Serum testosterone levels in male hypogonadism: Why and when to check—A review

Summary Aim Although “late onset hypogonadism”, a condition that includes low testosterone and symptoms, is common in men over the age of 40 years, diagnosis is not clear cut amongst non‐specialists. It is the aim of this review to provide an up to date picture of how this state should be diagnosed and managed. Methods We aim to describe how primary and secondary hypogonadism should be excluded before the diagnosis of late onset hypogonadism is reached. As laboratory testosterone measurements are essential the current pitfalls such as inappropriate sample collection and the use of population derived reference ranges are expanded. We review current evidence to determine associations between late onset hypogonadism and morbidity/mortality and benefits following testosterone replacement therapy. Results A review of the current evidence shows that late onset hypogonadism is associated with a worse metabolic state and increased mortality. Longitudinal studies have suggested that significant reductions in both symptoms and mortality are seen, especially in patients with type 2 diabetes. Discussion This review highlights the importance of diagnosing late onset hypogonadism due to its association with morbidity/mortality and benefits following testosterone replacement. Thus, after making recommendations to ensure correct diagnosis we speculate whether the time has come to move away from population derived testosterone levels towards evidence based action limits.

This review will focus on the diagnosis/management of primary/secondary HG in brief and late onset HG in depth to alleviate some of the prevailing confusion regarding both diagnosis and treatment.
Testosterone, the most important androgen produced by the testes, plays an integral role in men's health. 1 Androgensactatseveral sites in the sexual response system: within the CNS, peripheral nitrergicnervesandcorporacavernosa.Androgendeficiencymaybe associated with decreased libido, erectile dysfunction (ED) and insensitivity to phosphodiesterase type 5 (PDE5) inhibitor treatment. 2

| DIAGNOSIS OF SECONDARY HYPOGONADISM (PITUITARY/ HYPOTHALAMIC DISEASE)
Anteriorpituitarypathologycanbeduetodeficiencyorhypersecretory states, with clinical symptoms and signs attributed mainly due to altered levels of hormones produced by the end organs under its regulatory control. Deficiency state could be either generalised or axis specific and often caused by non-functional pituitary adenomas, craniopharyngiomas, metastases, inflammatory diseases, haemorrhage and infarction.
Haemochromatosis (interestingly also a cause of primary HG), sarcoidosis as well as rarer infiltrative disorders can also result in pituitary damage. When hypopituitarism is diagnosed without an underlying cause beingdetecteditisclassifiedas"idiopathic"hypopituitarism.
Luteinizing hormone (LH) secreted by the anterior pituitary in response to hypothalamic gonadotrophin releasing hormone (GnRH), stimulates the interstitial (Leydig) cells to produce testosterone.Although testosterone has some direct end-organ activity, its major effects appear mediated by active metabolites following peripheral conversion into oestradiol (brain and bone) and 5α-dihydrotestosterone (in other tissues). Increased testosterone levels result in negative feedback suppressing GnRH and LH. It is important that any suspicion of pituitary or hypothalamic disorder must be referred urgently to an endocrinologist.
Thus, it is essential to be aware of the symptoms and signs of pituitary/ hypothalamic disorders, some of which are non-specific and include hypotension, weight loss, hypoglycaemia, hypothyroidism and HG.
It is important to check LH, follicle-stimulating hormone (FSH) and prolactin to differentiate secondary (pituitary/hypothalamic) from primary HG. [3][4][5] Elevated FSH levels indicate Sertoli cell failure and low FSH is consistent with hypopituitarism. Elevated FSH levels are also associated with ageing in men. LH levels below the laboratory reference range are often consistent with secondary hypogonadism.
Consequently, measurement of FSH and LH may assist in differentiating primary from secondary HG. Both gonadotrophins are usually raised in primary HG (typically >10 iu/L) while testosterone may or may not be low, depending on the progression of the gonadal failure.
Hypothalamic/pituitary disease affecting the GnRH/LH/FSH axis usually presents as low testosterone (<9 nmol/L) without an appropriately increased gonadotrophin level or response on dynamic function testing. Referral to a specialist endocrinology clinic should be considered if the results are abnormal, inappropriate or difficult to interpret.
If hypopituitarism is suspected, the entire anterior pituitary profile should be investigated (it is perhaps best to consider the hormonal deficiencies by both region of secretion and individual axes which is not within the scope of this review), including prolactin (high with some tumours, drug treatments and stress, while low in other instances), 9 am cortisol, thyroid-stimulating hormone, free thyroxine, insulin-like growth factor-1, LH, FSH and repeat testosterone.A temporary hypogonadotrophinaemia may result as a response to severe stress. This can be differentiated from true hypopituitarism by the 9 am cortisol-in a stress response cortisol will be physiologically elevated. It is important to be aware that other conditions exist that demonstrate similar biochemical presentations to hypopituitarism. Diagnosing pituitary disease requires experience with knowledge of endocrine pathways as the clinical state of the patient has to be matched to the individual hormone levels followed byacascadeofdynamicfunctiontestsandimaging.Ashypopituitarism is diagnosed by examining the global endocrine function and specific pituitary axes, we present a simplified investigation pathway in Figure 1.
Suggested investigations for endocrine diseases are presented below.

| The consequences of hyperprolactinaemia
Hyperprolactinaemia is associated with ED, loss of libido and anorgasmia. 6 It is frequently accompanied by androgen deficiency since

Review criteria
The information for this review was gathered by appraisal of relevant publications following a search of the on-line medical database PubMed. Three of the co-authors (ML, AHH, SR) independently rated the relevance of the research papers and other related/cited articles. The remaining co-authors provided further information and editorial guidance.

Message for the clinic
• The interpretation of a testosterone level found to be low on a sample taken at 09.00, requires a serum prolactin, LH and FSH measurement in order to rule out secondary hypogonadism.Also,sexhormonebindingglobulin(SHBG) measurement may help.
• It is important that multiple medical disciplines are conversant with the management of male 'late onset hypogonadism (HG)' due to its high prevalence and diverse presenting symptoms. Decisions have to be taken regarding testosterone replacement treatment based on the evidence base.
• Anunderstandingoflongitudinalstudiesofmenwithlate onset hypogonadism following TRT across primary and secondary care has the potential to yield significant morbidity and mortality benefits for our patients. Amisdiagnosisofhyperprolactinaemiacanbeduetothepresence ofmacroprolactinor"big-big"prolactin.Thisisaheterogenouscomplex of prolactin and immunoglobulin and is the cause of apparent hyperprolactaemia in about 20% of cases. 9 Macroprolactin is detected and contributes to total prolactin measured by most commercial immunoassays, and its presence should be considered in all cases of mild to moderate hyperprolactinaemia. Macroprolactinaemia is diagnosed by re-assaying prolactin following precipitation with polyethylene glycol and most clinical laboratories have protocols in place to check for this when prolactin levels are above the method dependent cut off (approximately 700-1000 mU/L). 9 To avoid unnecessary investigations, baseline prolactin levels should be measured before initiating certain long-term treatments (eg, antipsychotics). Patients with persistent and unexplained hyperprolactinaemia should be referred to an endocrinologist.

| DIAGNOSIS OF PRIMARY HYPOGONADISM
This is also known as primary testicular failure and common causes include the following: • Klinefelter's Syndrome: the extra X chromosome results in abnormal development of the testicles leading to underproduction of testosterone.
• Undescended testicles: one or both of the testes may not descend at birth, although often correcting itself within the first few years of life without intervention. If the testes remain undescended reduced secretion of testosterone can manifest.
• Mumps orchitis: in the event of infection during adolescence or adulthood, long-term testicular damage may be evident with decreased testosterone production.
• Hemochromatosis: this can lead to both primary and secondary HG.
• Testicular trauma • Chemotherapy or radiation therapy: this can interfere with both testosterone and sperm production and although the effects of both treatments are often short-term, permanent infertility can manifest.

| CLINICAL ASSESSMENT OF HYPOGONADISM
The presence of sexual symptoms (eg, ED, loss of libido) and generalised symptoms of HG, such as decrease in beard or body hair growth, decrease in muscle mass, gynaecomastia, osteoporosis must be ascertained on history taking. Lower testosterone levels are more likely to be associated with more clinical features of HG.

| DIAGNOSIS OF LATE ONSET HYPOGONADISM
Late onset HG is defined as a combination of sexual symptoms and serum total testosterone levels <12 nmol/L. 10 Studies have shown this age-related male HG to affect metabolic prognostic parameters of ill health in type 2 diabetes (T2DM) and morbidity/mortality risk in adult men (eg, CVD and all-cause mortality). Lower testosterone levels appear to correlate with more severe ED. 11 The validated self-

| CAG repeats and the androgen receptor
The polymorphic number of CAG repeats within the androgen recep-

| Effect of other comorbidities on testosterone levels
Testosterone levels decline with age, but only between 6% and 12% of men have been described as developing late onset HG. [18][19][20] There are a large number of comorbid conditions which have be associated with low serum testosterone, 21 including heart failure, vascular disease, osteoporosis, dyslipidaemia, T2DM, metabolic syndrome, obesity and depression. 19,22,23 Serum testosterone levels differ by ethnic group. 24 The EMAS study 20 found that low testosterone levels were related to obesity and the metabolic syndrome (that increase with age), not just age alone, although sex hormone binding globulin (SHBG) levels increase with age with consequent decrease in free testosterone levels. 25 A total testosterone level <8 nmol/L has been proposed as a putative risk stratifier in the risk assesment for T2DM, CVD and all-cause mortality. 26 Patients with late onset HG should be considered for TRT and their symptoms monitored to assess benefit. There is a probable association between low vitamin D levels and the metabolic syndrome, including its classifying and associated factors, CVD and mortality. 27 However, the precise nature of this relationship in subgroups (eg, gender, age groups, ethnicity, etc.) is unclear. An association of secondary and compensated HG with vitamin D deficiency has also been reported. 28 Thus, far from being a benign consequence of ageing, HG has important and potentially harmful metabolic consequences including significantly increased CVD risk. 2,29 In men with T2DM, higher SHBG and lower free testosterone levels have been strongly associated with increased mortality. 25 The link between late onset HG, ED and CVD/ mortality is clear, 30 In older males, these levels can be used as a guide to when the testosterone level is borderline low. There are, however, a variety of causes of a low testosterone apart from age. If age-related ranges are not quoted, this could increase the numbers of apparently abnormal low results in older men, with an impact on further investigation and treatment.
However, there is significant evidence that reference ranges Ideally, if the circulating total testosterone level is low on first measurement (12 nmol/L or less), the test should be repeated after 2 weeks as testosterone is released in a pulsatile manner and the value obtained from a single test may be misleading. It is important to ensure that the sample was definitely taken in the morning and in the event of timing uncertainty, a further repeat on 9 am sample would be necessary. Moreover, serum testosterone levels in men should be ideally checked in the fasting state on a morning sample, but non-fasting levels are acceptable. 15,40 There is some difference across the literature 16 10,36 The reduced mortality following TRT has also been observed in an observational cohort of non-diabetic middleagedmenwithlowtotaltestosteronelevels(≤8.7nmol/L)andhigh chronic medical morbidity. 35 Asstatedpreviously,debateexistsastothethresholdtestosterone in men with HG that requires TRT. However, in our view overt HG (defined as the presence of clinical symptoms of HG (eg, sexual dysfunction) and total testosterone <8 nmol/L) most definitely requires treatment. In the presence of one or more borderline results, adding an SHBG (and serum albumin) to enable estimation of the circulating free testosterone can add supportive information to the total testosterone levels. Furthermore, SHBG should also be measured in men with conditionsthatmayaffectitsconcentration(seesection6.3).

| Free testosterone
Accuratemeasurementoffreetestosteroneistechnicallydifficultto achieve and basic calculations using total testosterone/SHBG (eg, free androgen index) have been discredited as assessments of free testos- Appversionalsoavailable("TCalc").
In practice, in older men with suspected HG (presenting with ED) with a low or borderline-low total testosterone level, SHBG measurement may be considered at the initial visit to reduce number of visits and prevent delays to treatment. In men with a total testosterone >12 nmol/L and symptoms, an estimated circulating free testosterone <0.225 nmol/L (<225 pmol/L) suggests the patient might benefit from a trial of TRT for hypogonadism. 14,15 Here, the estimated free testosterone potentially helps the decision to give TRT. A testosterone level between 13-28nmol/L in most cases suggests adequate gonadal function with no further testing required.

| Sex hormone binding globulin
Guidance states that offering testosterone replacement therapy TRT to men with total testosterone >12 nmol/L is not indicated. suggest secondary or primary HG should be referred to a specialist to elucidate the cause, assess the potential associated pituitary or testicular pathology and establish the appropriate replacement therapy.
Long-term follow-up and monitoring of treatment may be carried out in primary care depending on the underlying pathology and treatment.
In borderline cases where the pattern is unclear, it is recommended that the tests are repeated. Patients with total testosterone <8 nmol/L and/or estimated free testosterone <0.225 nmol/L (<225 pmol/L) on two consecutive occasions require investigation of the cause of their HG.

| Total testosterone 8-12 nmol/L, with variable estimated free testosterone, LH and FSH results:
Many of these men will be classified as having late onset HG. Most patients with a total testosterone between 8 and 12 nmol/L will not be hypogonadal if asymptomatic, and will not require further investigation and treatment. Patients experiencing symptoms of HG require further assessment, with TRT considered if not contraindicated. It is important to confirm symptomatic benefit with adequate testosterone replacement in these individuals. If no benefit is demonstrated despite adequate testosterone levels being attained, insensitivity states (estimation of CAG repeats) must be considered. In the case of sexual symptoms, it is essential that TRT is not discontinued prematurely.

| Total testosterone levels >12 nmol/L
Patients with two consecutive total testosterone >12 nmol/L and/ or estimated free testosterone >0.225 nmol/L (>225 pmol/L) do not warrant treatment. Re-testing after 6-12 months may be considered in some cases.

| Further laboratory testing
When assessing patients with potential late onset HG, it may be prudent to undertake some extra testing when assessing these patients. This could include glucose/HbA1c, liver function tests, urea and electrolytes, Full Blood Count (FBC) and serum prostate specific antigen (PSA) prior to consideringTRT. Patient counseling is recom-mendedtoexplaintheimplicationsofmeasuringserumPSA,including a discussion on the benefits and drawbacks of the test. 46 Two prostate pathologies are associated with testosterone levels: benign prostatic hyperplasia and prostate cancer. These pathologies tend to occur after 40 years of age just when testosterone levels are seen to decrease. The evidence is that there appears to be no increased risk of prostate cancer following TRT. 16 Prostate cancer is a dihydrotestosterone responsive tumour and much of the therapy targets testosterone/dihydrotestosterone production.
Examples of anti-androgen therapy include: (i) goserelin, a GnRH analogue to suppress gonadotrophins, producing a hypopituitary hypogonadal picture, (ii) cyproterone acetate, an androgen receptor antagonist having little effect on gonadotrophin/testosterone levels, (iii) Finasteride, a 5α-reductase inhibitor blocking the activation of testosterone to 5α-dihydrotestosterone.

| TREATMENT OF LATE ONSET HYPOGONADISM
Despite mounting evidence that TRT is associated with benefit in late onset HG, practice has been slow to take this into account. The response to treatment should be assessed at 3, 6, 9 and 12 months after the onset of treatment, and thereafter annually, including assessment of serum testosterone, haematocrit levels (as part of a FBC profile),serumPSAandothermetabolicparameters(eg,lipidprofile). 16 Bone mineral density (BMD) should be monitored only in men whereitwasabnormalbeforeinitiationofTRT.Anincreaseinlumbar spine BMD may already be detectable after 6 months of TRT and may continue for 3 more years. 52  treated with androgens had been thought to be at an increased risk for worsening of signs and symptoms, but there is no evidence at present that TRT either increases the risk of BPH or contributes to worsening of lower urinary tract symptoms (LUTS), and TRT may even improve LUTS in hypogonadal men with mild BPH. 48,53 Major concerns about the risks of TRT have focused primarily on the hyperstimulation of prostate growth and the induction of carcinoma of the prostate. The evidence is that there appears to be no increased risk of prostate cancer following TRT. 16

| CONCLUSION
The focus of this review is low testosterone levels associated with HG, a common phenomenon in middle aged and older men, i.e. late onset HG. Clinical training emphasises the importance of approaching most chronic pathologies from the clinical presentation and working backwards towards the aetiology before deciding on the management.
With many testosterone based myths present in healthcare circles, we felt that it was best to approach the topic from the hormone, central to much of HG.
The requesting of testosterone should be clinically driven.
Testosterone should be checked in the fasted state and when low should be repeated. Free testosterone must be calculated in certain circumstances. When low testosterone levels have been confirmed, the underlying aetiology will have to be determined: primary, secondary and late onset HG. Primary and secondary HG requires referral to appropriate specialists.
It is important that multiple medical disciplines understand the management of male late onset HG due to high prevalence and diverse presenting symptoms. Decisions have to be taken regarding TRT based on evidence which has been accumulating. We have presented some of this evidence and details of TRT and post-treatment clinical and laboratory monitoring.An understanding of longitudinal studies of men with late onset HG following TRT across primary and secondary care has the potential to yield significant morbidity and mortality benefits.

Professor Sudarshan Ramachandran, Dr Adrian Heald and Dr Mark
Livingston are occasional speakers for Besins Healthcare (UK) Ltd.
This company provided funding to allow this article to be available open-access. The sponsor had no role in the content or presentation of this manuscript.

ETHICAL APPROVAL
Not required.