hormone and stimulates testosterone production by the Leydig cells. The test is mainly used to differentiate between crypto-rchidism or ectopy of the testis (where a rise in testosterone levels is present, but diminished) and anorchia (where the testosterone rise is absent). On the first day of examination, basal blood samples are obtained between 8 am and 10 am; immediately thereafter a single injection of 5000 IU hCG is given intramuscularly. Further blood samples are obtained after 48 and/or 72 h. The rise of testosterone should be between 1.5 and 2.5-fold. Lower values indicate primary hypogonadism and higher values signal secondary hypo-gonadism. Anorchia and complete testicular atrophy are indicated by a failure to rise from baseline testosterone values in the expected range for a castrated man. A decreased reserve capacity of the Leydig cells is characteristic of an elderly man (23).

Anti-müllerian hormone (AMH), also known as müllerian inhibiting substance (MIS), is a testicular hormone secreted by immature Sertoli cells, and is responsible for the regression of müllerian ducts in male fetuses. The measurement of serum AMH is a sensitive and specific test for the detection of testes in prepubertal boys (24). A measurable value within the normal range for boys is predictive of testicular tissue, whereas an undetectable value is predictive of anorchia. Compared with the hCG test, the measurement of serum AMH is more sensitive and equally specific, and its predictive value for the absence of testicular tissue is higher in prepubertal boys (24). Serum concentrations of AMH differ quantitatively in prepubertal boys with abnormal and normal testes and, therefore, are also helpful for assessing the structural integrity of the testes (24). High levels of AMH are detected in patients with IHH; these are related to the absence of pubertal maturation of Sertoli cells and are similar to those in prepubertal boys (25). In IHH patients, hCG or testosterone treatment significantly reduces serum levels of AMH (25).

AMH levels are not affected by impaired spermatogenesis in general but are correlated with spermatogenic parameters in men with current or former maldescended testes. In these men, AMH might serve as a marker of Sertoli cell number, function, and/or maturation (26). AMH is not superior to FSH or inhibin-B as an endocrine predictor of the presence of testicular sperm in azoospermic men (27).

In men, insulin-like factor 3 (INSL3) is expressed in fetal and adult Leydig cells, and is responsible for the abdominal descent of the testes (28). Mutations of the gene for INSL3 and the gene of its receptor, LGR8/RXFP2, have been described in patients with maldescended testes (29). In males, INSL3 can be regarded as a specific marker of Leydig cells. Although INSL3 is not currently measured in routine andrological diagnostics, it might prove helpful for differential diagnosis of cryptorchidism versus anorchia in the future.

Inhibin-B is secreted from the testis as a product of Sertoli cells. It is involved in the regulation of pituitary FSH secretion. Inhibin-B levels show significant diurnal variation, with peak values in the early morning and nadirs in the late afternoon, followed by gradually increasing nocturnal values (30). Morning serum levels of inhibin-B are associated with FSH levels, sperm concentration and testicular volume in normal and infertile men (Fig. 9.3.2.2) (2, 31, 32). However, measurement of inhibin-B, FSH, or the combination of these parameters cannot accurately predict the presence of elongated spermatids in testicular biopsies of azoospermic patients. Additionally, inhibin-B and/or FSH measurements cannot predict the chances of successfully becoming a father after retrieval of elongated spermatids by testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) (2, 33).

Determination of 17β-oestradiol, aromatase activity, hCG, androstenedione, 5α-dihydrotestosterone (DHT), and 5α-reductase activity may be necessitated by particular findings, e.g. gynaecomastia, skeletal maturation disorders, suspected testicular tumour, or enzyme defects in testosterone biosynthesis and metabolism (Chapter 9.4 and Chapter 9.7). Molecular analyses of the androgen receptor gene and oestrogen receptor gene are indicated when androgen or oestrogen resistance is suspected (34–36).