The role of androgen receptor CAG repeat polymorphism and other factors which affect the clinical response to testosterone replacement in ...
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R D Stanworth and others AR CAG and response to 170:2 193–200
Clinical Study testosterone
The role of androgen receptor CAG repeat
polymorphism and other factors which
affect the clinical response to testosterone
replacement in metabolic syndrome and
type 2 diabetes: TIMES2 sub-study
R D Stanworth, S Akhtar1, K S Channer2 and T H Jones3,4
Correspondence
Department of Diabetes and Endocrinology, Derby Hospitals NHS Foundation Trust, Derby, UK, 1Department of should be addressed to
Human Metabolism, University of Sheffield, Sheffield, UK, 2Faculty of Health and Wellbeing, Sheffield Hallam T H Jones at Barnsley Hospital
University, Sheffield, UK, 3Barnsley Hospital, Diabetes and Endocrinology, Gawber Road, Barnsley, South Yorkshire Diabetes and Endocrinology
S75 2EP, UK and 4Academic Unit of Diabetes, Endocrinology and Metabolism, University of Sheffield Medical School, Email
Beech Hill Road, Sheffield, South Yorkshire S10 2RX, UK hugh.jones@nhs.net
European Journal of Endocrinology
Abstract
Context: The TIMES2 (testosterone replacement in hypogonadal men with either metabolic syndrome or type 2 diabetes)
study reported beneficial effects of testosterone replacement therapy (TRT) on insulin resistance and other variables in men
with diabetes or metabolic syndrome. The androgen receptor CAG repeat polymorphism (AR CAG) is known to affect
stimulated AR activity and has been linked to various clinically relevant variables.
Objective: To assess the role of AR CAG in the alteration of clinical response to TRT in the TIMES2 study.
Design: Subgroup analysis from a multicentre, randomised, double-blind, placebo-controlled and parallel group study.
Setting: Outpatient study recruiting from secondary and primary care.
Patients: A total of 139 men with hypogonadism and type 2 diabetes or metabolic syndrome, of which 73 received
testosterone during the TIMES2 study.
Intervention: Testosterone 2% transdermal gel vs placebo.
Main outcome measure: Regression coefficient of AR CAG from linear regression models for each variable.
Results: AR CAG was independently positively associated with change in fasting insulin, triglycerides and diastolic blood
pressure during TRT with a trend to association with HOMA-IR – the primary outcome variable. There was a trend to negative
association between AR CAG and change in PSA. There was no association of AR CAG with change in other glycaemic
variables, other lipid variables or obesity.
Conclusion: AR CAG affected the response of some variables to TRT in the TIMES2 study, although the association with
HOMA-IR did not reach significance. Various factors may have limited the power of our study to detect the significant
associations between AR CAG, testosterone levels and change in variables with testosterone treatment. Analysis of similar
data sets from other clinical trials is warranted.
European Journal of
Endocrinology
(2014) 170, 193–200
www.eje-online.org Ñ 2014 European Society of Endocrinology Published by Bioscientifica Ltd.
DOI: 10.1530/EJE-13-0703 Printed in Great Britain
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testosterone
Introduction
In vitro studies have demonstrated an inverse relationship other potentially beneficial effects including reduced total
between the length of androgen receptor CAG repeat and LDL-cholesterol and percentage body fat, seen in sub-
polymorphism (AR CAG) and transcriptional activity of group analyses (16).
the receptor after ligand activation (1, 2). Epidemiological We have analysed the effect of AR CAG in modulating
studies have identified associations of AR CAG with the effects of TRT on insulin resistance (HOMA-IR), lipids,
the prevalence of prostate cancer (3, 4), benign prostatic obesity, body composition and other clinical parameters
hypertrophy (5, 6), semen parameters of fertility (7, 8, 9), in the TIMES2 study.
bone mineral density (10), depression score (11) and
obesity (12, 13). This suggests that AR CAG may be
Subjects and methods
clinically relevant in the determination of phenotypic
outcomes. There is also data to suggest that serum TIMES2 study methods and derivation of
testosterone correlates with AR CAG (7, 13), reflecting population for this study
the participation of the AR in the negative feedback loop The TIMES2 study was a multicentre, randomised, double-
controlling serum testosterone. This compensatory blind and placebo-controlled study of 12 months,
response may diminish the effects of AR CAG on clinical duration with a primary endpoint of difference in change
variables in normal physiology. from baseline of insulin resistance assessed by HOMA-IR
Tissue androgen exposure in men treated with between testosterone and placebo groups after 6 and 12
testosterone replacement therapy (TRT) is a function of months. Secondary outcomes included changes in fasting
European Journal of Endocrinology
dose, preparation, absorption, distribution and metab- glucose, fasting insulin, HbA1c, lipids and anthropo-
olism rather than negative feedback. The loss of negative morphics. The trial received appropriate regulatory and
feedback means that effects of androgen are directly ethics approval and participants gave written informed
related to AR function for a given level of tissue androgen consent. Full study methods have been previously
exposure, therefore AR CAG may be of greater relevance in published (16).
testosterone-treated men. Variability in AR CAG could The study population comprised 220 men aged
theoretically necessitate different target levels for serum 40 or over, who fulfilled the International Diabetes
testosterone in men treated with testosterone. Federation (IDF) definition of the metabolic syndrome
Assessment of AR CAG as a modulator of androgen and/or type 2 diabetes. Additionally, participants were
effects in clinical trials is therefore justified but has hypogonadal with total testosterone !11 nmol/l or calcu-
received little attention. One study demonstrated that lated free testosterone (17) !255 pmol/l on two consecutive
change in prostate volume during TRT was inversely tests and hypogonadal symptoms. Initial dose of testoster-
related to AR CAG (14), which could be viewed as proof of one gel was 60 mg and this was titrated to keep the level of
concept that AR CAG modulates clinical response to TRT. testosterone between 17 and 52 nmol/l in the treated group.
The effect of AR CAG on changes in obesity and There was a high dropout rate in both testosterone
cardiovascular risk factors during TRT has not been studied and placebo groups, such that 157 men completed the
in clinical trials, although a retrospective audit of 66 men first 6 months of the study and 118 completed 12 months.
on open-label intramuscular testosterone undecanoate Reasons for withdrawal were adverse events (nZ26),
showed that reductions in blood pressure and increases in withdrawal of consent (nZ27), protocol violation
haematocrit were associated with shorter AR CAG (15). (nZ28) and loss of follow-up (nZ9). Study completion
The recently published TIMES2 (testosterone replace- was at least as prevalent in the testosterone group (75%)
ment in hypogonadal men with either metabolic syn- as the placebo group (68%). There were 125 men who
drome or type 2 diabetes) study has investigated the effects violated the protocol in the first 6 months, leaving an
of transdermal testosterone on insulin resistance, cardio- initial per-protocol group of 95.
vascular risk factors and symptoms in hypogonadal men The original TIMES2 study protocol included plans
with type 2 diabetes and/or metabolic syndrome (16). for assessment of AR CAG in all participants. Ethical
Testosterone resulted in significant reductions in the approval to assess AR CAG was not granted in one country.
primary outcome of insulin resistance as assessed by In addition, 27 participants withdrew consent during the
change in HOMA-IR after 6 months (16). Testosterone TIMES2 study and were deemed to have also withdrawn
also caused a significant reduction in lipoprotein-a and consent for this study. A further seven men left the trial
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testosterone
before any follow-up visit occurred. As a result, the study The primary independent factor of interest in each model
population for the analysis of AR CAG was 139, of which was AR CAG with other factors being baseline testo-
73 received testosterone and 66 received placebo gel. sterone, change in testosterone between baseline and
It was decided to assess data from the 6 month time point 6 months, baseline waist, change in waist, age and
of the main study in order to maintain group size and baseline variable x.
maximise statistical power for the genetic study. The aim of the study was to assess the effect of AR
CAG in the alteration of the clinical response to TRT,
so the testosterone-treated group was mainly of interest.
Genetic study
Change in variable x was found to be associated with
All eligible samples from the TIMES2 study for AR CAG baseline variable x during testosterone replacement in
measurement were analysed in the Departmental Labora- a number of cases. The same regression models were
tory, Academic Unit for Diabetes, Endocrinology and therefore applied to the placebo group – allowing analysis
Metabolism, University of Sheffield Medical School. of whether associations with baseline variable x reflected
DNA was extracted from peripheral lymphocytes in genuinely different responses to testosterone depending
whole blood and subjected to PCR to amplify the region of on baseline variable x or whether other characteristics
the AR gene containing AR CAG and AR GGC on separate of the population or data were likely to explain the
occasions. DNA was amplified in 25 ml reactions apparent association.
containing 22.5 ml PCR Master Mix (ABGene, Epsom,
UK), 0.5 ml 10 pmol each primer, 0.5 ml distilled water
and 1 ml DNA containing sample. The primers used for AR Results
European Journal of Endocrinology
CAG amplification were 5 0 -GCT GTG AAG GTT GCT GTT
Baseline characteristics of overall study population,
CCT CAT-3 0 and 5 0 -TCC AGA ATC TGT TCC AGA GCG
testosterone-treated and placebo groups
TGC-3 0 . Amplifications for AR CAG were performed using
an automated thermal cycler applying the following PCR Of 220 participants in the TIMES2 study, 139 were
conditions: 94 8C for 5 min, followed by 32 cycles of 1 min appropriate for the analysis of AR CAG after exclusion of
at 94 8C, 1 min at 58 8C and 1 min at 72 8C followed by those from countries in which assessment of AR CAG was
a 7 min final extension at 72 8C and denaturation for not given ethical approval and those who withdrew
5 min at 96 8C. consent during the study. Table 1 demonstrates that our
Following magnetic separation of PCR products, study population is well matched with the overall TIMES2
each sample was analysed by the capillary-based AB3730 study population. Table 2 compares baseline charac-
automated sequencer (Applied Biosystems), which pro- teristics in testosterone and placebo-treated groups.
duces electropherograms from which the DNA sequence Groups were well matched for all variables.
could be derived.
Assessment of normal distribution in
Statistical analysis the study population
Data were analysed at baseline and after 6 months’ study Q–Q plots for comparison of study data with standard
medication. In the event of the participants ceasing study normal distribution were constructed and detrended Q–Q
medication before 6 months had elapsed, the last
observation was carried forward. This was similar to the Table 1 Comparison of baseline parameters in the full TIMES2
primary analysis undertaken in the main TIMES2 study. study population vs the subpopulation involved in this study.
Statistical methods were undertaken using SPSS 15.0
TIMES2 AR CAG
(SPSS). Data were assessed for normal distribution using
Q–Q plots and detrended plots. Where normal distri- Parameter Full population Study population P value
bution was not confirmed, attempts were made to improve Total testosterone 9.4G3.0 9.5G2.9 0.755
normal approximation by logarithmic transformation. (nmol/l)
BMI (kg/m2) 32.1G6.0 31.8G5.9 0.643
These were sufficiently successful to allow data to be Waist (cm) 111.4G13.5 111.7G12.7 0.843
used in parametric testing in all cases. HOMA-IR (index) 5.4G3.6 5.5G3.6 0.798
Multiple linear regression models were constructed for Haematocrit (%) 43G4 43G4 0.645
PSA (ng/ml) 1.4G1.5 1.2G1.2 0.186
change in variable x during 6 months of study medication.
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testosterone
Table 2 Baseline variables in active and placebo groups. All values are expressed as meanGS.D.
Parameter Active Placebo P value
AR CAG 21.4G2.4 21.3G2.8 0.776
Total testosterone (nmol/l) 9.14G2.72 9.94G3.01 0.100
SHBG (nmol/l) 26.6G13.4 26.8G12.5 0.919
HOMA-IR index 5.89G3.68 5.09G3.59 0.210
Fasting insulin (pmol/l) 116.1G57.4 103.9G55.0 0.217
Fasting glucose (mmol/l) 8.05G3.23 7.63G2.79 0.423
HbA1c (%) 6.92G1.56 6.74G1.46 0.488
Total cholesterol (mmol/l) 4.52G1.17 4.88G1.14 0.073
HDL cholesterol (mmol/l) 1.16G0.31 1.24G0.28 0.122
LDL cholesterol (mmol/l) 2.66G0.98 2.89G1.05 0.177
Triglycerides (mmol/) 1.95G1.55 2.05G1.36 0.677
Lipoprotein-a (mmol/l) 0.349G0.383 0.404G0.349 0.380
PSA (ng/ml) 1.25G1.17 1.16G1.24 0.673
Haematocrit (%) 42.5G3.7 42.8G3.4 0.667
Waist (cm) 112.4G11.6 109.3G13.7 0.152
BMI (kg/m2) 32.7G6.3 31.0G5.5 0.096
Body fat mass (kg) 33.6G6.4 31.4G6.6 0.052
Systolic blood pressure (mmHg) 140.2G17.6 136.7G15.2 0.209
Diastolic blood pressure (mmHg) 83.4G10.1 81.6G8.7 0.264
P values for Student’s t-test for comparison of means of active and placebo groups with significance of P!0.05.
European Journal of Endocrinology
plots were further derived. These plots, shown in HbA1c (bZ0.220, PZ0.073). Post-hoc analysis compared
Supplementary data, see section on supplementary data those men who had testosterone O35 nmol/l (nZ24)
given at the end of this article, show that total during the study with the remainder of the group.
testosterone, AR CAG, age, waist circumference, body fat Student’s t-test did not show a significant difference in
percentage, HDL cholesterol, diastolic blood pressure, change in HbA1c between the groups (C0.30% for high
systolic blood pressure and haematocrit are approximately testosterone group vs C0.05%, PZ0.125).
normally distributed in this population. Conversely, Change in waist was significantly associated with
HOMA-IR, fasting insulin, fasting glucose, HbA1c, total changes in HOMA-IR (bZ0.259, PZ0.041) and fasting
cholesterol, LDL cholesterol, triglycerides, lipoprotein-a, insulin (bZ0.309, PZ0.011) but not fasting glucose or
BMI and PSA were not well matched. The Supplementary HbA1c (Table 3 and Supplementary data). Baseline waist was
data further shows improved matching of these variables not associated with change in insulin resistance, insulin,
with normal distribution after log transformation. glucose or HbA1c. There was no significant relationship
of age with changes in glycaemic variables, although
there was a trend with change in HOMA-IR (bZK0.248,
Effect of AR CAG on response to testosterone
PZ0.070) and fasting insulin (bZK0.218, PZ0.081).
replacement; insulin resistance, insulin,
Baseline fasting glucose was negatively correlated with
glucose and HbA1c
change in fasting glucose on testosterone level (Table 3),
Multiple linear regression analysis conducted in the but the similar relationship was seen in the placebo group
testosterone-treated group showed no significant associ- (Supplementary data).
ation of AR CAG with change in HOMA-IR after 6 months,
although there was a trend to an association between the
Effect of AR CAG on response to testosterone
two variables (Table 2, bZ0.237, PZ0.057) and there was a
replacement; body composition
positive association of AR CAG with change in fasting
insulin (Table 2, bZ0.268, PZ0.028). There was no Multiple linear regression analysis showed that AR CAG,
significant association of AR CAG with change in fasting baseline testosterone, change in testosterone during TRT
glucose or HbA1c. Serum testosterone was not signi- and age, all did not predict change in waist, BMI or body
ficantly related to change in HOMA-IR, fasting insulin or fat after 6 months (Table 3 and Supplementary data).
fasting glucose (Supplementary data). There was a trend Baseline values for the respective variables also failed to
towards an association of change in testosterone with predict response to testosterone.
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Table 3 Multiple linear regression analysis results. Models formed for each variable with AR CAG, baseline testosterone, change in
testosterone, baseline waist, change in waist and baseline variable. Association of AR CAG with each variable expressed on left side
of table. Results considered significant at P!0.05 and marked bold. Other significant associations shown in remainder of table. Non-
significant results not shown here (see Supplemental data for full results).
Association with AR CAG Other significant association (1) Other significant association (2)
Variable b P b P b P
HOMA-IR 0.237 0.057 Change in waist 0.259 0.041 – – –
Fasting insulin 0.268 0.028 Change in waist 0.309 0.011 – – –
Fasting glucose 0.091 0.472 Baseline glucose K0.298 0.022 – – –
HbA1c K0.087 0.489 – – – – – –
Waist K0.047 0.708 – – – – – –
BMI 0.058 0.649 – – – – – –
Body fat percentage 0.059 0.635 – – – – – –
Total cholesterol 0.052 0.644 Baseline total K0.483 !0.001 – – –
cholesterol
LDL cholesterol K0.109 0.332 Baseline LDL K0.474 !0.001 – – –
HDL Cholesterol 0.041 0.685 Baseline HDL K0.560 !0.001 – – –
Triglycerides 0.271 0.016 Baseline triglycerides K0.512 !0.001 – – –
Lipoprotein-a K0.071 0.572 – – – – – –
HCT K0.071 0.545 Baseline test K0.301 0.012 Baseline HCT 0.265 0.025
PSA K0.212 0.070 Age 0.254 0.045 Baseline PSA K0.314 0.010
Systolic BP 0.092 0.388 Baseline waist 0.231 0.044 Baseline Sys BP K0.561 !0.001
European Journal of Endocrinology
Diastolic BP 0.258 0.025 Baseline Dias BP K0.484 !0.001 – – –
Results considered significant at P!0.05 and marked bold. Other significant associations shown in remainder of table. Non-significant results not shown
here (see Supplementary data for full results).
Effect of AR CAG on response to testosterone Effect of AR CAG on response to testosterone
replacement; lipids and blood pressure replacement; PSA and haematocrit
Regression analysis conducted in the testosterone-treated AR CAG and change in testosterone were not associated
group showed that AR CAG was positively associated with change in haematocrit during testosterone replace-
with change in triglycerides (Table 3). There was no effect ment, although baseline testosterone was negatively
of waist or testosterone at baseline or after 6 months on associated (Table 3). Baseline haematocrit was positively
change in triglycerides, but there was a trend to negative associated with change in haematocrit, with the opposite
association with age (Supplementary data). There were pattern seen in placebo-treated men (Table 3 and
no associations of AR CAG, testosterone, waist or age Supplementary data).
with change in total cholesterol, LDL cholesterol, Age was positively associated with change in PSA
HDL cholesterol or lipoprotein-a (Table 3 and Supple- during TRT (Table 3). Baseline PSA was negatively related
mentary data). to change in PSA. The same pattern was not seen in the
AR CAG was positively associated with change in placebo group (Supplementary data). There was a trend to
diastolic but not systolic blood pressure (Table 3). There negative association between AR CAG and change in PSA.
was no significant relationship between testosterone and
change in blood pressure but there was a trend between
baseline testosterone and systolic blood pressure (Supple- Discussion
mentary data). Baseline waist was positively associated This study investigated the role of AR CAG in the
with change in systolic blood pressure (Table 3). modulation of the clinical effects of TRT in the TIMES2
Changes in blood pressure and each of the lipid study. AR CAG was not significantly associated with
fractions with the exception of lipoprotein-a were change in HOMA-IR, the primary outcome variable, but
associated with levels at baseline (Table 3), but similar there was a trend to positive association and AR CAG was
findings were apparent in the placebo group (Supple- significantly positively associated with change in fasting
mentary data). insulin, triglycerides and diastolic blood pressure. These
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associations imply a greater reduction in these parameters 12 months and the modified per-protocol group showed
during TRT in participants with shorter AR CAG (greater reductions in body fat percentage after 6 and 12 months
androgenic effect). The trend to negative association and reduced waist circumference in men with diabetes
between AR CAG and change in PSA suggests a possible after 12 months (16). The results of regression analysis
link of shorter AR CAG repeat with greater increases in PSA here show that change in waist circumference on TRT is
during testosterone replacement. a predictive factor for change in HOMA-IR and fasting
AR CAG has been shown to affect stimulated AR insulin and that this is independent of AR CAG and
transcriptional activity in vitro (1, 2). For the purposes of testosterone. The associations of AR CAG with trigly-
this study, we have made an assumption that AR CAG is cerides and diastolic blood pressure were not accompanied
linearly related to transcriptional activity and this is in line by relationships with change in waist. At least some
with in vitro data (1, 2). Our hypothesis is that AR CAG can beneficial effects of testosterone therefore seem to occur
be used as a marker of the effects of testosterone on various independently of change in adiposity.
parameters via actions at the AR. This may differ from the The lack of association of AR CAG and testosterone
overall physiological effects of testosterone due to con- with change in obesity in this study is in contrast to data
version of testosterone to active metabolites including linking AR CAG to waist circumference in men with
oestradiol as well as non-genomic actions of testosterone type 2 diabetes (13). Reductions in waist circumference in
occurring independently of the AR – a principle that has the TIMES2 study modified per-protocol analysis took
found support from data in epidemiological studies 12 months to develop in men with diabetes. It may be that
showing a variable relationship between testosterone changes in body composition take O6 months to evolve
and insulin resistance depending on AR CAG (18). The and that longer interventional studies would be needed
European Journal of Endocrinology
findings from our regression analysis would be consistent in order to gain concordance with the epidemiological
with testosterone effects at the AR, resulting in reductions data regarding the associations of AR CAG with adiposity.
in fasting insulin, HOMA-IR, triglycerides and diastolic Change in triglycerides was positively associated
blood pressure and increases in PSA. with AR CAG in this study, but triglycerides were not
The association of AR CAG with fasting insulin and associated with AR CAG in a cross-section of men with
trend to association with HOMA-IR suggests that the diabetes (22). Participants in the TIMES2 study attended in
reduction in HOMA-IR with testosterone in the TIMES2 a fasted state but this was not the case with the cross-
study (16) is mediated by a classical action of testosterone sectional study, which may have obscured an association
via the AR. Change in HOMA-IR after 6 months was the between triglycerides and AR CAG. Change in diastolic
primary outcome variable in the TIMES2 study and this is blood pressure was positively associated with AR CAG in
the first multicentre trial with adequate power to assess the this study. A correlation of systolic blood pressure with
effect of TRT on HOMA-IR. This adds to previous data from AR CAG in men with diabetes has previously been
small studies which have shown promising effects of TRT described (13) and AR CAG was associated with change
on glycaemic control or insulin resistance (19, 20). There in blood pressure during open label TRT (15). Most studies
was no change in HbA1c in the intention to treat group of TRT have not shown an effect on triglycerides or blood
from the TIMES2 study, but the study population included pressure. This could suggest that effects of testosterone via
a proportion of patients who were not diabetic or had the AR are balanced by opposite effects of testosterone or
well-controlled diabetes at baseline, which reduced the metabolites including oestradiol via other mechanisms.
statistical power to demonstrate any effect. HbA1c reflects Alternatively, it may be that changes in these variables
glycaemic control over the preceding 2–3 months and during testosterone treatment only occur with shorter,
therefore may not have had sufficient time for changes in more active AR CAG and that this is not a detectable
insulin resistance detected at 6 months to take full effect. change in the overall group.
These factors may also have reduced the possibility of Change in testosterone level during the trial was not
finding associations of change in HbA1c with AR CAG. significantly associated with change in any outcome
Testosterone has led to reductions in body fat or variable assessed. Testosterone levels of men whilst on
central adiposity in a number of trials, and meta-analysis transdermal testosterone gel are known to vary. It is also
has shown reductions in fat mass following TRT (21). likely that compliance with treatment was suboptimal in
The TIMES2 intention to treat analysis did not show some cases. The trial was not designed to optimise the
changes in body composition but the per-protocol regression analyses performed here and the purpose of
group showed a reduction in waist circumference after testosterone monitoring was primarily to allow dose
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titration. More frequent serum testosterone measures to erroneous associations. The cumulative effect of these
allow accurate representation of treatment effects and/or limitations means that this should be considered a pilot
use of alternative testosterone preparations with more study in terms of assessing the pharmacogenetics of
stable and predictable pharmacokinetics would have testosterone and AR CAG.
allowed more accurate assessment of androgen exposure A further limitation of the study was a high dropout
and may have produced positive results. Baseline testo- rate, which was similar in testosterone and placebo-treated
sterone was negatively associated with change in group, suggesting that this was a function of the study
haematocrit in the testosterone-treated men. This would requirements or the way that men were recruited rather
be in line with meta-analysis data suggesting that the than any problems relating to testosterone therapy.
effects of testosterone vary according to the baseline The high dropout rate contributed to a smaller group
testosterone level (21). size for AR CAG analysis than was anticipated and limited
In many cases, baseline results for a variable were study power. A significant proportion of patients had
associated with change in the same variable after protocol violations due to medication changes in the first
6 months. In most cases similar results were seen in the 6 months of the study – including alterations to medi-
placebo group, suggesting that factors other than response cation with effects on glycaemia, lipids and blood
to testosterone such as medication changes or short-term pressure. These medication changes and the use of other
fluctuations leading to regression to the mean during concurrent medications could have confounded our
follow-up were responsible. This was the case with total, results. The mode of testosterone administration and
LDL and HDL cholesterol, systolic and diastolic blood monitoring in the trial was not ideally suited to regression
pressure, triglycerides and fasting glucose. The finding of
European Journal of Endocrinology
analysis.
a positive association between baseline haematocrit and
Overall, the current study indicates that AR CAG does
change in haematocrit in the testosterone group and
influence the clinical response to TRT. This provides a
negative association in the placebo group indicates a clear
potential rationale to alter target treatment levels for
influence of baseline haematocrit in response to testoster-
patients on testosterone depending on AR CAG, although
one such that individuals with relatively high haematocrit
our data is not sufficient to justify such an approach.
levels at the start of TRT are more likely to have further
Similar analysis of data from other clinical trials would be
increases during treatment. This is an important clinical
useful to further assess this. Incorporation of this
practise point which should heighten the treating
pharmacogenetic approach into the power calculation
physician’s awareness that secondary polycythaemia is
for study size determination will be essential to optimise
more likely to occur in this group of patients. In the case
the data from such studies. Trials using long-acting
of PSA, the negative association of baseline result with
preparations such as intramuscular testosterone unde-
change in PSA, which is not replicated in the control
canoate would seem most appropriate.
group, suggests that those men with low PSA at baseline
are most likely to have larger rises in PSA with testosterone
treatment and this is counterintuitive.
Supplementary data
Measurement of AR CAG with a view to this analysis
This is linked to the online version of the paper at http://dx.doi.org/10.1530/
was included in the initial study protocol for the TIMES2 EJE-13-0703.
study, but the power calculation undertaken to determine
the required group size for the TIMES2 study was on the
basis of the primary outcome variable in the main study – Declaration of interest
change in HOMA-IR with 6 months TRT vs placebo. As T H Jones was deputy chief investigator for the TIMES2 and served as a
consultant to Prostrakan and received research grant support for the CAG
such the study was not adequately powered to allow
polymorphism analysis.
optimal analysis of the pharmacogenetic effects of AR CAG
in testosterone-treated individuals. As such, there is a high
chance of statistical type II error – a strong possibility that
Funding
our study has failed to detect some associations that T H Jones has received research grants and support, consulting fees, travel
should be present. In order to limit the risk of type II error, grants and honoraria for educational lectures from Bayer HealthCare and
honoraria from advisory boards and educational lectures from Lilly, Ferring
we have not applied statistical methods to allow for
and Clarus Pharmaceuticals. The TIMES2 study was sponsored and funded
multiple testing and this in turn also leads to an increased by Prostrakan. Prostrakan provided additional financial support for the
risk of type I error – the possibility that we have detected analysis of the CAG repeat polymorphism.
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bone density and bone metabolism in healthy males. Clinical
Author contribution statement Endocrinology 2001 55 649–657. (doi:10.1046/j.1365-2265.2001.01391.x)
T H Jones and K S Channer conceived and designed the TIMES2 protocol 11 Harkonen K, Huhtaniemi I, Makinen J, Hubler D, Irjala K,
including the sub-analysis of the CAG repeat polymorphism. S Akhtar Koskenvuo M, Oettel M, Raitakari O, Saad F & Pollanen P. The
performed the genetic study to derive AR CAG in all patients. Data and polymorphic androgen receptor gene CAG repeat, pituitary–testicular
statistical analysis was conducted by R D Stanworth who also wrote function and andropausal symptoms in ageing men. International
the paper. Journal of Andrology 2003 26 187–194. (doi:10.1046/j.1365-2605.2003.
00415.x)
12 Zitzmann M, Gromoll J, von Eckardstein A & Nieschlag E. The CAG
repeat polymorphism in the androgen receptor gene modulates body
Acknowledgements
fat mass and serum concentrations of leptin and insulin in men.
Many thanks to Prof. Stephen Walters at SCHARR for help with statistics.
Diabetologia 2003 46 31–39.
13 Stanworth RD, Kapoor D, Channer KS & Jones TH. Androgen
receptor CAG repeat polymorphism is associated with serum tes-
tosterone levels, obesity and serum leptin in men with type 2 diabetes.
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Received 14 February 2013
Accepted 28 October 2013
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