Testosterone threshold, assay and costs among laboratories for hypogonadism diagnosis

Introduction

Testosterone deficiency (hypogonadism) is a global health concern in adult men. According to the European Association of Urology (EAU), the incidence of hypogonadism is reported between 12.3 and 11.7 cases per 1,000 people per year. In men aged 40–79 years, the incidence of symptomatic hypogonadism varies between 2.1 and 5.7% (1).

The diagnosis of testosterone deficiency requires the presence of symptoms and/or signs combined with documentation of low serum testosterone concentrations. One of the challenges in evaluating men for the diagnosis of testosterone deficiency is that there is no universal consensus regarding the definition of a low serum value for total testosterone (TT).

The normal TT range is an ongoing discussion. While laboratories have no consensus, there are International Societies that do not conform to the normal range (2,3).

An additional source of confusion is that clinicians are often unaware of the thresholds proposed by various guidelines and rely exclusively on laboratory reference ranges to determine whether a test result is low or normal. A 2006 survey of 25 US clinical laboratories noted 17 different reference ranges for TT, with wide variability in both the lower and upper normal limits, despite efforts to enhance consistency among US laboratories (4).

Many different methods are available for TT measurement, most of which are adequate for the diagnosis of male hypogonadism. Wang et al. presented an article describing the TT reference range for adult men and its accuracy according to the type of assay used. They compared the results from 891 laboratories using 11 different assay methods with a TT range of 160–508 ng/dL. These results span the range between eugonadal and hypogonadal levels (2,5,6).

They also identified biases in chemiluminescence and electrochemiluminescence methods in the low direction of the serum TT values (2). Taieb et al. also reported underestimation of TT values when measured using electrochemiluminescence (7). Wang illustrated the importance of each laboratory in establishing a reference testosterone range for adult men for the diagnosis of hypogonadism (8,9).

Clinical experience in Mexico City suggests a similar situation, in which patients may present with testosterone results from one lab in which their result is categorized as low and may have a similar result from a second laboratory that categorizes the result as normal (10).

International societies have established a diagnosis of hypogonadism with an average of 300–350 ng/dL. The lower range of the American Urology Association (AUA) lower range varies from 300 ng/dL. The EAU established a limit of 350 ng/dL. The American Association of Clinical Endocrinology (EAA) lower limit of 320 ng/dL (Table 1) (11-13).

According to information published in 2020 by a national population census, Mexico City’s population is 9.21 million inhabitants, 47.8% represent male population (4.9 million) and 1.2 million men are older than 50 years (14-16).

Mexico City is one of the six megacities in Latin America and has experienced enormous advances in its overall quality of health care in the past 50 years. Mexico is the Latin American country with the highest overall Global Health Security Index Score. Mexico City is one of the most important cities in Latin America in terms of healthcare development (17-19).

The primary objective of this study was to determine the variability of TT reference ranges provided by laboratories in Mexico City and to compare them with guideline recommendations. A non-standardized testosterone threshold challenges proper diagnosis and treatment. Secondary objectives included determination of assay methods and cost. We present this article in accordance with the SURGE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-162/rc).

Methods

A survey was conducted by telephone to the most important public and private laboratories in Mexico City in August 2024. A total of 202 laboratories were contacted and 134 participated in the survey. The study was conducted in accordance to the Declaration of Helsinki and its subsequent amendments.

Data on laboratories offering TT quantification, lower and upper limits, measurement methods, and costs were collected. No incentives were provided for the laboratories included in this study.

Statistical analysis

Data were organized into a structured dataset with the following variables: laboratory type (public or private), measurement method, lower reference threshold, upper reference threshold and cost.

To clean the dataset, all testosterone threshold values were converted to ng/dL to ensure comparability.

Data was manually entered into a spreadsheet and cross-verified by the authors to ensure accuracy.

The average ranges of the lower and upper thresholds were calculated based on the collected data. A comparison between the measurement method and average normality threshold was established, along with a comparison between the method, normality range, and cost.

Finally, a comparison was made between the laboratory threshold for hypogonadism diagnosis and the AUA, EAU, and EAA lower TT limits for hypogonadism diagnosis.

Data regarding the types of androgen assays offered by the manufacturers were also collected.

Results

In total, 202 laboratories were included in the survey. A total of 134 laboratories offered TT quantification assays. Ten laboratories were public hospitals and 124 were private (97 laboratories and 27 hospitals). Forty-three out of 124 private laboratories shared the same manufacturer’s provider with the same assay methodology.

Reference range variability

In general, reference values for low TT ranged from 84 to 470 ng/dL, a variation of approximately 426.24%. The highest end of normal ranged from 400 to 1,719 ng/dL, a variation of approximately 487.29%.

The widest interval for normal TT values from a single laboratory was 262–1,593 ng/dL and the narrowest interval was 180–486 ng/dL.

Chemiluminescence, electrochemiluminescence, and immunochemistry were the three methods used to measure the TT values among the 134 laboratories. Furthermore, no laboratory has used a clinically determined target or threshold value to identify men with low TT, which is commonly performed in clinical trials for hypogonadism.

Laboratories using a chemiluminescence assay established a threshold for the lower limit of 130–470 ng/dL and upper limit of 480–1,719 ng/dL. The electrochemiluminescence threshold for the lower limit was 84–250 ng/dL and for the upper limit was 400–1,600 ng/dL.

The immunochemistry lower range was 132–813 ng/dL (Table 2).

TT upper limit presents a range from 400 to 1,719 ng/dL (variability range, 487.29%), whereas the lower limit range was 84 to 470 ng/dL (variability range, 426.24%).

Assay methods

TT assay methods were described by laboratories, 69 (51.11%) chemiluminescence, 25 (18.65%) electrochemiluminescence, one (0.74%) spectrophotometry, three (2.24%) immunochemistry assays, and 36 (26.8%) laboratories did not provide assay information (Tables 3,4).

Cost analysis

An average cost of 35 USD was established, ranging from 9 to 160 USD. Immunochemistry is the most expensive method, with an average cost of 52 USD, followed by chemiluminescence at 35 USD, and electrochemiluminescence at 32 USD.

Discussion

The results of this survey revealed enormous variability in the use of testosterone assays and reference ranges by clinical laboratories, and their significant discrepancy with international societies established a “threshold of normality”. The American Urological Association, EAU, and European Academy of Andrology have established their own normal TT ranges (1,11-13). This situation has made it more complicated to establish a diagnosis of hypogonadism based on lower limits (2,3).

In clinical practice, results below the societies lower ranges can be considered as “normal” with a clinical decision making for treatment without relevance and remains a confusing issue for clinicians and patients alike. The significance of this finding is the imminent underdiagnosis and undertreatment that could translate in men who might benefit from testosterone replacement therapy (TRT) not receiving treatment (8,9).

Centers for Disease Control and Prevention started a steroid hormones standardization project in 2007 to create testosterone measurement results that are traceable to a single accuracy standard, thereby ensuring comparability across analytical methods, time periods and laboratories. Nevertheless, the results of this survey revealed the wide variability in TT ranges reported by laboratories in Mexico City and poor knowledge of the internationally accepted threshold. The inclusion of the most important laboratories in Mexico City ensures a comprehensive and representative analysis of industry’s leading players.

Mexico City laboratories showed wide variability in both the limit range values. The upper threshold had a 487.29% variability, meanwhile, the lower threshold had a 426.24% variability.

Public laboratories in Mexico City share equipment for the assays; however, each laboratory reports a different range of normality. This translates to patients with TT levels under 250 ng/dL who are not diagnosed with hypogonadism.

Chemiluminescence is commonly employed for TT assays in both private and public laboratories, with electrochemiluminescence being the second most common assay.

For this purpose, it is relevant to note that 36 private laboratories could not describe the measurement method used.

In Mexico, hypogonadism is not considered a common health problem, and routine serum TT analysis is not common in clinical practice. In a country where the minimum wage is 14 USD per day, an assay with an average cost of 35 USD presents a financial challenge for patients who are not affiliated with Public Healthcare Institutions, and even so for patients who can access private laboratories, the necessity of retaking the assay because of a poor-quality test is almost certain (16-18).

Private laboratories were categorized according to the institution they served: hospitals or laboratories. We found that private hospitals prefer chemiluminescence for the assay and present a lower TT threshold of 130–249 ng/dL.

On the other hand, private laboratories have a wider range of options to perform the TT quantification assay, chemiluminescence was the most commonly used method (46, 47.42%), and with an average TT lower limit for hypogonadism diagnosis ranging from 150–470 ng/dL.

The laboratory, with the most expensive assay, also reported an altered normal range, establishing a lower threshold of 240 ng/dL. This means that a greater cost does not translates to a correct range of normality.

An interesting finding of this study was the presence of 27 TT ranges described as normal in laboratories in Mexico City. Even when using the same method, laboratories have no consensus on the threshold.

Twenty-five laboratories used electrochemiluminescence for TT measurements and presented six parameters of normality.

Sixty-six laboratories use chemiluminescence and present twenty different ranges of normality.

According to our findings, chemiluminescence has the closest low TT threshold compared to those reported by international societies and is also the cheapest.

This study presents some limitations. With a response rate close to 70%, only 134 of 202 laboratories offered TT quantification assays, which may not fully represent the field. Additionally, 36 laboratories could not provide complete information, potentially affecting the data’s accuracy, despite these factors, the findings still offer valuable insights into TT measurement assays, though further research could enhance understanding in this area.

Conclusions

There is a need for greater consistency in the reference ranges among laboratories, ideally adopting guideline threshold values to identify low TT levels.

There is no consensus among Mexico City laboratories regarding TT thresholds for normal ranges and even for hypogonadism diagnosis, according to International Societies. This situation is prone to occur across laboratories throughout Latin America and the world, which confuses clinicians with the diagnosis of hypogonadism.

There is a great necessity to start a standardization project that ensures comparability across assays methods and laboratories in Mexico City and all over the world.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the SURGE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-162/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-162/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-162/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-162/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance to the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Mulhall JP, Trost LW, Brannigann RE, et al. Evaluation and management of testosterone deficiency: AUA guideline. J Urol 2018;200:423. [Crossref] [PubMed]
2. Wang C, Catlin DH, Demers LM, et al. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2004;89:534-43. [Crossref] [PubMed]
3. Shin YS, Park JK. The Optimal Indication for Testosterone Replacement Therapy in Late Onset Hypogonadism. J Clin Med 2019;8:209. [Crossref] [PubMed]
4. Lazarou S, Reyes-Vallejo L, Morgentaler A. Wide variability in laboratory reference values for serum testosterone. J Sex Med 2006;3:1085-9. [Crossref] [PubMed]
5. Rosner W, Auchus RJ, Azziz R, et al. Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab 2007;92:405-13. [Crossref] [PubMed]
6. Swerdloff R, Wang C. Reflections on the T Trials. Andrology 2020;8:1512-8. [Crossref] [PubMed]
7. Taieb J, Mathian B, Millot F, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem 2003;49:1381-95. [Crossref] [PubMed]
8. Wang C. Challenges in the diagnosis of the right patient for testosterone replacement therapy. Eur Urol Suppl 2007;6:862-7. [Crossref]
9. Wang C, Swerdloff RS. Testosterone Replacement Therapy in Hypogonadal Men. Endocrinol Metab Clin North Am 2022;51:77-98. [Crossref] [PubMed]
10. Harvard Health. Testosterone: What it is and how it affects your health. Published June 22, 2023. Available online: https://www.health.harvard.edu/staying-healthy/testosterone--what-it-does-and-doesnt-do
11. Wang C, Nieschlag E, Swerdloff R, et al. Investigation, treatment and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA and ASA recommendations. Eur J Endocrinol 2008;159:507-14. [Crossref] [PubMed]
12. Sizar O, Leslie SW, Schwartz J. Male Hypogonadism. Treasure Island (FL): StatPearls Publishing; 2024.
13. Salonia A, Boeri C, Capogrosso P, et al. EAU Guidelines on Sexual and Reproductive Health - UROWeb. 2025. Available online: https://d56bochluxqnz.cloudfront.net/documents/full-guideline/EAU-Guidelines-on-Sexual-and-Reproductive-Health-2025.pdf
14. 14 de noviembre. Día mundial de la diabetes. Procuraduría Federal del Consumidor Gobierno. Gobierno de México, 2022. Available online: https://www.gob.mx/profeco/documentos/14-de-noviembre-dia-mundial-de-la-diabetes-319474
15. Rosas-Peralta M, Attie F. Cardiovascular disease. First cause of death in adults in Mexico and worldwide. Arch Cardiol Méx 2007. Available online: https://www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S1405-99402007000200001
16. Gobierno de México. Ciudad de México: Economy, employment, equity, quality of life, education, health and public safety. Data México. Secretaría de Economía. 2020. Available online: https://www.economia.gob.mx/datamexico/en/profile/geo/ciudad-de-mexico-cx
17. Schteingart M, Sobrino J, Ugalde V. Introduction: Mexico City as a Latin American megacity. In: Mexico City. Agenda Publishing; 2023:1-10.
18. The State of Public Health in Mexico. Mexico Initiatives. Available online: https://mexico.arizona.edu/revista/state-public-health-mexico
19. OECD Indicators. Health at a Glance 2023. 2023. Available online: https://www.oecd.org/en/publications/health-at-a-glance-2023_7a7afb35-en.html