The laboratory assessment of a patient with suspected hypogonadism includes measurements of the serum concentrations of follicle-stimulating hormone, luteinizing hormone, and testosterone. In most circumstances, this will be adequate to confirm whether the patient has androgen deficiency, and if so, whether the problem lies in the testes or in the hypothalamic-pituitary areas. In some patients, measurements of other testicular hormones and dynamic tests of the hypothalamic-pituitary-testicular axis may be required (Table Endocrine Tests in Male Reproductive Disorders).

Table Endocrine Tests in Male Reproductive Disorders

Basal hormones
Testosterone, FSH, and LH
Other hormones, binding proteins, and subunits
Free or bioavailable testosterone
Sex hormone binding globulin
Dihydrotestosterone
3α-androstanediol glucuronide
Estradiol
Prolactin
α- and β-subunits of FSH and LH
hCG
Dynamic tests
GnRH stimulation test
Clomiphene stimulation test
hCG stimulation test
Thyrotropin-releasing hormone (TRH) test

Abbreviations: FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone.

Testosterone (Total and Free or Bioavailable)

Testosterone concentrations are measured by radioimmunoassays (RIA), immunometric assays, or immunofluorometric assays (IFMA). Testosterone secretion has a circadian rhythm in man, with higher levels in the morning than evening. Since the normal ranges are based on morning values, blood samples for testosterone measurements should be drawn between 7 A.M. and 10 A.M. Automated assays for testosterone are frequently utilized by clinical laboratories. In general, these assays have variable and often poor levels of accuracy in the female and severely hypogonadal ranges. Liquid chromatography and tandem mass spectroscopy are part of the emerging technology for the most precise and accurate testosterone measurements. In most instances, measurement of total plasma testosterone will identify patients with androgen deficiency; however, since testosterone is bound to sex hormone binding globulin in the plasma, changes in sex hormone binding globulin concentrations will lead to changes in total testosterone concentrations. Increases in sex hormone binding globulin occur with hyperes-trogenemia and hyperthyroidism, phenytoin treatment, aging, anorexia nervosa, and prolonged stress. Decreases in sex hormone binding globulin are present with androgen treatment, GH excess, obesity, and hypothyroidism. sex hormone binding globulin-binding capacity in the plasma can be assessed by testing the binding of labeled androgens to sex hormone binding globulin after separation from other proteins (i.e., ligand displacement assays) or by RIA. In disorders with abnormal sex hormone binding globulin concentrations, the measurement of total testosterone may be misleading. For example, a patient with gross obesity may have low total testosterone concentrations, reflecting the low sex hormone binding globulin concentrations associated with obesity. To separate true hypogonadism from binding protein defects, it may be necessary to determine the free or bioavailable testosterone concentrations. Free testosterone concentrations are usually measured after equilibrium dialysis of a serum sample. About 3% of testosterone in blood is free, with the rest bound to sex hormone binding globulin (30%) or to albumin and other proteins (67%). The non-sex hormone binding globulin bound testosterone (i.e., free and albumin bound) is the bioavailable portion of circulating testosterone. Bioavailable testosterone can be estimated in the serum by RIA after removal of sex hormone binding globulin by ammonium sulfate or Concanavalin A-Sepharose. Salivary testosterone can also be used as an indicator of free testosterone since sex hormone binding globulin and other proteins are present in very low concentrations in the saliva.

Other Androgen Metabolites

The measurement of plasma dihydrotestosterone is generally not useful in the evaluation of testicular disorders other than 5α-reductase deficiency. In this disorder, usually presenting with ambiguous genitalia in infancy and varying degrees of virilization at puberty, measurement of serum dihydrotestosterone and testosterone will show an abnormally high testosterone-to-dihydrotestosterone ratio especially after the administration of human chorionic gonadotropins (hCGs).

Estradiol measurements are usually not necessary in the assessment of male reproductive disorders. Estradiol concentrations are elevated in patients with androgen resistance, estrogen-secreting neoplasms, Klinefelter’s syndrome, and hypogonadism associated with chronic liver disease and some cases of gynecomasria.

LH And Follicle-Stimulating Hormone

Measurements of plasma luteinizing hormone and follicle-stimulating hormone are important in classifying the anatomical level of the defect in hypo-gonadal patients. Since both gonadotropins are secreted in a pulsatile pattern, the collection of three samples, 15 to 20 minutes apart, may give more accurate assessment of the mean luteinizing hormone and follicle-stimulating hormone concentrations. Primary gonadal defects are characterized by low testosterone and high luteinizing hormone and follicle-stimulating hormone concentrations, whereas hypothalamic or pituitary disorders have low testosterone, luteinizing hormone, and follicle-stimulating hormone concentrations. The traditional RIA methods can distinguish clearly high follicle-stimulating hormone and luteinizing hormone (hypergonadotropic states) from normal concentrations but cannot clearly demarcate between normal and subnormal concentrations (i.e., hypogonadotropic states). The recently developed IFMA provide increased assay sensitivity from 0.5 to 0.05 IU / L for both gonadotropins. These new, sensitive gonadotropin assays allow the clinician to distinguish the low gonadotropin concentrations commonly observed in hypogonadotropic hypogonadism, delayed puberty, and after gonadotropin-releasing hormone analog treatment. Bioassays of both luteinizing hormone and follicle-stimulating hormone are also available but generally do not add more information to the sensitive immunoassays. In the rare patient with genetic mutations of the β-LH gene, however, determination of luteinizing hormone by bioassay will show low bioactive luteinizing hormone concentrations in the presence of elevated immunoreacrive luteinizing hormone concentrations. Rare patients with infertility have biologically inactive but immunoreacrive follicle-stimulating hormone.

Other Pituitary Hormones

Plasma prolacrin concentrations should be measured in patients with low testosterone and normal-to-low follicle-stimulating hormone and luteinizing hormone concentrations to exclude hyperprolac-tinemia. Measurements of the α- and beta-subunits of the gonadotropins may be useful in patients with pituitary tumors, with or without hypogonadism. Many pituitary tumors previously thought to be nonfunc-tioning secrete large amounts of α- and beta-subunits of luteinizing hormone and follicle-stimulating hormone. Moreover, plasma concentrations of α- and beta-subunits of the gonadotropins rise in response to TRH administration. In patients with germinomas, teratomas, and chorioepitheliomas, beta-hCG subunits may also be secreted and can serve as a tumor marker. Since beta-hCG cross-reacts with most conventional luteinizing hormone RIA, an elevated luteinizing hormone in a child with a germinoma may be due to cross-reaction from beta-hCG in the assay system. beta-hCG is usually not detected by the more specific two-site luteinizing hormone immunoassays.

Dynamic Tests of the Hypothalamic-Pituitary-Testicular Axis

Before the development of sensitive assays for the gonadotropins, dynamic tests to evaluate the hypotha-lamic-pituitary axis were developed. With the use of the more sensitive assays, these dynamic tests are reserved for occasional and unusual diagnostic problems.

Administration of the antiestrogen, clomiphene citrate (100 mg for seven days) causes a rise in plasma luteinizing hormone, follicle-stimulating hormone, and testosterone concentrations. Clomiphene citrate blocks the negative feedback of estrogens and may have a direct stimulatory action on the gonadotropins. This test has limited practical value in the clinical assessment of male reproductive disorders, however, and does not add information to the measurement of the basal concentrations of gonadotropins and testosterone.

The availability of gonadotropin-releasing hormone gave rise to the hope that the gonadotropin-releasing hormone test (administered as a 100 µg bolus to adults or 50 µg / m2 to children) would allow hypotha-lamic disorders to be distinguished from pituitary disorders; however, the gonadorropin response to a single dose of gonadotropin-releasing hormone is frequently suppressed in patients with hypothalamic disorders. Priming the gonado-trophs with repeated low dose gonadotropin-releasing hormone administration for a week followed by a bolus gonadotropin-releasing hormone injection has been shown to enhance the gonadorropin responsiveness in patients with hypothalamic disorders but not in those with pituitary disorders. In general, information obtained from gonadotropin-releasing hormone tests does not help in the diagnosis of tesricular disorders. It is sometimes used in patients with central precocious puberty in whom an exaggerated luteinizing hormone to follicle-stimulating hormone response is characteristic. Leydig cell function can be stimulated by a single injection of human chorionic gonadotropin (2000-5000IU), resulting in peak increases in plasma testosterone concentrations after 72 to 96 hours. The human chorionic gonadotropin test is useful in infants or children with cryptorchidism. A rise in testosterone in response to human chorionic gonadotropin indicates the presence of the testes and excludes anorchia. In patients with hypogonadism, elevated luteinizing hormone and low testosterone concentrations and the absence of a testosterone response to human chorionic gonadotropin suggest resistance to luteinizing hormone associated with Leydig cell hypoplasia or agenesis. A positive response to human chorionic gonadotropin indicates an abnormality of the endogenously secreted luteinizing hormone molecule. Bioassays of luteinizing hormone followed by molecular biology techniques to identify mutations of the luteinizing hormone gene may pinpoint the exact abnormality.

Multiple and frequent samplings for luteinizing hormone and follicle-stimulating hormone concentrations have been used to delineate the defect in gonadotropin-releasing hormone secretion in patients with IHH and aged men. Disorders of luteinizing hormone pulsatility, including absence of pulse, sleep-entrained pulses, and decreased frequency of amplitude of pulses have been defined. Sleep-entrained pulsatile secretion of luteinizing hormone is a hallmark of the onset of puberty and can be used to distinguish patients with early puberty from those with hypogonadotropic hypogonadism. Multiple blood samples, however, have to be taken at 10-minute intervals for a minimum of eight total hours to yield meaningful analyses of the pulsatile secretion of the gonadotropins. Because of the frequency and intensity of sampling, these investigative procedures are used mainly in clinical research studies.

Endocrine Tests in the Evaluation of Ambiguous Genitalia

In a neonate presenting with ambiguous genitalia, the chromosomal sex should be determined. In this section, the endocrine tests relevant to male pseudohermaphroditism (46,XY males with tesris and with abnormal external and / or internal genitalia) are briefly discussed.

Phenotypically male patients with 46,XX may appear as normal males with varying degrees of hypogonadism and pseudohermaphroditism and can be true hermaphrodites. These sex-reversed patients have a portion of the Y chromosome (i.e., SRY gene) located on the X chromosome. The common causes and key laboratory tests of male pseudohermaphroditism are listed in Table Laboratory Tests for the Diagnosis of Male Pseudohermaphroditism. For the diagnosis of the rare defect in infants with 3β-hydroxy steroid dehydrogenase / 17α-hydroxylase, 17,20 lyase deficiency, plasma measurements of the C21 steroid precursors such as dihydrogen androsterone, 17α-hydroxyprogesterone, and 17α-hydroxy pregnenolone before and after ACTH or human chorionic gonadotropin stimulation will confirm the diagnosis. Plasma C19 steroids (testosterone and androstenedione) will be low. XY patients with 17β-hydroxysteroid oxireductase deficiency usually have female or mildly virilized external genitalia at birth and present with some degree of virilizarion at puberty. Since the defect lies in the conversions of androstenedione to testosterone and estrone to estra-diol, the diagnosis is confirmed by finding low ratios of plasma testosterone to plasma androstenedione and estradiol to estrone, both at baseline and after human chorionic gonadotropin stimulation of Leydig cells.

Table Laboratory Tests for the Diagnosis of Male Pseudohermaphroditism

Cause Tests
Testosterone biosynthesis defects
P450 SCC: cholesterol side chain cleavage ↓ Basal and ACTH stimulated and mineralocorticoids ↓ Testosterone

↑ FSH

↑ LH

3β-Hydroxysteroid dehydrogenase ↑ Basal and ACTH stimulated 17α-pregnenolone, dehydroepiandrosterone, and dehydroepiandrosterone sulphate
P450 c17/17α-hydroxylase/17,20-lyase ↓ Testosterone

↑ FSH

↑ LH

↑ Deoxycorticosterone

↑ Corticosterone

Progesterone

↓ Renin

↑ Basal, ACTH and hCG stimulated 17α-pregnenolone and 17α-progesterone

17β-Hydroxysteroid oxidoreductase ↓ Testosterone

↓ Estradiol

↑ Basal and hCG-stimulated androstenedione and estrone

↑ FSH

↑ LH

Androgen resistance syndromes ↑ LH

↑ Testosterone

↑ Estradiol

Normal or ↑ FSH

Skin fibroblasts for androgen receptors: low or undetectable, unstable, abnormal receptor; abnormal androgen receptor gene

Defects in testosterone metabolism
5α-Reductase deficiency ↑ Basal and hCG-stimulated testosterone to dihydrotestosterone ratio

↓ Skin fibroblasts conversion of testosterone to dihydrotestosterone

Leydig cell hypoplasia or aplasia ↓ Testosterone

Absent testosterone response to hCG

Dysgenetic male pseudohermaphroditism
Gonadal dysgenesis, incomplete gonadal dysgenesis Chromosomal analysis
Vanishing testes (congenital anorchia) ↑ FSH

↑ LH

↓ Mullerian inhibiting factor

Absent testosterone response to hCG

Abbreviations: ACTH, adrenocorticotrophin; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropins; LH, luteinizing hormone.

The laboratory diagnosis of androgen resistance syndrome is made by measuring androgen receptor binding and function in genital fibroblast cultures. In patients with this disorder, androgen receptor binding may be low, undetectable, or unstable. Other variations in these disorders include abnormal ARs and postreceptor abnormalities. Using molecular biology techniques, single point mutations, multiple mutations, deletion, or premature stop codon defects have been identified to cause absent, quantitatively, or qualitatively abnormal ARs.

5α-reductase deficiency can be diagnosed by the measurement of the ratio of testosterone to dihydrotestosterone in plasma before and after human chorionic gonadotropin stimulation. Patients with this metabolic defect of testosterone cannot convert testosterone to dihydrotestosterone, thus resulting in high testosterone-to-dihydrotestosterone ratios.

In Leydig cell hypoplasia or aplasia, the Leydig cells are resistant to luteinizing hormone and human chorionic gonadotropin. The plasma testosterone concentrations are low and do not respond to exogenously administered human chorionic gonadotropin; the testes are small and atrophic, and testicular biopsy shows hypoplasia or absence of Leydig cells. In the vanishing testes syndrome, endocrine tests yield the same findings as in Leydig cell hypoplasia. Imaging by ultrasound, computed tomography scan, or magnetic resonance, however, fails to demonstrate the presence of testes. Recently, assays of mullerian-inhibiting substance have been introduced, and these levels have been found to be very low or undetectable in patients with anorchia. In testicular hypoplasia and genital anorchia, varying differentiation and development of the Mullerian and Wolffian duct structures and external genitalia are present, depending on the date of onset of the testicular regression.

The diagnosis of patients with mixed gonadal dysgenesis [45, XO / 46 XY; 45, XO / 47, XXY; 46, XYpi (partial loss of short arm of Y chromosome) and variants] depends on detailed chromosomal studies. The diagnosis of the persistence of Mullerian duct structures is typically made when a uterus or Fallopian tubes are found incidentally during laparotomy or surgery for the correction of a hernia in a phenotypic male who may also have a cryptorchid testis. The use of urethrograms or sonograms can help to visualize internal genitalia but are generally more useful in female pseudohermaphrodirism presenting as ambiguous genitalia.

Semen Tests

Testicular dysfunction is usually associated with impaired spermatogenesis, whether the primary defect is hypothalamic, pituitary, Leydig cell, Sertoli cell, or germ cell in origin. Such men often present to the physician with the complaint of infertility. The basic investigation of a male patient presenting with infertility is a semen analysis (Tables Laboratory Tests on Semen) Sperm antibodies and semen biochemistry are included in the basic tests but should be performed only when indicated. In general, the determination of semen volume, sperm count, motility, and morphology would be sufficient for the investigation of a patient with severe oligozoospermia (fewer than 5 million spermatozoa per mL semen) or asthenozoospermia (fewer than 10% motile) or teratozoospermia (fewer than 10% normal). In patients with normal or moderate impairment of semen parameters, further evaluation with specialized tests may be helpful to delineate specific defects of sperm function. Some of these tests may help to predict the success of assisted reproductive technologies such as in vitro fertilization. It is now known that approximately 20% of men with azoospermia or severe oligospermia harbor microdeletions in the long arm of the Y chromosome, most frequently in the AZF region.

Table Laboratory Tests on Semen

Basic
Semen volume
Sperm count, motility, morphology
Sperm antibodies
Semen biochemistry
Specialized
Sperm cervical mucus interaction tests
Sperm motion analysis, assessment of hyperactivated motility
Sperm acrosomal reactivity
Zona-free hamster oocyte sperm penetration test
Zona pellucida-binding tests (in vitro fertilization)
Sperm biochemistry

Routine Semen Analysis

Because of the marked inherent variability of semen parameters, at least two semen analyses at one- to two-week intervals should be assessed in the laboratory. The semen sample should be collected by masturbation. The procedures and methods for routine semen analyses should follow the World Health Organization Laboratory Manual for Human Semen and Sperm Cervical Mucus Interaction. Using such standardized methods allows comparison from laboratory to laboratory and quality control within the laboratory. If a sample shows no spermatozoa, it should be centrifuged and the pellet reexamined for the presence of spermatozoa before the diagnosis of azoospermia is given. The semen volume is usually between 2 mL and 5 mL. A low semen volume together with an acidic pH and azoospermia suggests genital tract obstruction caused by congenital bilateral absence of the vas deferens and seminal vesicles. The sperm count is assessed visually under a phase microscope with a white cell-counting chamber. Sperm motility is assessed visually and graded according to whether the spermatozoa has rapid progression, slow progression, no progression, or is nonmotile. A normal semen sample should have a sperm concentration of more than 20 million / mL or total sperm count of more than 40 million and 50% or more spermatozoa with progressive motility. A moderate decrease in sperm concentration to between 10 and 20 million / mL is compatible with fertility, provided sperm motility and morphology are normal. Although doubts have been raised about the value of routine semen analysis to distinguish between fertile and infertile men, studies have shown that concentration and morphology are variables capable of predicting fertility status especially in men with grossly abnormal semen parameters. In patients with unexplained infertility in whom routine semen analysis is normal, specialized tests of sperm function should be considered.

Until recently, sperm morphology has been classified using lenient criteria, with the lower limit for normal morphology being set at 50%. More recently, studies have indicated that by using more strict criteria, taking into consideration not only the shape of the spermatozoa but also the morphometric measurements (length, width, and length-to-width ratio) and the area occupied by the acrosome, sperm morphology was highly predictive of results of in vitro fertilization. Moreover, a multiple anomalies index wherein all abnormalities present in spermatozoa were scored (including midpiece and tail defects) showed a correlation with in vivo fertility. These studies suggest that stricter criteria with the inclusion of midpiece and tail morphology assessments may be more helpful for the diagnosis of male infertility.

The significance of the occurrence of leukocytes in semen is not clear and no direct relationship has been documented between the presence of leukocytes and genital infections. Semen cultures are usually performed if significant numbers of leukocytes (more than 1 million / mL) are present in the semen.

Sperm vitality can be assessed by supravital stains such as eosinnigrosin. Measurement of sperm vitality should be performed in patients with very low sperm motility. This will help to identify whether the nonmotile spermatozoa are living. The hypo-osmotic swelling test assesses the fluidity of the plasma membrane. Although not useful as a test of sperm fertilizing capacity, this test can assess sperm vitality.

Specialized Tests of Sperm Function

Specialized tests of sperm function are of great value in assessing patients with unexplained infertility. Assessment of sperm motion characteristics, sperm vitality, sperm autoantibodies, and sperm-cervical menses interaction, and human zona pellucida binding tests, zona-free hamster oocyte penetration tests, and the acrosome reaction tests are part of the highly specialized assessment of difficult cases of male factor infertility.

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