madman
Super Moderator
Sit and dwell on that!
Think of all those overmedicated men on T-therapy gassed up on FT well beyond their natty genetic set-point and unfortunately many off them off the hop!
You can blame all those run of the mill T clinics, kiddie forums loaded with all those numbskulls, throw those blast n cruisers in there too and put the icing on the cake the cesspool of so called gurus polluting gootube!
Gotta love all those kiddie forums loaded with the brainwashed sheep still pushing that more T is better mentality dishing out piss poor advice!
So much misinformation littered on the net its sickening.
As I have stated numerous times on the forum T is a threshold hormone!
Main point here being start low and go slow titrate the dose if need be until the threshold is crossed (bloodwork + symptoms).
Symptoms improved while at the same time minimizing/avoiding sides, keeping blood markers healthy and maintaining long-term health is key here.
Crossing the threshold turns the lights on but cranking the dimmer switch past that doesn't make them shine brighter. It's a ceiling effect, not a linear dose-response.
Especially when it comes to libido and erectile function.
The majority of symptoms will be improved once you achieve a healthy FT which for most would be aiming for a healthy/high-end trough 15-25 ng/dL.
Yes some will choose to run higher levels but the main benefit you are going to get here when driving up your FT sky-high is better gains in muscle/enhanced strength and recovery.
Plain and simple.
Every other symptom energy, mood, libido, erectile function let alone overall health (cardiovascular, brain, bone, tendons, immune system, lipids, and body composition) can easily be improved by achieving a healthy FT.
Even then I would put much more weight behind sleep, diet, exercise, thyroid/adrenals and stress (physical/mental) when it comes to energy, mood, libido and erectile function than supposedly needing a high/absurdly high FT.
This is a given!
Give your heads a shake peak--->trough (daily vs twice-weekly vs once weekly).
24/7 steady-state to boot!
Healthy natty males age 18-29 years mFT 2.5-97.5th percentile 6.39-25.3 ng/dL.
It's those natty outliers that fall in the 97.5% that are hitting a FT 25.3 ng/dL and this is a daily short-lived peak to boot!
Better yet look at where the median 11.71 ng/dL sits!
LMFAO!
Table 2: Age-stratified reference ranges for measured free testosterone in non-obese men. Abbreviatons: T = testosterone; FT = free testosterone. To convert the mFT results from pmol/L to ng/dL, divide the tabulated values by 34.67.
* In this study, we present the largest dataset of mFT levels to date, providing male reference ranges spanning a wide age range, using a gold standard quantification method and accepted CLSI methodologies for generating reference ranges.
* In routine clinical practice, FT concentrations are mostly derived from mathematical approximations using SHBG, total T and albumin levels (15 17). However, the gold standard method for FT determination remains direct measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) after separating the free fraction from the bound fraction through equilibrium dialysis (ED LC-MS/MS) (18). Nevertheless, most of available data on FT is based on calculated (cFT) rather than measured FT (mFT), as directly measuring FT is still technically demanding and time-consuming and is only available in a few advanced research or commercial laboratories (13,14,19 21).
* According to the free hormone hypothesis, only this small, unbound, fraction is biologically active, being capable of crossing the cell membrane to exert its intracellular functions (4). Meanwhile, the albumin-bound testosterone fraction may serve as a readily available buffer of testosterone maintaining the equilibrium between free and bound T. As such, the free T (FT) fraction may be a better measure of target tissue T exposure than total T. Circulating FT concentrations have been shown to better correlate with clinical symptoms of male hypogonadism (5 7). This holds especially true in situations where serum binding-protein concentrations or conditions are altered, such as in obesity, type 2 diabetes mellitus, during aging, or in carriers of specific SHBG single-nucleotide polymorphisms (8 10). In these circumstances, total T may be misleading and not accurately reflect androgen exposure, leading to an incorrect or missed diagnosis or flawed therapy monitoring
* Generally, cFT correlates well with mFT but various proposed algorithms for calculation of FT perform unequally, giving rise to disparate results for cFT. For example, for the most-commonly used Vermeulen formula, notwithstanding an excellent correlation, a positive bias of around 20% is observed between FT measured by ED LC-MS/MS and calculations (1).
* The established reference ranges are in line with previous reports of mFT (22,33,34) and may potentially be used as a reference data set for other methods. However, before these data can be more broadly implemented in clinical laboratories and clinical care, harmonization/standardization and cross-validation of existing mFT methods are required. Harmonization/standardization initiatives are crucial to move forward, given the increasing interest in mFT. Indeed, more clinical and commercial laboratories have been developing in-house mFT methods with improved throughput and shorter dialysis times (23,35,36). However, without the existence of a reference measurement system and reference measurement procedures, the performance of existing routine methods in terms of calibration and sample related effects cannot be assessed, nor can the calibration of the assays be traceable to a common reference point. This may result in large differences between methods.
Age-Stratified Reference Ranges for Directly Measured Serum Free Testosterone in Community-Dwelling and Healthy Men
Joeri Walravens1, Gido Snaterse1, Nick Narinx2, Tim Reyns3, Katleen Van Uytfanghe4, Dirk Vanderschueren2, Frederick Wu5, Jean-Marc Kaufman1, Leen Antonio2, Tom Fiers3, Bruno Lapauw1
Introduction
Serum concentrations of testosterone (T) in men are measured in the diagnosis of hypogonadism and to monitor substitution therapy. However, most of the total T pool in the human circulation is bound to several binding-proteins, mainly to sex hormone-binding globulin (SHBG) and albumin but also to corticosteroid-binding globulin (CBG) and orosomucoid, while only 1 to 2% of total T is present in unbound form (1 3). According to the free hormone hypothesis, only this small, unbound, fraction is biologically active, being capable of crossing the cell membrane to exert its intracellular functions (4). Meanwhile, the albumin-bound testosterone fraction may serve as a readily available buffer of testosterone maintaining the equilibrium between free and bound T. As such, the free T (FT) fraction may be a better measure of target tissue T exposure than total T. Circulating FT concentrations have been shown to better correlate with clinical symptoms of male hypogonadism (5 7). This holds especially true in situations where serum binding-protein concentrations or conditions are altered, such as in obesity, type 2 diabetes mellitus, during aging, or in carriers of specific SHBG single-nucleotide polymorphisms (8 10). In these circumstances, total T may be misleading and not accurately reflect androgen exposure, leading to an incorrect or missed diagnosis or flawed therapy monitoring. This is supported by in vivo experiments in transgenic mice expressing human SHBG, which showed that total T levels increased concomitantly with the expression of SHBG to normalize FT levels (11). Additionally, a clinical case has been reported of a man without measurable SHBG and low total T levels, but normal FT levels who nevertheless showed normal reproductive development and function (12). Consequently, several recent clinical guidelines have already recommended the determination of FT in men with conditions that alter SHBG levels or in men with borderline total T levels for the diagnosis and management of male hypogonadism (13,14).
In routine clinical practice, FT concentrations are mostly derived from mathematical approximations using SHBG, total T and albumin levels (15 17). However, the gold standard method for FT determination remains direct measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) after separating the free fraction from the bound fraction through equilibrium dialysis (ED LC-MS/MS) (18). Nevertheless, most of available data on FT is based on calculated (cFT) rather than measured FT (mFT), as directly measuring FT is still technically demanding and time-consuming and is only available in a few advanced research or commercial laboratories (13,14,19 21). Earlier work has also mainly focused on men aged above 40 years, resulting in a paucity of data in younger men. Generally, cFT correlates well with mFT but various proposed algorithms for calculation of FT perform unequally, giving rise to disparate results for cFT. For example, for the most-commonly used Vermeulen formula, notwithstanding an excellent correlation, a positive bias of around 20% is observed between FT measured by ED LC-MS/MS and calculations (1). Additionally, published mFT reference ranges thus far have only been established on a limited number of samples, did not sufficiently consider the effects of aging or BMI and the reported ranges vary substantially from one another (22,23). Indeed, both cFT and mFT have been shown to have inverse associations with age and BMI, potentially distorting reference ranges (20,23 25). Presently, the effect of BMI on FT reference ranges and which BMI cut-off is appropriate for the establishment of reference ranges remain unclear. Also, it is still unclear whether age-stratified reference ranges (Z-score approach) or reference ranges for a young, healthy population (T-score approach) are more appropriate and in case of the latter, which age category should be defined as "young". Depending on the clinical endpoint of interest, age-stratified reference ranges might serve as better diagnostic tools. Given these unknowns, it is unsurprising that there is ongoing skepticism regarding the validity and application of FT in clinical practice (26,27).
In this study, we aim to (I) establish age-stratified reference ranges for directly measured serum FT levels in a large cohort (minimum of 120 samples per age decade) of community-dwelling, adult men using a published gold standard method (1) and (II) assess the associations of FT levels with BMI and age.
mFT reference ranges
The reference range for mFT in non-obese men aged 18-39 years (n = 369) is 184-749 pmol/L. Age-stratified reference ranges across the whole population of non-obese men reflect the earlier described age-related decline in mFT, decreasing from 239-877 pmol/L in the category 18-29 years to 49-250 pmol/L at 80+ years (Figure 2; Table 2). In addition to this absolute decline, percentage mFT also markedly decreased with aging. Data for all men can be found in Supplementary Table 1 (32).
Table 3 illustrates T-score and proportion of non-obese men with mFT below the lower reference value for men aged 18-39 years per age category. The percentage of the population below the lower reference limit for young men showed an increasing trend across age categories, up to 76.3% for men aged 80 years and older. The mean mFT for men aged 18-39 years, which represent the zero value for T-scores was 390 ± 136 pmol/L. Mean T-scores decreased by on average 0.4 per decade, from 0.3 to -1.8 from ages 18-29 to 80+
Discussion
In this study, we present the largest dataset of mFT levels to date, providing male reference ranges spanning a wide age range, using a gold standard quantification method and accepted CLSI methodologies for generating reference ranges.
The established reference ranges are in line with previous reports of mFT (22,33,34) and may potentially be used as a reference data set for other methods. However, before these data can be more broadly implemented in clinical laboratories and clinical care, harmonization/standardization and cross-validation of existing mFT methods are required. Harmonization/standardization initiatives are crucial to move forward, given the increasing interest in mFT. Indeed, more clinical and commercial laboratories have been developing in-house mFT methods with improved throughput and shorter dialysis times (23,35,36). However, without the existence of a reference measurement system and reference measurement procedures, the performance of existing routine methods in terms of calibration and sample related effects cannot be assessed, nor can the calibration of the assays be traceable to a common reference point. This may result in large differences between methods. One such example is a recent publication reporting mFT levels twice as high as those reported in this and other publications, despite no apparent differences in the studied populations or analytical methods (23). Initiatives to set up a reference measurement system could greatly reduce variability between methods and improve comparability of results, aiding implementation of mFT in clinical practice (26). This is not only valid for mFT, but also important for cFT, as the various currently-available formulae can then be calibrated against the reference measurement procedure (15 17,37). Moreover, it would allow extensive validation of cFT calculations in situations in which the binding environment between T and SHBG may be altered, such as obesity, in which calculator performance may be suboptimal. In addition, it would also allow the development of new FT formulae if deemed necessary.
The feasibility to develop and implement a FT reference measurement procedure has been demonstrated in the past (38,39); fresh initiatives can be similar to the successful efforts of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee for Standardization of Thyroid Function Tests (C-STFT) on standardization of free thyroxine (40).
Further, we unequivocally confirmed that mFT levels decrease with age, both in median values as well as upper and lower limits of the reference ranges. This decrease was independent of total T and SHBG levels, suggesting that other aging-related factors may play a role in regulating FT levels. Earlier research has observed that LH levels consistently increase with age, suggesting that the age-related decrease in FT levels observed in this study, may be explained by an age-related testicular functional impairment (10). Given this age-related decrease in FT levels in men, it is still unclear which approach to implementing mFT reference ranges is the most clinically appropriate: an age-stratified (Z-score) approach or a reference sample-based (T-score) approach. For parameters that change over time, such as (free) T or bone mass, it might seem more appropriate to use reference ranges for a young, healthy population (T-score approach) (41). However, a substantial proportion of the older populations would be considered as having insufficient FT using a T-score approach, which may not necessarily reflect altered requirements or clinical manifestations of androgen deficiency. Consequently, we opted to provide both age-stratified reference ranges as well as reference ranges for young men aged 18-39 years (Table 2).
It is also unclear which BMI cut-off should be considered when establishing a reference population. Spline regression analysis showed a significant impact of BMI on mFT, and even more on total T, substantially decreasing androgen levels at higher BMI, suggesting careful consideration of the BMI cut-off when establishing reference ranges. The effect of BMI on total T levels was larger than on mFT, which may be indicative of mFT being a more robust marker of androgen exposure. For the here reported mFT reference ranges, the BMI cut-off was set at 30 kg/m², a cut-off commonly used to define obesity. Despite the substantial effect of BMI on mFT, allowing an upper BMI cut-off of 35 kg/m² resulted in only minor decreases in the reference ranges of, on average, -1.7% in our dataset (Supplementary Table 1) (32). This can likely be attributed to the substantial portion of samples (86.5%) from participants with a BMI < 30 kg/m2 in each age category combined with our robust analytical and statistical methodologies. Additionally, these reference ranges are only descriptive of a European, non-obese male population which was exclusively Caucasian. In the cohort of men aged 18 to 24 years, no participants had a BMI > 30 kg/m². Consequently, these reference ranges may not be generalizable to other populations of differing demographics, other ethnicities or men with obesity. For example, these reference ranges may not be generalizable to a US-based population, given the significant differences in demographic variables such as BMI. Furthermore, different ethnic groups may have different normal total testosterone and SHBG levels which can in turn result in variable mFT levels (42,43). This necessitates follow-up studies in other populations to verify the reference ranges proposed in this manuscript and determine to what degree population-specific reference ranges are required. Importantly, the performance of the reference ranges for the diagnosis and management of conditions, such as hypogonadism, has yet to be evaluated. More specialized studies are required to link mFT levels to total T and clinical symptoms. A limitation of this study is that we used a mix of cohorts of healthy men of up to 55 years and community-dwelling men above 55. However, these cohorts do originate from the same geographical region and are representative for this population, albeit with a bias towards more healthy individuals.
In summary, this study is the first to report age-stratified reference ranges for mFT, using a large number of samples from the general population, measured using a published gold standard for FT quantification and following accepted and robust methodology. When a FT measurement is indicated, these reference ranges can help clinicians to interpret the results and decide whether further investigation or treatment is warranted. However, care must be taken when applying these reference ranges to results from methods that are not comparable or from populations not represented in the current study sample. This study also shows the importance of considering age and BMI when measuring and interpreting (free) T.
Think of all those overmedicated men on T-therapy gassed up on FT well beyond their natty genetic set-point and unfortunately many off them off the hop!
You can blame all those run of the mill T clinics, kiddie forums loaded with all those numbskulls, throw those blast n cruisers in there too and put the icing on the cake the cesspool of so called gurus polluting gootube!
Gotta love all those kiddie forums loaded with the brainwashed sheep still pushing that more T is better mentality dishing out piss poor advice!
So much misinformation littered on the net its sickening.
As I have stated numerous times on the forum T is a threshold hormone!
Main point here being start low and go slow titrate the dose if need be until the threshold is crossed (bloodwork + symptoms).
Symptoms improved while at the same time minimizing/avoiding sides, keeping blood markers healthy and maintaining long-term health is key here.
Crossing the threshold turns the lights on but cranking the dimmer switch past that doesn't make them shine brighter. It's a ceiling effect, not a linear dose-response.
Especially when it comes to libido and erectile function.
The majority of symptoms will be improved once you achieve a healthy FT which for most would be aiming for a healthy/high-end trough 15-25 ng/dL.
Yes some will choose to run higher levels but the main benefit you are going to get here when driving up your FT sky-high is better gains in muscle/enhanced strength and recovery.
Plain and simple.
Every other symptom energy, mood, libido, erectile function let alone overall health (cardiovascular, brain, bone, tendons, immune system, lipids, and body composition) can easily be improved by achieving a healthy FT.
Even then I would put much more weight behind sleep, diet, exercise, thyroid/adrenals and stress (physical/mental) when it comes to energy, mood, libido and erectile function than supposedly needing a high/absurdly high FT.
This is a given!
Give your heads a shake peak--->trough (daily vs twice-weekly vs once weekly).
24/7 steady-state to boot!
Healthy natty males age 18-29 years mFT 2.5-97.5th percentile 6.39-25.3 ng/dL.
It's those natty outliers that fall in the 97.5% that are hitting a FT 25.3 ng/dL and this is a daily short-lived peak to boot!
Better yet look at where the median 11.71 ng/dL sits!
LMFAO!
Table 2: Age-stratified reference ranges for measured free testosterone in non-obese men. Abbreviatons: T = testosterone; FT = free testosterone. To convert the mFT results from pmol/L to ng/dL, divide the tabulated values by 34.67.
* In this study, we present the largest dataset of mFT levels to date, providing male reference ranges spanning a wide age range, using a gold standard quantification method and accepted CLSI methodologies for generating reference ranges.
* In routine clinical practice, FT concentrations are mostly derived from mathematical approximations using SHBG, total T and albumin levels (15 17). However, the gold standard method for FT determination remains direct measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) after separating the free fraction from the bound fraction through equilibrium dialysis (ED LC-MS/MS) (18). Nevertheless, most of available data on FT is based on calculated (cFT) rather than measured FT (mFT), as directly measuring FT is still technically demanding and time-consuming and is only available in a few advanced research or commercial laboratories (13,14,19 21).
* According to the free hormone hypothesis, only this small, unbound, fraction is biologically active, being capable of crossing the cell membrane to exert its intracellular functions (4). Meanwhile, the albumin-bound testosterone fraction may serve as a readily available buffer of testosterone maintaining the equilibrium between free and bound T. As such, the free T (FT) fraction may be a better measure of target tissue T exposure than total T. Circulating FT concentrations have been shown to better correlate with clinical symptoms of male hypogonadism (5 7). This holds especially true in situations where serum binding-protein concentrations or conditions are altered, such as in obesity, type 2 diabetes mellitus, during aging, or in carriers of specific SHBG single-nucleotide polymorphisms (8 10). In these circumstances, total T may be misleading and not accurately reflect androgen exposure, leading to an incorrect or missed diagnosis or flawed therapy monitoring
* Generally, cFT correlates well with mFT but various proposed algorithms for calculation of FT perform unequally, giving rise to disparate results for cFT. For example, for the most-commonly used Vermeulen formula, notwithstanding an excellent correlation, a positive bias of around 20% is observed between FT measured by ED LC-MS/MS and calculations (1).
* The established reference ranges are in line with previous reports of mFT (22,33,34) and may potentially be used as a reference data set for other methods. However, before these data can be more broadly implemented in clinical laboratories and clinical care, harmonization/standardization and cross-validation of existing mFT methods are required. Harmonization/standardization initiatives are crucial to move forward, given the increasing interest in mFT. Indeed, more clinical and commercial laboratories have been developing in-house mFT methods with improved throughput and shorter dialysis times (23,35,36). However, without the existence of a reference measurement system and reference measurement procedures, the performance of existing routine methods in terms of calibration and sample related effects cannot be assessed, nor can the calibration of the assays be traceable to a common reference point. This may result in large differences between methods.
Age-Stratified Reference Ranges for Directly Measured Serum Free Testosterone in Community-Dwelling and Healthy Men
Joeri Walravens1, Gido Snaterse1, Nick Narinx2, Tim Reyns3, Katleen Van Uytfanghe4, Dirk Vanderschueren2, Frederick Wu5, Jean-Marc Kaufman1, Leen Antonio2, Tom Fiers3, Bruno Lapauw1
Introduction
Serum concentrations of testosterone (T) in men are measured in the diagnosis of hypogonadism and to monitor substitution therapy. However, most of the total T pool in the human circulation is bound to several binding-proteins, mainly to sex hormone-binding globulin (SHBG) and albumin but also to corticosteroid-binding globulin (CBG) and orosomucoid, while only 1 to 2% of total T is present in unbound form (1 3). According to the free hormone hypothesis, only this small, unbound, fraction is biologically active, being capable of crossing the cell membrane to exert its intracellular functions (4). Meanwhile, the albumin-bound testosterone fraction may serve as a readily available buffer of testosterone maintaining the equilibrium between free and bound T. As such, the free T (FT) fraction may be a better measure of target tissue T exposure than total T. Circulating FT concentrations have been shown to better correlate with clinical symptoms of male hypogonadism (5 7). This holds especially true in situations where serum binding-protein concentrations or conditions are altered, such as in obesity, type 2 diabetes mellitus, during aging, or in carriers of specific SHBG single-nucleotide polymorphisms (8 10). In these circumstances, total T may be misleading and not accurately reflect androgen exposure, leading to an incorrect or missed diagnosis or flawed therapy monitoring. This is supported by in vivo experiments in transgenic mice expressing human SHBG, which showed that total T levels increased concomitantly with the expression of SHBG to normalize FT levels (11). Additionally, a clinical case has been reported of a man without measurable SHBG and low total T levels, but normal FT levels who nevertheless showed normal reproductive development and function (12). Consequently, several recent clinical guidelines have already recommended the determination of FT in men with conditions that alter SHBG levels or in men with borderline total T levels for the diagnosis and management of male hypogonadism (13,14).
In routine clinical practice, FT concentrations are mostly derived from mathematical approximations using SHBG, total T and albumin levels (15 17). However, the gold standard method for FT determination remains direct measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) after separating the free fraction from the bound fraction through equilibrium dialysis (ED LC-MS/MS) (18). Nevertheless, most of available data on FT is based on calculated (cFT) rather than measured FT (mFT), as directly measuring FT is still technically demanding and time-consuming and is only available in a few advanced research or commercial laboratories (13,14,19 21). Earlier work has also mainly focused on men aged above 40 years, resulting in a paucity of data in younger men. Generally, cFT correlates well with mFT but various proposed algorithms for calculation of FT perform unequally, giving rise to disparate results for cFT. For example, for the most-commonly used Vermeulen formula, notwithstanding an excellent correlation, a positive bias of around 20% is observed between FT measured by ED LC-MS/MS and calculations (1). Additionally, published mFT reference ranges thus far have only been established on a limited number of samples, did not sufficiently consider the effects of aging or BMI and the reported ranges vary substantially from one another (22,23). Indeed, both cFT and mFT have been shown to have inverse associations with age and BMI, potentially distorting reference ranges (20,23 25). Presently, the effect of BMI on FT reference ranges and which BMI cut-off is appropriate for the establishment of reference ranges remain unclear. Also, it is still unclear whether age-stratified reference ranges (Z-score approach) or reference ranges for a young, healthy population (T-score approach) are more appropriate and in case of the latter, which age category should be defined as "young". Depending on the clinical endpoint of interest, age-stratified reference ranges might serve as better diagnostic tools. Given these unknowns, it is unsurprising that there is ongoing skepticism regarding the validity and application of FT in clinical practice (26,27).
In this study, we aim to (I) establish age-stratified reference ranges for directly measured serum FT levels in a large cohort (minimum of 120 samples per age decade) of community-dwelling, adult men using a published gold standard method (1) and (II) assess the associations of FT levels with BMI and age.
mFT reference ranges
The reference range for mFT in non-obese men aged 18-39 years (n = 369) is 184-749 pmol/L. Age-stratified reference ranges across the whole population of non-obese men reflect the earlier described age-related decline in mFT, decreasing from 239-877 pmol/L in the category 18-29 years to 49-250 pmol/L at 80+ years (Figure 2; Table 2). In addition to this absolute decline, percentage mFT also markedly decreased with aging. Data for all men can be found in Supplementary Table 1 (32).
Table 3 illustrates T-score and proportion of non-obese men with mFT below the lower reference value for men aged 18-39 years per age category. The percentage of the population below the lower reference limit for young men showed an increasing trend across age categories, up to 76.3% for men aged 80 years and older. The mean mFT for men aged 18-39 years, which represent the zero value for T-scores was 390 ± 136 pmol/L. Mean T-scores decreased by on average 0.4 per decade, from 0.3 to -1.8 from ages 18-29 to 80+
Discussion
In this study, we present the largest dataset of mFT levels to date, providing male reference ranges spanning a wide age range, using a gold standard quantification method and accepted CLSI methodologies for generating reference ranges.
The established reference ranges are in line with previous reports of mFT (22,33,34) and may potentially be used as a reference data set for other methods. However, before these data can be more broadly implemented in clinical laboratories and clinical care, harmonization/standardization and cross-validation of existing mFT methods are required. Harmonization/standardization initiatives are crucial to move forward, given the increasing interest in mFT. Indeed, more clinical and commercial laboratories have been developing in-house mFT methods with improved throughput and shorter dialysis times (23,35,36). However, without the existence of a reference measurement system and reference measurement procedures, the performance of existing routine methods in terms of calibration and sample related effects cannot be assessed, nor can the calibration of the assays be traceable to a common reference point. This may result in large differences between methods. One such example is a recent publication reporting mFT levels twice as high as those reported in this and other publications, despite no apparent differences in the studied populations or analytical methods (23). Initiatives to set up a reference measurement system could greatly reduce variability between methods and improve comparability of results, aiding implementation of mFT in clinical practice (26). This is not only valid for mFT, but also important for cFT, as the various currently-available formulae can then be calibrated against the reference measurement procedure (15 17,37). Moreover, it would allow extensive validation of cFT calculations in situations in which the binding environment between T and SHBG may be altered, such as obesity, in which calculator performance may be suboptimal. In addition, it would also allow the development of new FT formulae if deemed necessary.
The feasibility to develop and implement a FT reference measurement procedure has been demonstrated in the past (38,39); fresh initiatives can be similar to the successful efforts of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee for Standardization of Thyroid Function Tests (C-STFT) on standardization of free thyroxine (40).
Further, we unequivocally confirmed that mFT levels decrease with age, both in median values as well as upper and lower limits of the reference ranges. This decrease was independent of total T and SHBG levels, suggesting that other aging-related factors may play a role in regulating FT levels. Earlier research has observed that LH levels consistently increase with age, suggesting that the age-related decrease in FT levels observed in this study, may be explained by an age-related testicular functional impairment (10). Given this age-related decrease in FT levels in men, it is still unclear which approach to implementing mFT reference ranges is the most clinically appropriate: an age-stratified (Z-score) approach or a reference sample-based (T-score) approach. For parameters that change over time, such as (free) T or bone mass, it might seem more appropriate to use reference ranges for a young, healthy population (T-score approach) (41). However, a substantial proportion of the older populations would be considered as having insufficient FT using a T-score approach, which may not necessarily reflect altered requirements or clinical manifestations of androgen deficiency. Consequently, we opted to provide both age-stratified reference ranges as well as reference ranges for young men aged 18-39 years (Table 2).
It is also unclear which BMI cut-off should be considered when establishing a reference population. Spline regression analysis showed a significant impact of BMI on mFT, and even more on total T, substantially decreasing androgen levels at higher BMI, suggesting careful consideration of the BMI cut-off when establishing reference ranges. The effect of BMI on total T levels was larger than on mFT, which may be indicative of mFT being a more robust marker of androgen exposure. For the here reported mFT reference ranges, the BMI cut-off was set at 30 kg/m², a cut-off commonly used to define obesity. Despite the substantial effect of BMI on mFT, allowing an upper BMI cut-off of 35 kg/m² resulted in only minor decreases in the reference ranges of, on average, -1.7% in our dataset (Supplementary Table 1) (32). This can likely be attributed to the substantial portion of samples (86.5%) from participants with a BMI < 30 kg/m2 in each age category combined with our robust analytical and statistical methodologies. Additionally, these reference ranges are only descriptive of a European, non-obese male population which was exclusively Caucasian. In the cohort of men aged 18 to 24 years, no participants had a BMI > 30 kg/m². Consequently, these reference ranges may not be generalizable to other populations of differing demographics, other ethnicities or men with obesity. For example, these reference ranges may not be generalizable to a US-based population, given the significant differences in demographic variables such as BMI. Furthermore, different ethnic groups may have different normal total testosterone and SHBG levels which can in turn result in variable mFT levels (42,43). This necessitates follow-up studies in other populations to verify the reference ranges proposed in this manuscript and determine to what degree population-specific reference ranges are required. Importantly, the performance of the reference ranges for the diagnosis and management of conditions, such as hypogonadism, has yet to be evaluated. More specialized studies are required to link mFT levels to total T and clinical symptoms. A limitation of this study is that we used a mix of cohorts of healthy men of up to 55 years and community-dwelling men above 55. However, these cohorts do originate from the same geographical region and are representative for this population, albeit with a bias towards more healthy individuals.
In summary, this study is the first to report age-stratified reference ranges for mFT, using a large number of samples from the general population, measured using a published gold standard for FT quantification and following accepted and robust methodology. When a FT measurement is indicated, these reference ranges can help clinicians to interpret the results and decide whether further investigation or treatment is warranted. However, care must be taken when applying these reference ranges to results from methods that are not comparable or from populations not represented in the current study sample. This study also shows the importance of considering age and BMI when measuring and interpreting (free) T.
