madman
Super Moderator
* Immunoassays show acceptable accuracy for total T but are unreliable for free T measurement [19,20].
* As previously noted, the main limitation of free T evaluation in clinical practice is the poor accuracy of the available immunoassay methods [19]. To address this issue, several calculation models based on SHBG, albumin, and total T have been developed. Among these, the Vermeulen formula is considered the most reliable approximation of circulating free T, despite a reported 20–30% overestimation [32,33]. The Vermeulen formula was the most frequently cited equation in the European laboratory survey [21]. A further limitation of free T assessment relates to potential inaccuracies in SHBG measurement.
* Another critical issue concerns single-nucleotide polymorphisms (SNPs), which are common in the general population and may affect SHBG levels and binding capacity, thereby influencing free T calculation.
* Results indicated that SHBG-related SNPs may influence total T assessment but not free T when measured by LC-MS/MS. A trend toward higher calculated free T was observed in carriers of the rare rs6259 polymorphism, associated with elevated SHBG levels, whereas bioavailable T remained unaffected [35].
* Based on these findings, the strong recommendation to perform SHBG measurement and free T calculation was maintained in all conditions characterized by altered SHBG levels (Tables 2 and 3).
* the EAU SRH Guidelines Panel further considers it highly advisable to assess SHBG and free T in all symptomatic patients.
* In this setting, limited evidence suggests that a threshold of 220 pmol/l for free T can be considered as a reliable threshold. However, recent data based on direct free T measurement via LC-MS/MS indicated differences in free T levels according to age [38]. Therefore, more studies are required to better clarify the most appropriate threshold for free T in routine practice.
Despite variations in proposed T thresholds, all major guidelines agree that diagnosis of hypogonadism requires both low circulating T and relevant symptoms [1,8–17]. However, discrepancies persist regarding biochemical assessment, preanalytical conditions (fasting and morning sampling), and whether total or free T should be measured (Table 1). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is considered the reference method for total T [18], but high cost limits its routine use. Immunoassays show acceptable accuracy for total T but are unreliable for free T measurement [19,20]. Consequently, guidelines recommend calculating free T only when sex hormone–binding globulin (SHBG) alterations are suspected (Table 2).
Although not without methodological limitations, a recent internet-based survey conducted across 27 European countries revealed substantial variability in laboratory practices, including widespread reliance on immunoassays for total T measurement, limited adoption of LC-MS/MS, and inconsistent preanalytical protocols [21]. These findings underscore the need for harmonized recommendations. This paper aims to provide updated guidance from the European Association of Urology (EAU) Sexual and Reproductive Health (SRH) Guidelines Panel, offering a critical interpretation of technical aspects in biochemical confirmation of male hypogonadism during aging, to support standardized clinical and research strategies for T therapy (TTh).
3. The guidelines
3.1. Biochemical T measurement in routine clinical practice
Based on the 2026 update of the EAU SRH Guidelines, the previous recommendation supporting the measurement of total T under fasting conditions remains unchanged (Table 3). This is supported by evidence showing that both total and calculated free T levels decrease within 30 min following oral glucose administration during an oral glucose tolerance test [23]. These results are independent across the different spectra of glucose tolerance tests and have been corroborated by other studies [24]. The available data indicate that measuring circulating T in nonfasting conditions can lead to an underestimation of total T by >30% compared with its true value [24]. Current evidence also supports the presence of a circadian rhythm in T secretion, with higher levels observed in the morning even among older men [25,26].
3.2. Total T threshold in routine clinical practice
Although acknowledging the potential limitations of the reference, which nonetheless remains crucial for illustrating the real-world scenario and the heterogeneity of diagnosis approaches, data from the European laboratory survey confirmed a wide variability in reference ranges for normal T levels across different immunoassay methods [21]. It should be noted that the normative data underpinning commercial immunoassay reference ranges are uncertain with regard to the reference population used and the quality of preanalytical factors [29]. Therefore, while reaffirming the need for a rigorous technical measurement methodology that incorporates all previously discussed and agreed-upon aspects [23– 25,27,28] and in alignment with other major scientific societies in the field, the EAU SRH Guidelines Panel has maintained its recommendation to adopt a widely recognized total T threshold that is appropriately integrated with the clinical symptomatic profile for diagnosing male hypogonadism. Similar considerations apply to age-related total T thresholds [21,29]. Consequently, the EAU SRH Guidelines Panel reiterates its previous position and advocates for thresholds of normalcy that primarily rely on symptomatic outcomes observed in men undergoing TTh [1] (Table 3).
3.3. Free T and SHBG evaluation in routine clinical practice
Both cross-sectional [30] and longitudinal data [31] from the European Male Aging Study have demonstrated that free T assessment improves diagnostic accuracy for symptomatic male hypogonadism, particularly in older and obese men. As previously noted, the main limitation of free T evaluation in clinical practice is the poor accuracy of the available immunoassay methods [19]. To address this issue, several calculation models based on SHBG, albumin, and total T have been developed. Among these, the Vermeulen formula is considered the most reliable approximation of circulating free T, despite a reported 20–30% overestimation [32,33]. The Vermeulen formula was the most frequently cited equation in the European laboratory survey [21]. A further limitation of free T assessment relates to potential inaccuracies in SHBG measurement. Previous studies have shown that when SHBG levels fall within the reference range, calculated free T offers no diagnostic advantage. Conversely, markedly elevated or reduced SHBG levels may lead to misinterpretation of total T [34]. Accordingly, most current guidelines recommend SHBG measurement and free T calculation only in the presence of conditions that are known to alter SHBG concentrations (Table 2).
Another critical issue concerns single-nucleotide polymorphisms (SNPs), which are common in the general population and may affect SHBG levels and binding capacity, thereby influencing free T calculation. In this context, a recent large population-based study assessed hormonal profiles in 999 healthy men [35]. Total T was measured by LC-MS/MS in all patients; free T was calculated using the Vermeulen formula and was directly measured by LC-MS/MS in a subset of 314 men. Multiple SNPs were analyzed. Results indicated that SHBG-related SNPs may influence total T assessment but not free T when measured by LC-MS/MS. A trend toward higher calculated free T was observed in carriers of the rare rs6259 polymorphism, associated with elevated SHBG levels, whereas bioavailable T remained unaffected [35].
Based on these findings, the strong recommendation to perform SHBG measurement and free T calculation was maintained in all conditions characterized by altered SHBG levels (Tables 2 and 3). Although definitive scientific evidence cannot be provided, considering the strong association between metabolic derangements and reduced SHBG and total T levels [14,36] as well as the potential influence of genetic factors [35,37], which are not always predictable, the EAU SRH Guidelines Panel further considers it highly advisable to assess SHBG and free T in all symptomatic patients.
In this setting, limited evidence suggests that a threshold of 220 pmol/l for free T can be considered as a reliable threshold. However, recent data based on direct free T measurement via LC-MS/MS indicated differences in free T levels according to age [38]. Therefore, more studies are required to better clarify the most appropriate threshold for free T in routine practice.
4. Conclusions
With the primary aim of ensuring consistency in methodological and technical aspects that may otherwise render the diagnosis of adult male hypogonadism complex and less accurate, this summary presents the EAU SRH Guidelines Panel’s rationale, strongly reaffirming previous recommendations, with the sole exception of the revision of the recommended time frame for blood sampling, moving the upper limit forward from 11:00 AM to 10:00 AM. Furthermore, the currently available evidence is insufficient to support a strong recommendation for routine assessment of albumin and SHBG levels, or for the calculation of free T in all symptomatic individuals. Nonetheless, the EAU SRH Guidelines Panel acknowledges that such evaluations may be considered in men presenting with clinical conditions known to alter circulating SHBG concentrations. Emerging evidence increasingly supports a broader evaluative approach, extending beyond individuals with established SHBG-altering conditions, to include even otherwise apparently healthy patients.
* As previously noted, the main limitation of free T evaluation in clinical practice is the poor accuracy of the available immunoassay methods [19]. To address this issue, several calculation models based on SHBG, albumin, and total T have been developed. Among these, the Vermeulen formula is considered the most reliable approximation of circulating free T, despite a reported 20–30% overestimation [32,33]. The Vermeulen formula was the most frequently cited equation in the European laboratory survey [21]. A further limitation of free T assessment relates to potential inaccuracies in SHBG measurement.
* Another critical issue concerns single-nucleotide polymorphisms (SNPs), which are common in the general population and may affect SHBG levels and binding capacity, thereby influencing free T calculation.
* Results indicated that SHBG-related SNPs may influence total T assessment but not free T when measured by LC-MS/MS. A trend toward higher calculated free T was observed in carriers of the rare rs6259 polymorphism, associated with elevated SHBG levels, whereas bioavailable T remained unaffected [35].
* Based on these findings, the strong recommendation to perform SHBG measurement and free T calculation was maintained in all conditions characterized by altered SHBG levels (Tables 2 and 3).
* the EAU SRH Guidelines Panel further considers it highly advisable to assess SHBG and free T in all symptomatic patients.
* In this setting, limited evidence suggests that a threshold of 220 pmol/l for free T can be considered as a reliable threshold. However, recent data based on direct free T measurement via LC-MS/MS indicated differences in free T levels according to age [38]. Therefore, more studies are required to better clarify the most appropriate threshold for free T in routine practice.
Despite variations in proposed T thresholds, all major guidelines agree that diagnosis of hypogonadism requires both low circulating T and relevant symptoms [1,8–17]. However, discrepancies persist regarding biochemical assessment, preanalytical conditions (fasting and morning sampling), and whether total or free T should be measured (Table 1). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is considered the reference method for total T [18], but high cost limits its routine use. Immunoassays show acceptable accuracy for total T but are unreliable for free T measurement [19,20]. Consequently, guidelines recommend calculating free T only when sex hormone–binding globulin (SHBG) alterations are suspected (Table 2).
Although not without methodological limitations, a recent internet-based survey conducted across 27 European countries revealed substantial variability in laboratory practices, including widespread reliance on immunoassays for total T measurement, limited adoption of LC-MS/MS, and inconsistent preanalytical protocols [21]. These findings underscore the need for harmonized recommendations. This paper aims to provide updated guidance from the European Association of Urology (EAU) Sexual and Reproductive Health (SRH) Guidelines Panel, offering a critical interpretation of technical aspects in biochemical confirmation of male hypogonadism during aging, to support standardized clinical and research strategies for T therapy (TTh).
3. The guidelines
3.1. Biochemical T measurement in routine clinical practice
Based on the 2026 update of the EAU SRH Guidelines, the previous recommendation supporting the measurement of total T under fasting conditions remains unchanged (Table 3). This is supported by evidence showing that both total and calculated free T levels decrease within 30 min following oral glucose administration during an oral glucose tolerance test [23]. These results are independent across the different spectra of glucose tolerance tests and have been corroborated by other studies [24]. The available data indicate that measuring circulating T in nonfasting conditions can lead to an underestimation of total T by >30% compared with its true value [24]. Current evidence also supports the presence of a circadian rhythm in T secretion, with higher levels observed in the morning even among older men [25,26].
3.2. Total T threshold in routine clinical practice
Although acknowledging the potential limitations of the reference, which nonetheless remains crucial for illustrating the real-world scenario and the heterogeneity of diagnosis approaches, data from the European laboratory survey confirmed a wide variability in reference ranges for normal T levels across different immunoassay methods [21]. It should be noted that the normative data underpinning commercial immunoassay reference ranges are uncertain with regard to the reference population used and the quality of preanalytical factors [29]. Therefore, while reaffirming the need for a rigorous technical measurement methodology that incorporates all previously discussed and agreed-upon aspects [23– 25,27,28] and in alignment with other major scientific societies in the field, the EAU SRH Guidelines Panel has maintained its recommendation to adopt a widely recognized total T threshold that is appropriately integrated with the clinical symptomatic profile for diagnosing male hypogonadism. Similar considerations apply to age-related total T thresholds [21,29]. Consequently, the EAU SRH Guidelines Panel reiterates its previous position and advocates for thresholds of normalcy that primarily rely on symptomatic outcomes observed in men undergoing TTh [1] (Table 3).
3.3. Free T and SHBG evaluation in routine clinical practice
Both cross-sectional [30] and longitudinal data [31] from the European Male Aging Study have demonstrated that free T assessment improves diagnostic accuracy for symptomatic male hypogonadism, particularly in older and obese men. As previously noted, the main limitation of free T evaluation in clinical practice is the poor accuracy of the available immunoassay methods [19]. To address this issue, several calculation models based on SHBG, albumin, and total T have been developed. Among these, the Vermeulen formula is considered the most reliable approximation of circulating free T, despite a reported 20–30% overestimation [32,33]. The Vermeulen formula was the most frequently cited equation in the European laboratory survey [21]. A further limitation of free T assessment relates to potential inaccuracies in SHBG measurement. Previous studies have shown that when SHBG levels fall within the reference range, calculated free T offers no diagnostic advantage. Conversely, markedly elevated or reduced SHBG levels may lead to misinterpretation of total T [34]. Accordingly, most current guidelines recommend SHBG measurement and free T calculation only in the presence of conditions that are known to alter SHBG concentrations (Table 2).
Another critical issue concerns single-nucleotide polymorphisms (SNPs), which are common in the general population and may affect SHBG levels and binding capacity, thereby influencing free T calculation. In this context, a recent large population-based study assessed hormonal profiles in 999 healthy men [35]. Total T was measured by LC-MS/MS in all patients; free T was calculated using the Vermeulen formula and was directly measured by LC-MS/MS in a subset of 314 men. Multiple SNPs were analyzed. Results indicated that SHBG-related SNPs may influence total T assessment but not free T when measured by LC-MS/MS. A trend toward higher calculated free T was observed in carriers of the rare rs6259 polymorphism, associated with elevated SHBG levels, whereas bioavailable T remained unaffected [35].
Based on these findings, the strong recommendation to perform SHBG measurement and free T calculation was maintained in all conditions characterized by altered SHBG levels (Tables 2 and 3). Although definitive scientific evidence cannot be provided, considering the strong association between metabolic derangements and reduced SHBG and total T levels [14,36] as well as the potential influence of genetic factors [35,37], which are not always predictable, the EAU SRH Guidelines Panel further considers it highly advisable to assess SHBG and free T in all symptomatic patients.
In this setting, limited evidence suggests that a threshold of 220 pmol/l for free T can be considered as a reliable threshold. However, recent data based on direct free T measurement via LC-MS/MS indicated differences in free T levels according to age [38]. Therefore, more studies are required to better clarify the most appropriate threshold for free T in routine practice.
4. Conclusions
With the primary aim of ensuring consistency in methodological and technical aspects that may otherwise render the diagnosis of adult male hypogonadism complex and less accurate, this summary presents the EAU SRH Guidelines Panel’s rationale, strongly reaffirming previous recommendations, with the sole exception of the revision of the recommended time frame for blood sampling, moving the upper limit forward from 11:00 AM to 10:00 AM. Furthermore, the currently available evidence is insufficient to support a strong recommendation for routine assessment of albumin and SHBG levels, or for the calculation of free T in all symptomatic individuals. Nonetheless, the EAU SRH Guidelines Panel acknowledges that such evaluations may be considered in men presenting with clinical conditions known to alter circulating SHBG concentrations. Emerging evidence increasingly supports a broader evaluative approach, extending beyond individuals with established SHBG-altering conditions, to include even otherwise apparently healthy patients.
