madman
Super Moderator
Abstract
Testosterone (T), the principal androgen secreted by the testes, plays an essential role in male health. Male hypogonadism is diagnosed based on a combination of associated clinical signs and symptoms and laboratory confirmation of low circulating T levels. In this review, we have highlighted factors, both biological and analytical, that introduce variation into the measurement of serum T concentrations in men; these need to be considered when requesting T levels and interpreting results. There is an ongoing need for analytical standardization of T assays and harmonization of pre and post-analytical laboratory practices, particularly in relation to the laboratory reference intervals provided to clinicians. Further, there is a need to share with service users the most up-to-date and evidence-based action thresholds for serum T as recommended in the literature. Estimation of free testosterone may be helpful. Causes of secondary hypogonadism should be considered. A comprehensive approach is required in the management of male hypogonadism, including lifestyle modification as well as medication where appropriate. The goal of treatment is the resolution of symptoms as well as the optimization of metabolic, cardiovascular, and bone health. The advice of an endocrinologist should be sought when there is doubt about the cause and appropriate management of hypogonadism.
2. Causes of Male Hypogonadism
COVID-19 Infection and Testosterone
3. Factors Affecting the Measurement of Total Testosterone Levels
3.1. Biological Variation of T Levels
T circulates in both protein-bound and non-protein-bound (free) forms. In men, ~50%is loosely bound to albumin, 44% is bound to sex hormone-binding globulin (SHBG),4% is bound to other proteins (e.g., cortisol-binding protein) and 2% is free (non-protein bound) [39]. Serum total T levels, like many hormones, can be influenced by various biological factors (Table 3) that contribute to fluctuations in T levels within individuals (which vary significantly). It is important to be aware of these variables, which can be broadly classified into either physiological or analytical factors, to avoid misdiagnosing hypogonadism. The physiological factors within a male that play an important role are summarised in Table 3 [39,40].
3.2. Analytical Variation
3.2.1. Total Testosterone Assays
Serum total T assays clearly play an important role in the clinical evaluation of male hypogonadism. In UK clinical biochemistry laboratories, total T levels in adult males are routinely measured using non-radioactive methods on automated analyzers, often with commercial immunoassays (>80% [41], also termed ‘direct-assays’) but sometimes with mass spectrometry (MS); however, significant inter-assay variation was observed between different immunoassays and different MS platforms in a UK-wide survey of NHS clinical laboratories, with intra-assay variability being another technical constraint [41]. The direct assays are so-called because there is no extraction of T from any binding protein included in the method procedure, which uses antibodies to directly bind T for subsequent quantitation in the sample. This lack of extraction can leave the method more prone to interference (e.g., from other similar cross-reacting molecules) and, at lower T levels, aberrations in the levels of serum-binding proteins can lead to measurement inaccuracies. Liquid chromatography–tandem MS (LC-MS/MS) is considered a better method due to its potentially higher specificity and sensitivity; however, in reports from the United Kingdom National External Quality Assurance Scheme (UK NEQAS) for Steroid Hormones, some of the mass spectrometry assays are actually being outperformed by the better immunoassays. UK NEQAS data from 2021 shows between-laboratory imprecision of 5–10% for all T assays (covering a concentration range = 0.5–35 nmol/L); there is also a range of biases between the different methods, which is an issue when trying to apply universal reference ranges or cut-off thresholds in the diagnosis and management of hypogonadism. Consequently, wide variability has been reported in the reference ranges provided by UK laboratories, both between the laboratories and different methods used and even amongst users of the same method, with the lower limit of normal (LLN) ranging from 4.9–11 nmol/L [41].
Varying the LLN and upper limit of normal (ULN) have potential consequences for men with all types of hypogonadism in terms of whether or not to initiate TRT and adjustment to therapy (e.g., lowering the dose), respectively. Moreover, the quality of the data used to calculate these normative ranges for the commercial immunoassays is questionable in terms of both controlling the pre-analytical factors described above in sampling protocols (as far as practically possible) and the applicability of the derived intervals to the population sampled; this degrades the clinical value in their use for diagnosing hypogonadism.
3.2.2. Sex Hormone-Binding Globulin (SHBG) Assays and Calculated FreeTestosterone (cFT)
Like T levels, SHBG is measured using immunoassays in the UK, and these assays are prone to similar analytical variability and differing manufacturer biases, leading to inconsistent results between the methods. Interestingly, SHBG has now been shown to be associated with symptoms of hypogonadism and mortality [42]. According to the free hormone hypothesis, only unbound T is bioactive and thus able to bind to androgen receptors in the target tissues [43]. The estimation of calculated free testosterone (cFT) is considered useful in patients with conditions that alter SHBG levels and when the total T levels are close to the LLN (see Table 4) to avoid the under/over-diagnosis of hypogonadism. Variation equations have been used to derive cFT, most commonly the Vermeulen equation in the UK [41]. It is worth noting that the equation is only an estimation of free testosterone and incorporates the test results of albumin, SHBG, and total testosterone in the calculation(combining variability for three tests). Equilibrium dialysis followed by MS is considered the reference method for estimating FT, but it is laborious and time-consuming, and lacks standardization [44]. This method is not available in the UK for routine clinical practice, and other direct measurement methods tend to be inaccurate and are not recommended. In a national audit of UK clinical laboratories [41], none were offering direct measurement of free or bioavailable testosterone.
4. Laboratory Evaluation/Diagnosis of Male Hypogonadism
5. Therapeutic Intervention and Thresholds for Monitoring TRT inMale Hypogonadism
6. Conclusions
In this review, we have highlighted factors, both biological and analytical, that introduce variation into the measurement of serum total T levels in men; these need to be considered when requesting T levels and interpreting patient results. Inconsistencies have been reported between clinical laboratories, and there is an ongoing need for analytical standardization of T assays and harmonization of pre- and post-analytical laboratory practices, particularly in relation to the laboratory reference intervals provided to clinicians. Further, there is a need to share with service users the most up-to-date and evidence-based action thresholds for T as recommended in the literature. A recent expert joint statement on testing and interpretative recommendations by the SfE and Association for Clinical Biochemistry and Laboratory Medicine (ACB) [56,86] is a welcome attempt to address the existing gaps between clinical and laboratory medicine associations (as well as the national external quality assurance provider for clinical laboratories) to improve the current situation for patients requiring measurement of their T levels. Future work should consider how to develop harmonized T reference intervals/treatment thresholds for all clinical laboratories, leading to reduced variation and clinical confusion in the approach to diagnosis and treatment of male hypogonadism; ideally, this would be done by ensuring the total T results from the different methods and manufacturers are more closely aligned (akin to what was done with glycated hemoglobin A1c (HbA1c) standardization), which is not an easy undertaking without the introduction of universal reference standards. Programs such as that of the Centers for Disease Control and Prevention(CDC) reference laboratory for standardizing hormone measurements, including for total T ([87]; https://www.cdc.gov/labstandards/pdf/hs/HoSt_Brochure.pdf; accessed on10 November 2023), and the availability of reference materials will help to accomplish this, but not whilst it remains voluntary. Other less robust approaches would be to (1) use assay-specific treatment thresholds or (2) establish harmonized reference ranges/action thresholds for T (as described by Travison et al. [88]) that can be applied across laboratories by cross-calibrating T assays to a reference method (such as LC–tandem mass spectrometry)and standard calibrator(s) in a healthy, non-obese male population. Pre- and post-analytical harmonization of laboratory function relating to the measurement of T also should be considered to address the biological/sample collection aspects and the evidence-based advice provided by laboratories to clinicians.
Testosterone (T), the principal androgen secreted by the testes, plays an essential role in male health. Male hypogonadism is diagnosed based on a combination of associated clinical signs and symptoms and laboratory confirmation of low circulating T levels. In this review, we have highlighted factors, both biological and analytical, that introduce variation into the measurement of serum T concentrations in men; these need to be considered when requesting T levels and interpreting results. There is an ongoing need for analytical standardization of T assays and harmonization of pre and post-analytical laboratory practices, particularly in relation to the laboratory reference intervals provided to clinicians. Further, there is a need to share with service users the most up-to-date and evidence-based action thresholds for serum T as recommended in the literature. Estimation of free testosterone may be helpful. Causes of secondary hypogonadism should be considered. A comprehensive approach is required in the management of male hypogonadism, including lifestyle modification as well as medication where appropriate. The goal of treatment is the resolution of symptoms as well as the optimization of metabolic, cardiovascular, and bone health. The advice of an endocrinologist should be sought when there is doubt about the cause and appropriate management of hypogonadism.
2. Causes of Male Hypogonadism
COVID-19 Infection and Testosterone
3. Factors Affecting the Measurement of Total Testosterone Levels
3.1. Biological Variation of T Levels
T circulates in both protein-bound and non-protein-bound (free) forms. In men, ~50%is loosely bound to albumin, 44% is bound to sex hormone-binding globulin (SHBG),4% is bound to other proteins (e.g., cortisol-binding protein) and 2% is free (non-protein bound) [39]. Serum total T levels, like many hormones, can be influenced by various biological factors (Table 3) that contribute to fluctuations in T levels within individuals (which vary significantly). It is important to be aware of these variables, which can be broadly classified into either physiological or analytical factors, to avoid misdiagnosing hypogonadism. The physiological factors within a male that play an important role are summarised in Table 3 [39,40].
3.2. Analytical Variation
3.2.1. Total Testosterone Assays
Serum total T assays clearly play an important role in the clinical evaluation of male hypogonadism. In UK clinical biochemistry laboratories, total T levels in adult males are routinely measured using non-radioactive methods on automated analyzers, often with commercial immunoassays (>80% [41], also termed ‘direct-assays’) but sometimes with mass spectrometry (MS); however, significant inter-assay variation was observed between different immunoassays and different MS platforms in a UK-wide survey of NHS clinical laboratories, with intra-assay variability being another technical constraint [41]. The direct assays are so-called because there is no extraction of T from any binding protein included in the method procedure, which uses antibodies to directly bind T for subsequent quantitation in the sample. This lack of extraction can leave the method more prone to interference (e.g., from other similar cross-reacting molecules) and, at lower T levels, aberrations in the levels of serum-binding proteins can lead to measurement inaccuracies. Liquid chromatography–tandem MS (LC-MS/MS) is considered a better method due to its potentially higher specificity and sensitivity; however, in reports from the United Kingdom National External Quality Assurance Scheme (UK NEQAS) for Steroid Hormones, some of the mass spectrometry assays are actually being outperformed by the better immunoassays. UK NEQAS data from 2021 shows between-laboratory imprecision of 5–10% for all T assays (covering a concentration range = 0.5–35 nmol/L); there is also a range of biases between the different methods, which is an issue when trying to apply universal reference ranges or cut-off thresholds in the diagnosis and management of hypogonadism. Consequently, wide variability has been reported in the reference ranges provided by UK laboratories, both between the laboratories and different methods used and even amongst users of the same method, with the lower limit of normal (LLN) ranging from 4.9–11 nmol/L [41].
Varying the LLN and upper limit of normal (ULN) have potential consequences for men with all types of hypogonadism in terms of whether or not to initiate TRT and adjustment to therapy (e.g., lowering the dose), respectively. Moreover, the quality of the data used to calculate these normative ranges for the commercial immunoassays is questionable in terms of both controlling the pre-analytical factors described above in sampling protocols (as far as practically possible) and the applicability of the derived intervals to the population sampled; this degrades the clinical value in their use for diagnosing hypogonadism.
3.2.2. Sex Hormone-Binding Globulin (SHBG) Assays and Calculated FreeTestosterone (cFT)
Like T levels, SHBG is measured using immunoassays in the UK, and these assays are prone to similar analytical variability and differing manufacturer biases, leading to inconsistent results between the methods. Interestingly, SHBG has now been shown to be associated with symptoms of hypogonadism and mortality [42]. According to the free hormone hypothesis, only unbound T is bioactive and thus able to bind to androgen receptors in the target tissues [43]. The estimation of calculated free testosterone (cFT) is considered useful in patients with conditions that alter SHBG levels and when the total T levels are close to the LLN (see Table 4) to avoid the under/over-diagnosis of hypogonadism. Variation equations have been used to derive cFT, most commonly the Vermeulen equation in the UK [41]. It is worth noting that the equation is only an estimation of free testosterone and incorporates the test results of albumin, SHBG, and total testosterone in the calculation(combining variability for three tests). Equilibrium dialysis followed by MS is considered the reference method for estimating FT, but it is laborious and time-consuming, and lacks standardization [44]. This method is not available in the UK for routine clinical practice, and other direct measurement methods tend to be inaccurate and are not recommended. In a national audit of UK clinical laboratories [41], none were offering direct measurement of free or bioavailable testosterone.
4. Laboratory Evaluation/Diagnosis of Male Hypogonadism
5. Therapeutic Intervention and Thresholds for Monitoring TRT inMale Hypogonadism
6. Conclusions
In this review, we have highlighted factors, both biological and analytical, that introduce variation into the measurement of serum total T levels in men; these need to be considered when requesting T levels and interpreting patient results. Inconsistencies have been reported between clinical laboratories, and there is an ongoing need for analytical standardization of T assays and harmonization of pre- and post-analytical laboratory practices, particularly in relation to the laboratory reference intervals provided to clinicians. Further, there is a need to share with service users the most up-to-date and evidence-based action thresholds for T as recommended in the literature. A recent expert joint statement on testing and interpretative recommendations by the SfE and Association for Clinical Biochemistry and Laboratory Medicine (ACB) [56,86] is a welcome attempt to address the existing gaps between clinical and laboratory medicine associations (as well as the national external quality assurance provider for clinical laboratories) to improve the current situation for patients requiring measurement of their T levels. Future work should consider how to develop harmonized T reference intervals/treatment thresholds for all clinical laboratories, leading to reduced variation and clinical confusion in the approach to diagnosis and treatment of male hypogonadism; ideally, this would be done by ensuring the total T results from the different methods and manufacturers are more closely aligned (akin to what was done with glycated hemoglobin A1c (HbA1c) standardization), which is not an easy undertaking without the introduction of universal reference standards. Programs such as that of the Centers for Disease Control and Prevention(CDC) reference laboratory for standardizing hormone measurements, including for total T ([87]; https://www.cdc.gov/labstandards/pdf/hs/HoSt_Brochure.pdf; accessed on10 November 2023), and the availability of reference materials will help to accomplish this, but not whilst it remains voluntary. Other less robust approaches would be to (1) use assay-specific treatment thresholds or (2) establish harmonized reference ranges/action thresholds for T (as described by Travison et al. [88]) that can be applied across laboratories by cross-calibrating T assays to a reference method (such as LC–tandem mass spectrometry)and standard calibrator(s) in a healthy, non-obese male population. Pre- and post-analytical harmonization of laboratory function relating to the measurement of T also should be considered to address the biological/sample collection aspects and the evidence-based advice provided by laboratories to clinicians.
