body compositional and cardiometabolic effects of testosterone

1

Body compositional and cardiometabolic effects of testosterone therapy in obese men

with severe obstructive sleep apnea: a randomised placebo-controlled trial

Camilla M Hoyos1 BAppSc (Hons), MPH (Hons), PhD, Brendon J Yee1,2,3 MBChB,

FRACP, PhD, Craig L Phillips1,4 PhD, Elizabeth A Machan1 BAppSc (Hons), Ronald R

Grunstein2,3, MB BS, FRACP, MD, PhD, Peter Y Liu1,5 MB BS (Hons), FRACP, PhD*.

Endocrine and Cardiometabolic Research Group1 and Sleep and Circadian Research Group2, NHMRC Centre for Integrated Research and Understanding of Sleep (CIRUS) Woolcock Institute of Medical Research, University of Sydney and Royal Prince Alfred Hospital3 and Royal North Shore Hospital4 and Division of Endocrinology, Department of Medicine, David Geffen School of Medicine at UCLA, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute

5

Word Count=3847

Running Title: Testosterone on Cardiometabolic Health

Keywords: Androgen Deficiency; Testosterone supplementation; Obstructive Sleep Apnea; Body

Composition; Cardiometabolic Health

*To whom correspondence should be addressed

Peter Y. Liu, Division of Endocrinology, Department of Medicine, David Geffen School of Medicine

at UCLA, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, 1000 W

Carson Street Torrance CA 90502

Tel: +1-310-222-1867

Fax: +1-310-533-0627

[email protected]

of 31 Accepted Preprint first posted on 30 July 2012 as Manuscript EJE-12-0525

Copyright © 2012 European Society of Endocrinology.

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Abstract

Background: The combination of male gender, obstructive sleep apnea (OSA) and obesity

magnifies cardiometabolic risk. There has been no systematic study evaluating whether

testosterone therapy can improve cardiometabolic health in obese men with OSA by

improving body composition, visceral abdominal fat and insulin sensitivity.

Objective: To assess body compositional and cardiometabolic effects of testosterone

treatment in obese men with severe OSA.

Design: An 18-week randomized, double blind, placebo-controlled, parallel group trial in 67

men.

Methods: Participants (age=49 ± 12years, apnea hypopnea index (AHI) =39.9 ± 17.7

events/hour, body mass index=31.3 ± 5.2 kg/m2) were placed on a hypocaloric diet and

received intramuscular injections of either 1000mg testosterone undecanoate (n=33) or

placebo (n=34) for 18 weeks. Outcomes were the changes in body composition (total muscle

mass; total and abdominal fat (total body dual-energy X-ray absorptiometry and

computerised tomography), weight, insulin sensitivity (homeostasis model assessment),

abdominal liver fat (computerised tomography), arterial stiffness (pulse wave analysis),

resting metabolic rate and respiratory quotient (indirect calorimetry), blood lipids and

metabolic syndrome from baseline to week 18.

Results: After 18 weeks, testosterone treatment increased insulin sensitivity (-1.14 units, -

2.27 to -0.01 95%CI, p<0.05), reduced liver fat (0.09 Hounsfield attenuation ratio, 0.009 to

0.17 95%CI, p=0.03) and increased muscle mass (1.6kg, 0.69 to 2.5 95%CI, p=0.0009) to a

greater extent than placebo. Other measures of body composition and regional adiposity as

well as the number of participants with metabolic syndrome did not change. Testosterone also

decreased arterial stiffness (augmentation index) by 3.2% (-6.01% to -0.46% 95%CI, p=0.02)

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and decreased the respiratory quotient (-0.04, -0.08 to -0.001 95%CI, p=0.04) after 18 weeks

compared to placebo.

Conclusion: Eighteen weeks of testosterone therapy in obese men with OSA improved

several important cardiometabolic parameters but did not differentially reduce overall weight

or the metabolic syndrome. Longer term studies are required.

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Introduction

Obesity occurs in 32% of all men in the USA 1 and many of these men develop obstructive

sleep apnea (OSA) 2, 3. The resulting intermittent hypoxia amplifies the cardiometabolic risk

of obesity by promoting atherogenesis through mechanisms that include inhibition of

triglyceride clearance, inactivation of lipoprotein lipase and induction of insulin resistance 4, 5

6. Indeed, all cause 7, 8 and cardiovascular 8, 9 mortality is amplified when untreated OSA is

combined with coexisting obesity and male gender . Understanding the mechanisms and

methods by which cardiometabolic risk may be reduced in this specific obese population is

therefore important.

Testosterone treatment increases muscle and decreases fat in hypogonadal and eugonadal

males 10, as well as in men with relative androgen deficiency related to age 11 or HIV 12. In

addition, testosterone therapy decreases visceral abdominal fat (VAF) 13-15

and improves

insulin sensitivity 16-20 in some, but not all 21-26 studies. Specific characteristics of the

populations studied may contribute to these discrepancies. Improved body composition

would be expected to improve cardiometabolic health including insulin sensitivity, VAF,

non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome. No single study has

assessed the effect of testosterone on both radiographically measured VAF and liver fat 16

.

Few studies have assessed the impact of testosterone therapy on the metabolic syndrome 16.

Conversely, biochemical androgen deficiency is associated with increased cardiovascular

events 26 and testosterone therapy has been shown to improve arterial stiffness, an early

marker of atherosclerosis, in men with coronary artery disease or advanced age 27, 28.

Biochemical androgen deficiency is common in men with OSA 29, 30

, is more severe with

increasing hypoxemia and can be partly reversed by continuous positive airway pressure

therapy 31. Biochemical androgen deficiency is also common in obese men 29, especially with

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increasing adiposity 32

and can be partly reversed with weight loss 33, 34

. These data suggest

that men who are obese or who have OSA have some degree of relative androgen deficiency

29. Whether testosterone therapy is beneficial in these men requires direct evaluation by

randomised placebo controlled trials in this population.

Prior research has suggested that testosterone therapy may improve cardiometabolic risk in

older men with chronic heart failure 18

, abdominally obese men 14, 15

, and men with the

metabolic syndrome and/or diabetes 16, 17, 20, 35, 36, but may increase cardiovascular risk in frail

older men with limited mobility 37. Since the effect of testosterone therapy on

cardiometabolic risk has not been systematically studied in obese men with OSA who are at

magnified cardiometabolic risk and who have some degree of relative androgen deficiency,

we undertook a randomised placebo-controlled trial to directly determine this. We measured

change in anthropometry, abdominal visceral fat, total body fat and lean muscle, basal

metabolic rate, insulin sensitivity, blood lipids, and metabolic syndrome status. These

assessments included more precise measures of cardiometabolic risk including

radiographically determined liver fat and tonometry determined arterial stiffness. The effects

of testosterone therapy on sleep and breathing in this study have recently been published 38

.

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Subjects and Methods

Participants and Study Design

The entry criteria has been previously described in our report of respiratory effects 38. In

brief eligible participants were adult obese men with OSA, defined as aged 18 years or over,

body mass index >30kg/m2 and apnea hypopnea index (AHI) >10 events/hr by in-laboratory

polysomnography.

This was a randomised double-blind, placebo controlled, parallel group study. Eligible

participants were randomly assigned to receive intramuscular injections of either Reandron™

(Testosterone undecanoate 1000mg in 4ml castor oil vehicle, Bayer Schering) or 4ml oil

vehicle placebo at 0, 6 and 12 weeks. All participants were concurrently enrolled in a

lifestyle modification program in which a dietitian prescribed a 2500kJ (600kcal) daily deficit

diet based on a macronutrient content of <30% fat (with emphasis on reducing saturated fat in

the diet), 15% protein, and at least 55% carbohydrate. Advice on behavioural modification

and physical activity was also provided 38. At randomisation, subjects received a number in

ascending order that corresponded with a pre-numbered and pre-packaged treatment kit. A

computer-generated randomisation list was created using a block size of four. Subjects were

assigned to active medication or placebo in a 1:1 ratio. All researchers involved in the

conduct of the study were blinded to the treatment allocation for the duration of the study.

All subjects provided written informed consent and both the patient information sheet and

study protocol were approved by the Sydney South West Area Health Service Human

Research and Ethics Committee (Royal Prince Alfred Hospital Zone). The study is registered

with the Australia New Zealand Clinical Trials Network, number ACTRN12606000404527.

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Measurements

Anthropometry

Height and weight as well as waist, hip, neck, mid-arm and mid-thigh circumferences were

measured according to anthropometric recommendations 39 at each visit by a single observer.

Imaging

Body composition included measures of abdominal, liver and total fat and total lean muscle

mass by computerised tomography (CT) and dual-energy X-ray absorptiometry (DXA)

scanning which were performed at 0 and 18 weeks. Two 10mm contiguous images of both

the upper (T12) and lower (L4) abdomen were obtained by CT (16 slice light speed extra, GE

healthcare, Madison, Wisconsin, USA). The volumes of subcutaneous and visceral abdominal

fat (SCAF and VAF, respectively) in the abdomen were quantified using a validated software

tool (Hippofat™; CNR Institute of Clinical Physiology, Pisa, Italy) 40. Total fat and lean mass

were measured by total body DXA (Lunar Prodigy GE healthcare, Madison, Wisconsin,

USA).

Liver fat was estimated by liver and spleen Hounsfield unit attenuations (E-FILM™, Merge

Healthcare Milwaukee, Wisconsin, USA) from unenhanced CT slices in several homogenous

areas (≥ 10 cm2) that were adjacent to the T12/ L1 intervertebral space and free of

vasculature. The liver to spleen attenuation ratio (L/S ratio) was used to estimate liver fat 41.

A higher ratio indicates a lower liver fat content. Arterial stiffness and central blood pressure

were measured at 0, 6 and 18 weeks in the supine position by pulse wave analysis (PWA,

Sphymo-Cor; AtCor Medical; Sydney, NSW, Australia), a method that our group has

previously implemented in another study 41. A validated transfer algorithm was used to

calculate the aortic augmentation index (AIx), which is the ratio of the augmentation pressure

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(due to the reflected component of the pulse pressure wave) to pulse pressure, expressed as a

percentage. Resting metabolic rate and the respiratory quotient were determined by indirect

calorimetry (Delta Trak II Metabolic Monitor, Datex-Ohmeda Inc., Madison, Wisconsin,

USA).

Blood

Fasting venous blood was collected in the early morning at each visit. Standard biochemistry

and hematology panels, insulin, leptin, sex hormone-binding globulin and prostate-specific

antigen were all analysed by platform assays while low-density lipoprotein (LDL)-cholesterol

was calculated by the Friedewald equation 42. Blood glucose was determined by the

hexokinase method (Modular P®, Roche Diagnostics, Castle Hill, Australia) from blood

collected in sodium fluoride (Vacutainer; Becton Dickinson, Rutherford, NJ). Insulin

sensitivity was calculated by the homeostasis model assessment (HOMA-IR) method 43.

Luteinizing hormone and follicle-stimulating hormone concentrations were measured by

commercially available Delfia assays (Perkin-Elmer Life Sciences, Rowville, Australia) and

total testosterone by mass spectrometry (API-5000 triple-quadruple; Applied

Biosystems/MDS SCIEX, Ontario, Canada). Free testosterone was calculated using a mass

equation 44. Within assay coefficients of variation were less than 10% for all assays.

Determination of Metabolic Syndrome and Other Measurements

The metabolic syndrome was defined according to international consensus guidelines 45 and

the National Cholesterol Education Program’s Adult Treatment Program Panel Criteria III

using the previously described anthropometric, blood pressure and blood parameters 46

. A

score was given to each individual criterion and then summed together. Metabolic syndrome

was defined by a total score ≥3. At all visits physical activity was assessed by the physical

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activity scale for the elderly questionnaire and handgrip strength by dynamometer (Jamar, JA

Preston Co. Jackson, MI) as previously described 47. Lower urinary tract symptoms were

assessed at the same time by International Prostate Symptom Score 48. At each visit subjects

were asked to report adverse events, including injection-related adverse events such as

bruising, stinging or pain. Adverse events were categorized according to the Medical

Dictionary for Regulatory Activities (MedDRA) System Organ Class classification.

Statistical Analysis

The randomisation code was not broken until all data were collected and the database cleaned

and locked. The outcome variables for continuous variables were the calculated differences

from baseline at 6, 12 and 18 weeks. The between-group differences of all changes were

assessed by linear regression. Outcomes with repeated measures were assessed by mixed

model analyses and tested treatment, time and the interaction of treatment and time. Analyses

were adjusted by factors that, despite randomisation, significantly differed between groups at

baseline and were also a priori confounders of the outcome of interest. Normality of residuals

was confirmed.

The presence (or absence) of metabolic syndrome was assessed at every visit. The change in

the proportion of participants with or without the metabolic syndrome from baseline was

analysed separately at each subsequent visit by examining the treatment by syndrome

interaction. Repeated measurements were modelled in these analyses which utilised

generalised estimating equations which were then confirmed by Bayesian methods.

Additional analysis explored the influence of baseline testosterone on treatment effects in

order to assess potential thresholds of relative androgen deficiency which may identify

subgroups in whom testosterone may have enhanced effects. Baseline total and free

testosterone were included in separate regression models as a dichotomised factor using

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predefined cut points for total testosterone (8, 11 and 13nmol/L) and free testosterone (160,

220 and 280pmol/L) recently shown to be useful in the definition of late-onset hypogonadism

32, which included the definition stated in the current guidelines (total testosterone, 8nmol/L)

49. The statistical significance of the interaction terms of treatment and each dichotomised

variable were examined.

Analyses were performed using SAS version 9.2 (SAS Institute, Cary, North Carolina). Data

were considered significantly different at p<0.05 (two sided) and are presented as mean

differences ± standard errors, mean ± standard deviation or mean (95% CI), as indicated.

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Results

Eighty-one men were screened of whom 67 were randomised to receive either testosterone

(n=33) or matching placebo (n=34). Thirteen participants discontinued leaving 26 men in the

testosterone group and 28 men in the placebo group who completed the entire 18-week

treatment period. The flow of participants through the study has been previously published 38.

Baseline characteristics were comparable except testosterone-treated men had lower waist

circumference (table 1). Pre-existing cardiovascular disease was reported in 3 participants:

acute myocardial infarction (n=1, placebo), rheumatic valvular disease (n=1, placebo) and

transient ischemic attack (n=1, testosterone). There were no differences in the use of lipid-

lowering medications between the groups at baseline (table 1). All participants remained on

these medications for the duration of the study and no other participants started lipid-

lowering treatment during the course of the study. Medical Dictionary for Regulatory

Activities (MedDRA) defined adverse events were similar between the two groups (table 2).

Hormones and cardiometabolic effects

Testosterone therapy increased blood testosterone concentrations and suppressed

gonadotropins, often to the detection limit of the assay (table 3). Arterial stiffness (i.e.

augmentation index) was significantly less in the testosterone treated group compared with

the placebo group overall (table 3) and at 18 weeks (figure 1A). Central augmentation

pressure was also improved after testosterone compared to placebo (table 3). There were no

changes in heart rate, systolic or diastolic blood pressure in either group after 18 weeks of

treatment (table 3).

Testosterone treatment decreased insulin resistance (HOMA-IR) (figure 1B) and liver fat

(figure 1C) compared to placebo (table 3). Testosterone significantly increased LDL-

cholesterol compared to placebo (table 3). There were no changes in any other blood lipids,

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the total to high-density lipoprotein (HDL) cholesterol ratio or liver transaminases (table 3).

The respiratory quotient significantly decreased with testosterone compared to placebo

treatment indicating a shift to fat, rather than carbohydrate, utilisation (table 3). However

resting energy expenditure did not significantly improve more with testosterone than with

placebo (table 3). Testosterone therapy did not significantly alter the net development of

metabolic syndrome at week 6 (p=0.93), week 12 (p=0.33) nor week 18 (p=0.96) using a

consensus definition of metabolic syndrome 45. Analogous findings were obtained using an

alternate definition of metabolic syndrome 46.

Body Composition

Weight, body mass index and waist circumference as well as total fat, SCAF and VAF

significantly decreased over time due to the weight-loss program (p<0.0001), but there were

no significant differences between groups at any single timepoint or overall (table 3). In

contrast, testosterone therapy significantly increased lean muscle mass, compared with

placebo (figure 1D and table 3). There were no significant between-group differences in any

other anthropometric measurement ( table 3).

Prostate and hematological outcomes

Testosterone increased serum prostate-specific antigen and hematocrit compared to placebo,

but no participant met predefined withdrawal criteria and there was no change in self-

reported prostate symptoms in either group (table 3). Hemoglobin also increased with

testosterone compared to placebo (table 3). There were no other significant between-group

differences in blood hematology or other routine tests of renal and liver function (data not

shown).

Other efficacy outcomes and exploratory analysis

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Dominant and non-dominant hand strength increased with time (both p<0.05) but there were

no differences between groups (table 3). Physical activity was significantly increased

compared with placebo at week 6 (p=0.03); however there was no overall between-group

difference (table 3).

Exploratory analyses using total testosterone dichotomised by cutpoints of 8, 11 and 13 nM

and free testosterone dichotomised by thresholds of 160, 220 and 280pmol/L were performed

and the interaction between treatment and the dichotomised variable examined. The

interaction term of treatment and any dichotomised factor for testosterone were not

statistically significant for any measures of body composition. This indicates that the

testosterone treatment effects on body composition were not influenced by baseline

testosterone concentrations.

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Discussion

This is the first randomised placebo controlled study to examine the body compositional and

cardiometabolic effects of testosterone treatment in obese men with OSA. Indeed only two

previous studies, one of which we conducted, have examined pharmacological methods to

alter body composition in men with OSA 50, 51 and neither study was randomised nor

contained a no-treatment control. This study therefore adds to the very limited literature

examining mechanisms and methods by which cardiometabolic health and body composition

can be altered in men with OSA. We demonstrate that 18 weeks of testosterone therapy does

not further reduce weight, BMI or metabolic syndrome but does improve several important

cardiometabolic risk factors as well as lean muscle mass and respiratory quotient, in obese

men with OSA. This is a specific population in whom both relative androgen deficiency and

increased cardiometabolic risk have been documented. Recently published data from this

study has shown that testosterone mildly worsens sleep-disordered breathing in a time-limited

manner, irrespective of initial testosterone concentrations 38.

We demonstrate that testosterone therapy reduces liver fat and increases insulin sensitivity in

obese men with OSA. These changes in liver fat or insulin sensitivity should independently

decrease cardiovascular risk 52

. To our knowledge this is the first study of testosterone

therapy to accurately measure liver fat by CT imaging although there are now ongoing

studies examining the effect of transdermal testosterone treatment on liver fat in

hypogonadal men with (clinicaltrials.gov number: NCT01560546) and without diabetes

(NCT00700024). Transaminases did not change after testosterone treatment which is in

agreement with a previous 6 month randomised controlled trial in older men 53

although a

non-controlled study did find significant reduction in transaminases after 1 year 54. Imaging

techniques for the quantification of liver fat are sensitive and validated methods whereas

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transaminases may not detect changes in liver fat and can be influenced by a variety of

biological circumstances 55. Previous randomised placebo-controlled studies show that

testosterone increases insulin sensitivity in 3 populations of men: eugonadal abdominally

obese men, by euglycemic hyperinsulinemic clamp 13-15; biochemically androgen deficient

men with type II diabetes and/or the metabolic syndrome, by HOMA-IR 17, 35, 56-58 including a

meta-analysis of men with the metabolic syndrome 20

, and; elderly patients with chronic heart

failure, by HOMA-IR 18, 59. Another meta-analysis of diabetic men reported significant

reductions in HbA1c, fasting glucose and triglycerides, which may have resulted in an

improvement in insulin resistance although this was not directly assessed 36. Over the longer

term, these changes would be expected to reverse metabolic syndrome and NAFLD, although

we did not show a reduction in metabolic syndrome after 18 weeks. Only two other

randomised placebo controlled trials of testosterone therapy have examined effects on

metabolic syndrome, albeit in non-OSA populations 57, 58. Both reported reductions in

metabolic syndrome but were of longer-duration (1-2 years) and both specifically recruited

men with metabolic syndrome. However no studies thus far have targeted reversal of NAFLD

despite an independent association between low testosterone levels and hepatic steatosis, a

marker for NAFLD 60, 61. The prevalence of NAFLD is increasing particularity in patients

with cardio-metabolic risk factors and is associated with increased risk of liver damage and

possibly future CVD 52, 62 . Therefore a reduction in NAFLD by lowering liver fat could have

an important impact on disease burden. Our findings in conjunction with analogous data in

other populations suggest the utility of performing a longer term study.

Other cardiovascular risk factors were also generally improved after testosterone

supplementation. Arterial stiffness as measured by the augmentation index was significantly

reduced in the testosterone group compared with the placebo group. Similar findings have

been reported in men with chronic heart failure and low testosterone levels 27

and in men with

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acquired hypogonadism 28

. Augmentation pressure, an additional marker of arterial stiffness,

was also significantly improved at 18 weeks in the testosterone group compared to placebo.

These measures are validated predictors of cardiovascular risk 63. Testosterone therapy also

significantly altered substrate utilisation to favour oxidation of fat, rather than carbohydrate,

as shown by a significant reduction in the respiratory quotient. Such changes should promote

fat loss if persistent over the longer term, although this was not observed within the 18 week

treatment period. There was no change in the total to HDL cholesterol ratio, a standard

measure of coronary heart disease risk 64, although a small increase of uncertain clinical

relevance in LDL cholesterol was observed. In other studies, testosterone therapy has been

reported to either decrease 65, 66 or not change LDL cholesterol 53, 56, 67-69. There were no

changes to lipid-lowering therapy during the course of the study suggesting that the changes

in LDL cholesterol were not as a result of alterations in concomitant medications.

On balance, the improvements in insulin sensitivity, liver fat and arterial stiffness would

seem to outweigh the possible changes in LDL cholesterol, suggesting that testosterone

therapy generally improves cardiometabolic risk in obese men with OSA. Whether this

improvement persists beyond 18 weeks would need to be verified with longer-term studies.

Furthermore, the factors which ultimately prove to be decisive in modulating whether or not

cardiovascular events occur in response to testosterone therapy have not yet been determined,

but advanced age and immobility are two possibilities 37. However, baseline testosterone

concentrations do not appear to be relevant in this population. Such an analysis was possible

because participants were specifically selected to have both obesity and OSA, and therefore

to have relative androgen deficiency 29, rather than recruited on the basis of baseline

testosterone concentrations.

Testosterone increased lean muscle mass in this population which is consistent with previous

studies in both eugonadal and hypogonadal men 10, 11

. We did not observe a further reduction

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in total fat mass in contrast with previous randomised controlled studies, but many of these

were not performed in obese populations and therefore did not include lifestyle modification

10, 11. Indeed, the differential effect on muscle and fat we observed could also be explained by

the lifestyle modification program administered which was focussed on diet rather than

exercise. Furthermore, since chronic intermittent hypoxia directly influences the uptake of

triglycerides in animal models 5, testosterone effects on fat mass may differ between those

with and without OSA.

Unsurprisingly we did not detect a significant reduction in total weight compared with

placebo since testosterone would increase muscle and reduce fat resulting in attenuated net

changes. This finding is consistent with most 14, 15, 23, 70

but not all 35

previous randomised

controlled studies. We did not observe a reduction in VAF with testosterone compared to

placebo which may also be explained by the lifestyle modification program administered to

all participants and/or the influence of chronic intermittent hypoxia. Furthermore, this finding

is consistent with the hypothesis that changes in insulin sensitivity occur before changes in

VAF 15

. Nevertheless, previous randomised placebo-controlled studies using computer

imaging to estimate VAF have generally shown no change in VAF after 6-24 months of

testosterone treatment 21-24. One notable exception is a study of non-obese older men with age

related hypogonadism which reported a between-group reduction in VAF that was driven by

an increase in the placebo group 70. Testosterone has also been shown to reduce VAF in

abdominally obese men with low to normal testosterone levels after 8 and 9 months

treatment, despite no changes in any other body composition measurement including total

lean mass 13-15. It could be that abdominally obese men may be more susceptible to changes

in VAF compared to our population who were selected for generalised obesity as defined by

a BMI≥30kg/m2.

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It is currently unclear whether or not low testosterone levels are causally related to CVD or

whether it is simply a marker of illness 71, 72. In this study of obese men with OSA, a

population who may be at a greater risk of CVD, testosterone therapy improved some

markers of cardiometabolic health despite no reduction in weight, fat mass or OSA

parameters. However longer-term studies of the effect of testosterone therapy of hard

cardiometabolic endpoints in this particular population are required for confirmation.

There are some possible limitations that should be acknowledged. The duration of

testosterone treatment may have been too short to result in changes in some of the outcomes

measures 73. As all the participants received a weight loss program, we cannot extrapolate

these findings to men not receiving a weight loss program. However, all obese men with

OSA (such as ours) or without OSA ought to be placed on a weight loss program, so our

scientific study design appeared to be the most appropriate to answer a pragmatic clinical

question. Our findings also pertain to the use of intramuscular testosterone undecanoate.

Different modes of administration may have different results 74. Finally the cut points used to

examine the influence of baseline testosterone levels on the effects treatment on body

composition were developed for late-onset hypogonadism, not necessarily for obese middle-

aged men with OSA, nevertheless these cut points seemed to be a reasonable starting point.

The role of testosterone replacement in obese men with OSA in clinical practice needs to

consider all potential risks and benefits, including prostate, hematological and respiratory

effects 38, 75. Nevertheless, this mechanistic report of solely cardiometabolic effects show that

18 weeks of testosterone therapy may potentially improve certain important parameters of

cardiometabolic health and provides a basis on which future larger and longer term studies

can be implemented.

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Acknowledgements

We thank Roo Killick, Kerri Melehan, Farid Saad and the Data and Safety Monitoring

Committee which consisted of Professor Michael Hensley (chair), Associate Professor Val

Gebski and Professor Stephen Twigg. We also thank the men who participated in the study,

the sleep physicians and the study co-ordinators.

Conflicts of Interest

There is no conflict of interest to disclose.

Role of the funding source

Supported by the National Health and Medical Research Council of Australia (NHMRC)

through a project grant (512499), a Centre for Clinical Research Excellence in

Interdisciplinary Sleep Health (571421) and fellowships to CH, CP, RRG and PYL (512057,

571179, 202916 and 1025248, respectively). Bayer Schering supplied study drug, matching

placebo and $20,000 to date.

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67. Kenny AM, Prestwood KM, Gruman CA, Fabregas G, Biskup B & Mansoor G. Effects of transdermal testosterone on lipids and vascular reactivity in older men with low bioavailable testosterone levels. J

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& Berman N. Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Journal of Clinical Endocrinology & Metabolism 2000

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Figure Legend

Figure 1. Changes from baseline in efficacy outcomes. Data are mean and SEM of the change from baseline.

Overall between group differences are indicated by the p value, determined by linear regression and adjusted

for baseline waist circumference.

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Table 1. Baseline participant characteristics (mean±SD).

Testosterone (n=33)

Placebo (n=34)

p value*

Age (years) 48 ± 1.6 49 ± 1.6 0.50

Hormonal

LH (U/L) 3.3 ± 2.2 3.4 ± 1.4 0.82

FSH (U/L) 4.3 ± 3.7 4.5±2.5 0.76

Testosterone (nmol/L) 13.2 ± 5.3 13.4±5.1 0.77

Free Testosterone (nmol/L) 0.29 ± 0.02 0.28±0.07 0.81

SHBG (nmol/L) 22.2 ± 8.0 21.9±8.6 0.90

Leptin (ng/L) 15.0 ± 8.0 17.9±10.4 0.15

Body Composition

Height (cm) 175.7 ± 6.3 177.0 ± 6.6 0.41

Weight (kg) 108.0 ± 15.3 114.7 ± 17.8 0.10

BMI (kg/m2) 34.9 ± 4.3 36.6 ± 4.9 0.15

Waist circumference (cm) 115.7 ± 8.8 120.7 ± 11.1 0.04

Hip circumference (cm) 115.8 ± 9.2 118.9 ± 10.1 0.19

Neck circumference (cm) 43.9 ± 3.0 44.1 ± 2.6 0.79

Total Lean Mass (kg) 63.1 ± 6.4 65.5 ± 7.8 0.20

Total Fat Mass (kg) 37.9 ± 8.3 40.0 ± 7.6 0.33

SCAF (cm3) 805.0 ± 273.9 868.2 ± 301.1 0.42

VAF (cm3) 367.4 ± 127.9 432.1 ± 156.1 0.10

Cardiometabolic

Metabolic Syndrome n (%) 17 (53) 15 (48) 0.80

Systolic blood pressure (mmHg) 129.1 ± 12.1 128.0 ± 14.3 0.73

Diastolic blood pressure (mmHg) 82.3 ± 11.8 82.9 ± 11.8 0.85

Heart rate (bpm) 71.4 ± 10.1 73.3 ± 8.5 0.41

AIx (%) 20.0 ± 1.5 20.5 ± 1.2 0.77

CAP (mmHg) 7.4 ± 0.7 7.1 ± 0.6 0.78

PPA 1.3 ± 0.1 1.3 ± 0.02 0.87

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Testosterone (n=33)

Placebo (n=34)

p value*

REE (kcal) 0.85 ± 0.05 0.83 ± 0.06 0.17

RQ 2006.7 ± 316.7 2033.0 ± 287.2 0.73

Liver Fat (HU) 0.9 ± 0.06 0.7 ± 0.3 0.12

Insulin (µIU/mL) 105.0 ± 83.2 109.4 ± 99.4 0.84

Glucose (mmol/L) 5.5 ± 0.9 5.6 ± 1.7 0.76

HOMA-IR (unit) 3.84 ± 0.57 4.47 ± 1.01 0.59

Hyperlipidemia-no. (%) 6 (18) 4 (12) 0.51

Statin Therapy- no. (%) 5 (15) 4 (12) 0.73

Total Cholesterol (mmol/L) 5.3 ± 1.0 5.2 ± 0.9 0.71

Total Cholesterol/HDL-C ratio 4.8 ± 0.3 4.6 ± 0.2 0.68

HDL-C (mmol/L) 1.2 ± 0.3 1.2 ± 0.3 0.97

LDL-C (mmol/L) 3.2 ± 0.9 3.2 ± 0.9 0.96

Triglycerides (mmol/L) 2.0 ± 0.2 2.1 ± 0.2 0.79

AST (U/L) 26.3 ± 8.7 26.2 ± 8.6 0.55

ALT (U/L) 42.9 ± 21.2 39.9 ± 20.0 0.99

Prostate and Haematology

PSA (ng/ml) 1.0 ± 0.7 1.0 ± 0.6 0.99

IPSS (score between 1-7) 5.0 ± 0.68 4.5 ± 0.85 0.63

HCT (L/L) 0.45 ± 0.03 0.46 ± 0.03 1.0

HB (g/L) 154.7 ± 9.7 153.0 ± 8.0 0.46

Values are mean ± SD or number (percent) as specified. *The p values were calculated by Student’s t-test or

Fisher’s Exact Test as appropriate

LH=luteinizing hormone; FSH=follicle-stimulating hormone; SHBG= sex hormone-binding globulin;

BMI=body mass index; SCAF=subcutaneous abdominal fat; VAF=visceral abdominal fat; AIx=augmentation

index; CAP=central augmentation pressure; PPA=peripheral pulse amplification; REE= Resiting Energy

Expenditure; RQ=Respiratory Quotient; HU=Hounsfield units; HOMR-IR=homeostasis model assessment of

insulin resistance; HDL-C=high-density lipoprotein cholesterol; LDL-C=low-density lipoprotein cholesterol;

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3

ALT=Alanine transaminase; AST=Aspartate transaminase; PSA=prostate-specific antigen; IPSS=International

prostate symptom score; HCT=hematocrit; HB=hemoglobin

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Table 2. Adverse events

†p values from Fisher’s exact test

Adverse Event Category Number of events p value†

Testosterone

(n=33)

Placebo

(n=34)

All- n (%) 24 (73) 24 (71) 1.0

Administration site- n (%) 7 (21) 4 (12) 0.34

Cardiac- n (%) 1 (3) 0 (0) 0.49

Respiratory- n (%) 12 (36) 11 (32) 0.80

Musculoskeletal- n (%) 7 (21) 7 (21) 1.0

Renal and Urinary- n (%) 1 (3) 0 0.61

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1

Table 3. Changes from baseline in testosterone and placebo treated groups.

Unadjusted mean change

from baseline

Adjusted between-group

mean differences (95% CI)

Testosterone

(n=33)

Placebo

(n=34)

p

value*

Hormones and SHBG

LH (U/L) -2.90 0.52 -2.98 (-3.84 to -2.11) <0.0001

FSH (U/L) -3.53 0.23 -3.37 (-4.43 to -2.30) <0.0001

Testosterone (nmol/L) 3.83 0.69 5.73 (3.63 to 7.84) <0.0001

Free Testosterone

(nmol/L)

0.11 0.004 0.11 (0.06 to 0.16) <0.001

SHBG (nmol/L) 0.37 1.85 -1.35 (-3.29 to 0.59) 0.17 Leptin (ng/mL) -5.66 -4.45 -1.27 (-4.74 to 2.19) 0.47

Body Composition

Weight (kg) -1.54 -2.15 0.85 (-0.33 to 2.03) 0.16

BMI (kg/m2) -0.50 -0.71 0.30 (-0.10 to 0.69) 0.14

Waist Circumference (cm) -3.26 -3.51 0.25 (-1.27 to 1.76) 0.75

Hip Circumference (cm) -0.82 -1.42 0.61 (-1.28 to 2.49) 0.52

Waist-Hip Ratio -0.02 -0.019 -0.002 (-0.02 to 0.02) 0.81

Total Lean Mass (kg) 1.2 -0.4 1.6 (0.69 to 2.5) 0.0009

Total Fat Mass(kg) -3.1 -2.9 -0.02 (-1.4 to 1.4) 0.98 SCAF (cm3) -70.1 -48.3 -21.8 (-67.1 to 23.5) 0.34

VAF (cm3) -29.6 -44.4 23.1 (-23.4 to 70.0) 0.32

Cardiometabolic Systolic blood pressure

(mmHg)

1.7 0.5 2.3 (-3.0 to 7.6) 0.39

Diastolic blood pressure

(mmHg)

0.64 0.69 0.32 (-4.4 to 5.1) 0.89

Heart rate (bpm) -2.0 -2.6 1.3 (-3.3 to 5.8) 0.58

AIx (%) -0.79 2.20 -3.24 ( -6.01 to -0.46) 0.02

CAP (mmHg) -0.27 0.94 -1.28 (-2.56 to -0.004) <0.05

PPA 0.03 -0.02 0.04 (-0.0002 to 0.09) 0.06

REE (kcal) 81.6 24.1 42.6 (-69.3 to 154.5) 0.45 RQ -0.04 0.01 -0.04 (-0.08 to -0.001) 0.04

Liver Fat (HU) 0.11 0.05 0.09 (0.009 to 0.17) 0.03

HOMA-IR (units) -1.30 -0.37 -1.14 (-2.27 to -0.01) <0.05 Insulin (µIU/mL) -29.1 -9.33 -23.6 (-51.8 to 4.57) 0.10

Glucose (mmol/L) -0.36 -0.05 -0.22 (-0.71 to 0.26) 0.36

Total Cholesterol (mmol/L)

0.01 -0.10 0.12 (-0.14 to 0.38) 0.35

Total Cholesterol/HDL-C

ratio

0.13 0.001 0.18 (-0.14 to 0.50) 0.26

HDL-C (mmol/L) -0.06 -0.04 -0.02 (-0.09 to 0.05) 0.57

LDL-C (mmol/L) 0.21 -0.06 0.27 (0.03 to 0.54) 0.03

Triglycerides (mmol/L) -0.29 -0.16 -0.01 (-0.45 to 0.42) 0.95

ALT (U/L) -2.79 -1.14 -1.65 (-4.87 to 1.57) 0.31

AST (U/L) 1.68 1.63 0.04 (-4.20 to 4.28) 0.98

Prostate and Haematology

PSA (ng/mL) 0.21 -0.03 0.24 (0.10 to 0.38) 0.001

IPSS (score 1-7) 0.20 -0.06 0.01 (-1.63 to 1.65) 0.99

HCT (L/L) 0.03 -0.0004 0.03 (0.02 to 0.04) <0.0001

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2

*Between group differences (and 95% confidence intervals) and p values are adjusted by waist circumference.

Mean differences from baseline in the testosterone and placebo groups are unadjusted.

CI=confidence interval; LH=luteinizing hormone; FSH=follicle-stimulating hormone; SHBG=sex hormone-

binding globulin; BMI=body mass index; SCAF=subcutaneous abdominal fat; VAF=visceral abdominal fat;

AIx= augmentation Index; CAP=central augmentation pressure; PPA=pulse pressure amplification;

REE=resting energy expenditure; RQ=respiratory quotient; HU=Hounsfield units; HOMR-IR=homeostasis

model assessment of insulin resistance; HDL-C=high-density lipoprotein cholesterol; LDL-C=low-density

lipoprotein cholesterol; ALT= Alanine transaminase; AST=Aspartate transaminase; PSA=prostate-specific

antigen; IPSS=International prostate symptom score; HCT=hematocrit; HB=hemoglobin; PASE=physical

activity scale for the elderly.

HB (g/L) 6.73 0.30 6.48 (3.72 to 9.23) 0.0001

Other efficacy Outcomes

Muscle Strength Non-

Dominant Arm (kg)

1.6 0.6 0.83 (-1.19 to 2.84) 0.41

Muscle Strength Dominant

Arm (kg)

2.1 1.0 0.82 (-1.49 to 3.13) 0.48

Physical Activity (PASE

score)

43.7 6.2 -35.0 (-6.54 to 76.5) 0.10

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1

Weeks

0 18

Lean

Mass (

kg

)

-0.5

0.0

0.5

1.0

1.5D

p=0.0009

C

p=0.03

0 6 12 18

HO

MA

-IR

(u

nit

s)

-2

-1

0B

p<0.05

0 6 12 18

Au

gm

en

tati

on

In

dex

(%

)

-4

-2

0

2

4A

p=0.02

Weeks

0 18

Liv

er

Fat

(HU

)

0.00

0.05

0.10

0.15

Figure 1.

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body compositional and cardiometabolic effects of testosterone

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