Gonadotropin
Plasma and serum are the samples of choice for gonadotropin and androgen measurement.
The evaluation of gonadotropins, FSH, and LH, provides essential information on the origin of testicular dysfunction. They are currently measured by immunofluorometric or electrochemiluminescence assays, characterized by a high diagnostic sensitivity (Table 1). The values obtained by different methods vary considerably, and each technique has a different reference range.
Table1. Reference values of the main hormones
Since gonadotropin levels change throughout the day, it is recommended that any pathologic or questionable results be evaluated on a second blood collection.
Testosterone
Circulating testosterone is carried 40% by sex hormone binding globulin (SHBG) and 58% by albumin, while 2% is free and represents the biologically active fraction. Since the binding of testosterone to SHBG is very stable, only testosterone bound to albumin is bioavailable. “Bioavailable testosterone” refers to the proportion of free and albumin-bound testosterone that can be cleaved and made available in target tissues. Daily testosterone fluctuations require venous sampling between 8 and 10 a.m. However, several studies showed that the circadian rhythm fades with age and, therefore, in the elderly, this time window loses its significance.
The assay of total testosterone (including free and protein- bound portion) is performed by electrochemiluminescent immunometric method (Table 2) and liquid chromatography- tandem mass spectrometry (LC-MS/MS). Currently, LC-MS/MS is the best method, as it provides an accurate and precise estimate of total testosterone levels with high diagnostic sensitivity. However, alterations in SHBG levels may affect total testosterone levels. In such a case, the assay of free testosterone, i.e., the free portion representing approximately 2% of circulating testosterone, is recommended. Several methods have been proposed to measure the free fraction of testosterone. Equilibrium dialysis is the gold standard for quantifying the biologically active form of testosterone; however, it is a technique scarcely available in most clinical laboratories.
Table2. Conditions associated with altered circulating SHBG levels
For this reason, it is recommended to use computational equations allowing free testosterone evaluation using SHBG, albumin, and total testosterone. The values obtained using the Vermeulen formula show a high correlation with those obtained by equilibrium dialysis, representing a simple and reliable indicator (Fig. 1). Alternatively, free testosterone can be measured by enzyme immunoassay (Table 2). Testosterone is also detectable in saliva at a concentration comparable to free testosterone in serum; however, its assay requires careful quality control because it may be subject to several artifacts.
Fig1. Equation for calculating free and bioavailable testosterone (Vermeulen’s formula). (Copyright EDISES 2021. Reproduced with permission)
Inhibin B
The evaluation of inhibin B reflects the functionality of the seminiferous tubules. Reduced inhibin B concentrations are observed if spermatogenesis is impaired (e.g., after chemotherapy). The inhibin B assay is performed by ELISA (Enzyme-Linked ImmunoSorbent Assay).
Prolactin
High prolactin levels may be associated with hypogonadotropic hypogonadism. Increased prolactin can be detected in prolactin-secreting tumors (prolactinomas). Pituitary Magnetic Resonance Imaging (MRI) is essential in prolactinoma (macroprolactinomas or macroprolactinomas). The prolactin assay is performed by the electrochemiluminescent immunometric method.
Dynamic Hormonal Investigations
Stimulation Test with GnRH
In the past, the GnRH test was used in the differential diagnosis of hypogonadotropic hypogonadism, but it is currently no longer recommended because the assessment of testosterone and gonadotropins is sufficient to make the diagnosis. The functional test of stimulation with GnRH, measuring LH levels basal, 30 and 60 minutes after intravenous administration of 100 μg of GnRH, is used in the differential diagnosis of delayed puberty. An LH concentration >10 IU/L, 30 minutes after GnRH administration, allows the diagnosis of constitutional pubertal delay. Functional testing is helpful in patients preparing for pituitary surgery or with lesions in the hypothalamic-pituitary region.
Human Chorionic Gonadotropin Stimulation Test
Like LH, human chorionic gonadotropin (hCG) stimulates testosterone synthesis in Leydig cells. The hCG stimulation test is recommended to assess Leydig cell function, especially in young men with congenital anorchidism (an autosomal recessive syndrome with male karyotype and variable phenotype in the absence of testicular tissue) and to differentiate this clinical condition from cryptorchidism (failure of one or both testes to descend into the scrotal sac). The test involves the administration of 5000 IU of hCG intramuscularly and the dosage of testosterone at baseline and after 72 hours. Leydig cell function is considered normal if testosterone levels increase by 1.5–2 μg/L. This test is not recommended in adults because testosterone and gonadotropin assays are sufficient for diagnosis. In young men with congenital anorchidia, no increase in testosterone levels is observed after stimulation with hCG. Subjects with gonadotropin deficiency respond poorly to hCG administration due to a loss of sensitivity of Leydig cells.
