The Research Ethics Committee of King Faisal Specialist Hospital and Research Center (KFSHRC) approved the study. Testosterone, d3-testosterone, as well as ammonium acetate, acetonitrile, and methyl tert-butyl ether were purchased from Sigma-Aldrich MO, USA; IsoScience, PA, USA; and Fisher Scientific, NJ, USA, respectively. The stability of testosterone in extracted and unextracted plasma and salivary samples was tested under common clinical laboratory conditions. We report a specific, sensitive, and fast ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for determining clinically relevant testosterone level in human plasma and saliva. Given its high sensitivity and specificity, LC-MS/MS has emerged as the preferred method for the quantification of steroid hormones in human serum 13,14. The AMR was 2.5–1000 ng/dL (0.09–34.7 nmol/L), requiring 0.5 mL of male serum and 1.0 mL of female serum. A reference laboratory in the United States reported a high-turbulence flow HPLC, with the online SPE method achieving an assay linear range of 2 to 2000 ng/dL and a LOQ of 0.3 ng/dL . The databases also do not include testosterone formulations administered in a clinic setting, so our results underrepresent the scope of the observed TT prescribing practices. Our study’s main strength is the utilization of state PDMP databases with mandatory inclusion of all dispensed prescriptions regardless of payor, in contrast to commercial databases and is the first to report new TT prescribing data since 2018. While TT did not show an increased risk of major adverse cardiac events (MACE) in a high-risk population , most research has been limited to topical gels and not the more widely used injectable formulations that have shown a higher incidence of MACE, hospitalizations, and death . LH serum levels showed a significant decline over the years, adjusting for subjects’ age, while no trend emerged considering FSH. Categorical data such as the proportion of serious adverse events will be reported as a risk ratio with 95% CIs. To be included in the review, studies will be required to (1) include women aged 18–75 years and (2) to have directly compared systemic testosterone therapy with identical placebo or a blinded comparator therapy. In 2003, Starcevic et al. published the first LC-MS/MS method for the measurement of endogenous testosterone levels in human serum . Currently, there are various approaches to treating patients with testosterone insufficiency, including the use of testosterone pellets and formulations combined with aromatase inhibitors, which need more studies for a better understanding of their effects . Our understanding of the relative advantages of both physiological and pharmacological therapies for aging men is greatly improved by the effects of testosterone treatment and exercise on factors such as strength, aerobic fitness, and body composition . However, these statistical approaches rely on aggregate data, which restricts the use of more rigorous methodological statistical techniques. Indeed, biases related to the retrospective design of the study and the heterogeneity of subjects could not be eliminated. The question remains whether the progressive decline in testosterone and more generally testicular function is an adaptation to overpopulation. While the role of environmental pollution and population density cannot be excluded, the mechanism behind the decrease in hypothalamic-pituitary gonadal function in healthy men described here remains unknown. In this context, factors known to potentially cause so-called functional hypogonadism, such as lifestyle habits and stress 43,44,45,46, should be considered when interpreting the hormonal trends observed in our data. The discrepancy between the trends observed in LH and FSH levels over time suggests that the underlying mechanism could involve the hypothalamic rather than pituitary function. A positive pressure manifold (Biotage) was used to load the mixture onto the cartridge. Two hundred microliters of serum samples were spiked with 25 µL of the IS solution and gently mixed with the sample-conditioning solution (200 µL). Support liquid extraction cartridge, Isolute SLE+ 400 µL, was procured from Biotage (Charlotte, NC, USA). Ultra-low steroid hormone serum was sourced from Golden West Biologicals (Temecula, CA, USA). Testosterone (1.0 mg/mL), epitestosterone (1.0 mg/mL), and 2,3,4-13C3testosterone (100 µg/mL) were purchased from Cerillant (Round Rock, TX, USA). It is significant to emphasize that since this study reviews already published studies pertaining to patient data, ethical approval is not necessary. The likelihood of sarcopenia (age-related muscle loss) and diminished muscle strength rises as testosterone levels naturally fall with age; however, research indicates that testosterone therapy may mitigate these effects in older men, enhancing physical performance, sexual drive, and muscle mass . Descriptive, observational, and experimental studies including healthy men-more especially, those assessing the effects of testosterone therapy-were required for inclusion. The decline in testosterone serum levels across years of observation was also confirmed when considering variables related to population change (Chi-squared 177.0, p 2 48.9%). Similarly, a decrease in LH serum levels was confirmed in three out of six groups (Table 5 and supplementary material, page 17–19). Considering the limited number of studies, the analysis of testosterone trend measured by MS requires more data over a longer observation period. Additional confounders, such as the laboratory methodology used to measure serum testosterone levels and the country of origin of the study population, were also extracted.