1. Introduction
Testosterone (T), the principal androgen secreted by the testes, plays an essential role in male health [
1]. It is important for the development and maintenance of adult male secondary sexual characteristics. Male hypogonadism is diagnosed based on a combination of associated clinical signs and symptoms (
Table 1), and laboratory confirmation of low circulating T levels and decreased fertility [1, 2]; further testing is then required to elucidate the underlying aetiology. It has a prevalence estimated at 6-12% in the general population, increasing with age [
3], but may be found in up to 40% of men with type 2 diabetes mellitus (T2D) (with overt and borderline hypogonadism at 17% and 25%, respectively) [
4,
5,
6]. In older males, there is an overlap between the non-specific effects of ageing and late-onset hypogonadism [
7]. Longitudinal studies have demonstrated both hypogonadism and erectile dysfunction (ED) to be independently associated with increased total and cardiovascular disease (CVD)-related mortality, thus highlighting the importance clinically of this diagnosis [
8,
9,
10].
The British Society for Sexual Medicine (BSSM) and the European Association of Urology (EAU) guidelines on sexual dysfunction recommend that all men with ED should have, as a minimum standard, an initial measurement of T, and, in those with a poor response to phosphodiesterase type 5 inhibitors (PDE5i), T should be rechecked [2, 5]. In men with T2D, NICE guidance [
11] recommends an annual check/assessment for ED due to its prevalence in this group of >70%; accordingly, this should identify hypogonadism in up to 40% of patients. The BSSM, the Society for Endocrinology (SfE), the American Urological Association (AUA) and the American Association of Clinical Endocrinologists (AACE) [5, 12–14], and other national and international guidelines, recommend screening of T levels men with T2D, obesity (waist circumference >102 cm or BMI >30 kg/m
2) and metabolic syndrome, which will lead to increased detection of candidates for testosterone replacement therapy (TRT). Clinicians across diverse medical specialties (e.g., diabetes, endocrinology, urology, sexual medicine, general practice) are increasingly checking T levels driven in relation to a growing understanding of the risks associated with male hypogonadism. Prevailing clinical guidance on the diagnosis and management of hypogonadism in men should be supported by the clinical laboratory with accurate and precise analytical methodologies for the measurement of T levels, and other appropriate hormones and proteins, as this will have a direct impact on treatment decisions for patients (for example, the initiation and monitoring of TRT).
4. Laboratory Evaluation/Diagnosis of Male Hypogonadism
Diagnosis of hypogonadism in men is based upon the identification of its non-specific features through clinical assessment and blood testing. Serum total T is the most widely accepted biomarker to establish biochemically the presence of hypogonadism. When requesting serum T levels in men, the following categories are useful reasons for testing: 1) diagnosis of primary hypogonadism; 2) diagnosis of secondary hypogonadism: pituitary/hypothalamic disease; 3) late onset hypogonadism (also known as testosterone deficiency, adult-onset hypogonadism, and functional hypogonadism); and 4) for improving patient fertility [
1].
As there is a circadian variation (diurnal rhythm) in the secretion of T, with peak levels in the early morning, specimens taken to measure total T should be taken in the morning between 7.00 AM and 11:00 AM, which is especially important in men aged <40 years [
22]. This diurnal variation, however, is substantially blunted in older men, and in men with lower T levels [
23], but it may still be evident (even in elderly subjects), supporting the morning blood test recommendation in all age groups [
24]. In night/shift workers, T should be measured within 3 hours of waking up, because the diurnal rhythm is primarily driven by sleeping patterns, and not endogenously by circadian factors. Laboratory confirmation of hypogonadism in male shift workers is complicated and warrants specialist referral.
Evaluation of hypogonadism should not be made during acute illnesses. T levels are influenced by insulin, with a 75 g glucose load shown to lower T by 25% [
24]. Fasting T levels were reported to be up to 30% higher in healthy subjects compared to those taken in a non-fasting state [24, 25]. As such, the EAU guidelines [
2] now recommend that T is measured in a fasting state, although the evidence base for this is still inadequate and this does create practical difficulties for routine blood tests, which have generally moved to using non-fasting samples (e.g., checks for diabetes and dyslipidemia). However, Livingston et al. [
26], found no significant effect of fasting in a real-world UK clinical laboratory study of samples of 213 patients with suspected hypogonadism. Until further evidence to support this is available, the recent BSSM guideline [
5] supports measuring T in the fasting state for an initial test, but suggests a pragmatic approach is taken by clinicians since most patients do not routinely go around in a fasting state; consequently, insistence on fasting samples may introduce a barrier to patient investigation and a non-fasting early morning sample is considered acceptable.
When circulating T levels are borderline or low on first measurement, the test should be repeated on at least two occasions (ideally after a period of four weeks) as T is released in a pulsatile manner and the result of a single assay may be misleading), and also to check serum SHBG and albumin (required to estimate FT or bioavailable T levels, e.g. using the Vermeulen equation available at
http://www.issam.ch/freetesto.htm). Where total T levels are between 8–12 nmol/L, the FT level should be checked [2, 5], and serum LH and FSH levels should also be measured. Measurement of LH, FSH and prolactin will help to differentiate secondary from primary hypogonadism [
2]. This is not as clear cut in older men [
27]. In the case of known or suspected abnormal SHBG levels, FT should also be estimated [
5].
Serum prolactin levels are recommended when both LH and FSH levels are low. A very low total T (<5.2 nmol/L), and low LH and FSH are more likely to be associated with hyperprolactinaemia, pituitary tumour or other pituitary pathology. Regarding other investigations, in men with T levels <5.2 nmol/L and increased prolactin levels or reduced LH and FSH levels, pituitary magnetic resonance imaging (MRI) should be performed to exclude a pituitary adenoma/empty sella [22, 28]. Hyperprolactinaemia is associated with ED, loss of libido/sexual interest and anorgasmia, and should be ruled in/out by blood testing in all men with these findings. It is frequently accompanied by androgen deficiency because high prolactin levels suppress LH production and, consequently, cause hypogonadism. A moderate elevation of prolactin levels (<1000 mU/L) is unlikely to cause ED. There can be many causes for hyperprolactinaemia, both medical and physical, including stress, drugs (such as neuroleptics and anti-emetics), prolactin-secreting pituitary tumour (identification of these cases is very important), hypothyroidism and chronic renal failure. The presence of macroprolactin or ‘big–big’ prolactin, a heterogenous complex of prolactin and immunoglobulin A (150–170 kDa) which cross-reacts in the total prolactin assay, can lead to over-investigation of hyperprolactinaemia; this benign condition is the apparent cause of hyperprolactinaemia in about 20% of cases [29, 30]. The presence of macroprolactin should be considered in all cases of mild-to-moderate elevations in serum prolactin as it is measured in all commercial immunoassays, to a varying extent. Macroprolactinaemia is detected by re-assaying prolactin after precipitation with polyethylene glycol (PEG). Protocols to detect macroprolactin are in place in most clinical laboratories when prolactin levels are above a method-dependent cut-off (usually at levels of ~600–700 mU/L). Patients with persistent and unexplained hyperprolactinaemia should be referred to an endocrinologist.
At present, there is no definitive reference range or LLN threshold value for serum T that can be used to reliably and accurately identify men with hypogonadism; in part, this is because hypogonadal symptoms manifest at varying levels between individuals, and because of the variation in results between T immunoassays and their associated reference ranges. Thus, diagnostic and therapeutic T threshold concentrations represent a spectrum across the biological continuum and are dependent on the clinical context [
31]. However, the following threshold values function as action cut-off values for clinical practice rather than reference ranges. Patients with suggestive clinical features and two consecutive morning levels <8 nmol/L are likely to have hypogonadism. Although there are no studies directly comparing different testosterone cut-off levels for intervention, total testosterone <8 nmol/L correlates well with sexual symptoms of male hypogonadism and there is strong evidence in this group for a high prevalence of complications of hypogonadism and symptomatic improvement with treatment. Most of the current guidelines also agree with this action limit and the premise that further assessment for the aetiology of hypogonadism is required in these men.
The non-specific symptoms found in hypogonadism and variation in what T levels are considered ‘normal’ make the diagnosis challenging clinically [
33]. Guidelines agree that total T >12 nmol/L is unlikely to represent hypogonadism. One exception would be when the LH level is raised and there is a concern about subclinical/compensated primary hypogonadism, or androgen receptor cytosine, adenine, guanine (CAG) repeat polymorphism [
34]. This latter point relates to the androgen receptor (AR) mediating the peripheral effects of testosterone. The main mechanism of action for the AR is to direct regulation of gene transcription. Exon 1 of the AR gene contains a polymorphic sequence of CAG repeats, which varies in number from 10 to 35, and which encodes polyglutamine stretches of the AR transactivation domain [
34]. The evidence suggests that the number of CAG repeats in the coding region of the androgen receptor gene is negatively correlated with the transcriptional activity of the AR [
35]; stanworth2011dyslipidaemia]. Recently it was reported that CAG repeat number may partially influence the risk of mortality in older men [
36] and in men with T2D [
37]. Thus, it may be that future evaluation of androgen status will include determining the CAG repeat number as well as total and free testosterone.
In a national survey of UK clinical biochemistry laboratories [
18], the responses showed considerable variation in practice in the measurement and reporting of male T levels, including the laboratory reference ranges provided. Reference intervals based on population distributions are often misinterpreted as the ‘normal’ range and can lead to confusion in the absence of clear treatment guidelines [
32], particularly as treatment ambiguity often arises when T levels are borderline and, unfortunately, a borderline range is not acknowledged by a majority of laboratories [
18]. We would recommend that clinicians become familiar with the T assays utilised in their local laboratory and the associated reference intervals given, whilst also having an appreciation that reference ranges represent 95% of the normal population; these ranges may also have been derived by the commercial manufacturer of the T assay being used, and in a different patient population, with samples potentially not collected under standardised conditions. Reference ranges for total T are not designed to replace evidence-based action thresholds.
Improvements in the standardisation of T assays and the consistency of reporting between laboratories is required. If abnormal results are found and confirmed, discussion with, or referral to, a specialist endocrinology clinic should be considered. Many patients with hypogonadism can be treated in primary care, but where a pituitary or hypothalamic disorder is suspected, the advice of an endocrinologist should always be sought. Furthermore, the advice of an endocrinologist is necessary where there is doubt about the cause and appropriate management of the hypogonadism.
5. Therapeutic intervention and thresholds for monitoring TRT in male hypogonadism
A comprehensive approach is required in the management of male hypogonadism, including exercise and diet modification, as well as medication where appropriate. Testosterone treatment is only one potential option in the older man with low serum T in the context of holistic management where successful lifestyle measures (especially optimisation of body weight) and careful optimisation of comorbidities have important health benefits and may, by themselves be sufficient to normalise their serum T [
31]. Weight loss and lifestyle change should always form part of the management, but a significant elevation in T is not usually seen unless more than 5%–10% of weight loss is achieved. TRT is also recommended in men with HIV and chronic renal disease. Screening for low T (
Table 2) is recommended, especially in the presence of hypogonadal symptoms in all other populations (including those with CVD, chronic pulmonary diseases, cirrhosis, rheumatoid arthritis and cancer), because although such conditions are potentially associated with an increased prevalence of low T, there is a lack of evidence for benefit of TRT in asymptomatic individuals [
22].
The goal of treatment is in the restoration of symptoms including a sense of well-being (energy levels and mood), libido and sexual function, prevention/improvement of already established osteoporosis and optimization of bone density, restoration of muscle strength and improvement in mental acuity and metabolic parameters [
27]. Accurate and precise determination of T levels in men, taking into consideration the biological and analytical factors described earlier when taking samples and interpreting results, will directly impact decisions about the initiation of TRT. Significant benefits have already been shown in hypogonadal men following TRT [
38] in the Testosterone Trial cohort, including improvement in sexual function, quality-of-life, vitality, physical performance, mood, depression, bone mineral density and anaemia. In males aged >40 years with a total T level of ≤8.7 nmol/L (≤250.7 ng/dL), TRT was found to improve symptoms and significantly reduce mortality in men with T2D [
39]. Two further longitudinal studies confirmed this in men with low total T levels and T2DM/underlying elevated cardiovascular risk, although using different cut-off points (10.4 nmol/L (299.7 ng/dL) [
40], and total T of 12 nmol/L (345.8 ng/dL) and cFT of 0.25 nmol/L (7.2 ng/dL) [5, 41, 42]. Improvement in sexual function, improved erection, and restored/enhanced PED5I responsiveness was also seen in those given TRT [42, 43]. Moreover, there is evidence of a decrease in insulin resistance by TRT in men with T2D and with chronic heart failure [44, 45].
In terms of the diagnostic and treatment thresholds for intervention in hypogonadal symptomatic men, the guidelines of the BSSM and International Society for Sexual Medicine (ISSM) both cite a total T level <12 nmol/L or cFT <225 pmol/L (<0.225 nmol/L) based on two separate morning (<11AM) samples as usually requires TRT [
5]. Total T levels >12 nmol/L or FT of >225 pmol/L (>0.225 nmol/L) do not require T Therapy. Levels between 8–12 nmol/L may require a trial of TRT (for a minimum of 6 months, based on improvement in symptoms). Evidence also supports treatment of men with total T concentrations <14 nmol/L in symptomatic men with pre-diabetes, aiming to prevent progression to overt T2DM. In those with appropriate symptoms, cFT levels (<225 pmol/L, 0.225 nmol/L) provides supportive evidence for TRT, and they were found to closely relate to the clinical symptoms and all-cause mortality in the EMAS study [
46]. Raised LH levels and T below normal, or in the lower quartile of the reference range, indicates inadequate testicular function prompting consideration of TRT dependent on the severity of symptoms [
5]. For those started on TRT, typically there will be a perceived benefit after 3 months; however, if there is no impact on symptoms after 3-6 months, then the diagnosis needs to be re-evaluated, and discontinuation of TRT considered [
5].
Men with total T <8.0 nmol/L (<230.5 ng/dL) or cFT <0.180 nmol/L (<5.2 ng/dL) usually require TRT, while those with total T between 8.0–12.0 nmol/L (230.5–345.8 ng/dL) may require TRT, depending on the presence of symptoms associated with hypogonadism. The timeline for improvement in symptoms following initiation of testosterone supplementation is variable, but generally shorter following prescription of testogel than depot testosterone (normally testosterone undecanoate). There is evidence of under prescribing of testosterone in primary care with marked variation between general practices in testosterone prescribing with an indication that the variation was largely related to general practitioner choice [
47].
Quality of life (QoL) is a summation of psychological variables, which contribute to the subjective perception that life is worthwhile [
48]. Positive effects of TRT on QoL have been seen in larger cohorts of hypogonadal men of up to more than 1.000 patients in uncontrolled ‘real-life’ settings or registries [49, 50]. Effects on muscle mass [51, 52] and bone mass take much longer to be manifest [
53]. Specifically Snyder et al. [
53] reported that 12-months of treatment with of 1% testosterone gel in men >65 years of age and serum T levels <9.5 nmol/L (275 ng/dL) resulted in a significant increase in volumetric bone mineral density. Lumbar spine bone mineral density (BMD) starts to increase after 6 months of treatment and may continue for 3 years of treatment [
54].
It is recommended that there is monitoring of serum testosterone level 3-4 weeks after initiation of testogel supplementation and before the 4th injection of depot testosterone, aiming for a trough serum T level within the laboratory reference range, with titration of testosterone dose accordingly. Once established on a specific dose of testosterone replacement, monitoring is undertaken annually and will include a check of full blood count (FBC), focusing on haematocrit and haemoglobin level and prostate specific antigen (PSA).
Contraindications to TRT are locally advanced or metastatic breast and prostate carcinoma, elevated haematocrit >48%, severe chronic heart failure (New York Heart Association Stage IV) and untreated obstructive sleep apnoea [
55]. All guidelines report that TRT is to be avoided in men who desire fertility in the next 6-12 months [
56]. If there is uncertainty about the safety of testosterone replacement, referral to a specialist Endocrinology clinic is recommended.
A number of studies have focused on the cardiometabolic benefits of TRT in hypogonadal men in the context reports that low levels predict an increase in all-cause mortality during long-term follow-up [
40]. In the TIMES 2 study [
57], the efficacy of transdermal 2% testosterone gel was evaluated over 12 months in hypogonadal men with T2D and/or metabolic syndrome. TRT reduced insulin resistance in the overall population and in T2D individuals. Glycaemic control was significantly better in the testosterone treated group than the placebo group, with improvements also seen in total and LDL cholesterol, lipoprotein a (Lpa), body composition. In a subsequent study [
58] TRT in the form of testosterone undecanoate was independently associated with reduced mortality in men with T2D. PDE5i use was associated with decreased mortality in all patients those not on testosterone replacement, suggesting independence of effect. Regarding diabetes prevention, in the T4DM study [
59], men aged 50–74 years, with a waist circumference of 95 cm or higher, a serum T concentration of 14·0 nmol/L or lower but without and impaired glucose tolerance (oral glucose tolerance test [OGTT] 2-h glucose 7·8–11·0 mmol/L) or newly diagnosed T2D were randomised to receive an intramuscular injection of testosterone undecanoate (1000 mg) or placebo for 2 years. At 2 years, 2-hour glucose of 11·1 mmol/L or higher on OGTT was reported in 21% of 413 participants with available data in the placebo group and 12% of 443 participants in the testosterone group (relative risk 0·59, 95% CI 0·43 to 0·80). Thus, TRT for 2 years reduced the proportion of participants with T2D beyond the effects of a lifestyle programme.
Some concern has been expressed regarding the cardiovascular safety following testosterone replacement. Thus, while most studies demonstrate either benefit or no increase in cardiovascular events, a few have reported higher CVD events in men on TRT; specifically, the retrospective cohort study reported in 2013 by Vigen et al [
60] and Finkle et al. [
61] who examined 55,593 insurance claims and compared the incidence rate of myocardial infarction in the 12 months prior to, and 3 months after, the initial prescription of TRT, suggested that TRT was likely to increase cardiovascular (CV) risk. However in a definitive landmark multicentre study recently published [
62], 5246 men 45 to 80 years of age who had pre-existing or a high risk of cardiovascular disease testosterone levels of less than 10.4 nmol/L (300 ng/dl) were randomly assigned to receive daily transdermal 1.62% testosterone gel. A primary cardiovascular end-point event occurred in 7.0% in the testosterone group and in 7.3% in the placebo group. Thus, there was no excess of cardiovascular events.