Prospective, Longitudinal Study of 800 Recreational Athletes With Current and Former Use of Performance and Image Enhancing Drugs (PIED), Focusing on Androgenic Anabolic Steroids (AAS) - Rigshospitalet
Conditions: Anabolic Steroid Overdose, Infertility, Cardiovascular Complication, Illicit Drug Use, Fitness, Mental Health Issue
Sponsor: Caroline Michaela Kistorp

VEK nr. H-21035759 
Protocol version 3, 19-05-22 
Page 1 of 28 
 
STUDY PROTOCOL 
1 
Cross-sectional study of 800 recreational athletes with current and former use of performance and 
2 
image enhancing drugs (PIED), focusing on androgenic anabolic steroids (AAS) 
3 
Short title: FItness DOping in DenmarK 
4 
 
5 
Project group  
6 
- 
Caroline Kistorp1,  
7 
- 
Jon Rasmussen1,  
8 
- 
Niels H. Brandt-Jacobsen1,  
9 
- 
Yeliz Bulut1, 
10 
- 
Morten Schou2,  
11 
- 
Jan Frystyk3 
12 
- 
Michael Kjær4 
13 
 
14 
Institutions: 
15 
1Department of Medical Endocrinology, Centre of Cancer and Organ Diseases, University Hospital 
16 
of Rigshospitalet, Denmark (Main centre) 
17 
2Department of Cardiology, Herlev and Gentofte University Hospital, Denmark  
18 
3Department of Endocrinology (Body Identity Clinic), Odense University Hospital, Kløvervænget 6, 
19 
5000 Odense C, Denmark 
20 
4Department of Sports Medicine, Bispebjerg and Frederiksberg University Hospital.  
21 
 
22 
Sponsor and principal investigator 
23 
Caroline Kistorp, MD, PhD, Professor, 
24 
Department of Medical Endocrinology, Center of Cancer and Organ Disease, Rigshospitalet, 
25 
University Hospital of Copenhagen, Denmark 
26 
Ole Måløvs Vej 26, Opg. 26, 5th floor 
27 
DK-2200 Copenhagen, Denmark 
28 
Telephone: +45 22 47 56 99 / +45 35 45 96 42 
29 
Email: caroline.micheala.kistorp@regionh.dk 
30 
 
31 
Investigator 
32 
Jon Bjarke Jarløv Rasmussen, MD, PhD, Specialist registrar, Post.doc 
33 
 
34 
Collaborating institutions 
35 
- 
Body Identity Clinic (BIC), Odense, Denmark 
36 
- 
Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark 
37 
- 
Copenhagen University Hospital - Herlev and Gentofte, Copenhagen, Denmark 
38 
- 
Centre for Preventative Doping Research at the German Sport University Cologne, Germany 
39 
(Mario Thevis) 
40 
 
41 

VEK nr. H-21035759 
Protocol version 3, 19-05-22 
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Research Ethical Committee 
1 
The Regional Scientific Ethical Committee of the Region of Copenhagen 
2 
 
3 
4 

VEK nr. H-21035759 
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Content 
1 
Background .......................................................................................................................................... 6 
2 
Epidemiology of illicit use of anabolic androgenic steroids ............................................................ 6 
3 
Health-related complications of illicit AAS use .............................................................................. 6 
4 
AAS-induced male hypogonadism .................................................................................................. 6 
5 
Objective .............................................................................................................................................. 8 
6 
Study Methodology .............................................................................................................................. 9 
7 
Participants ........................................................................................................................................... 9 
8 
Inclusion criteria (AAS Cohort) ....................................................................................................... 9 
9 
Inclusion criteria (Control cohort) ................................................................................................. 10 
10 
Exclusion criteria ........................................................................................................................... 10 
11 
Procedures .......................................................................................................................................... 10 
12 
Dual x-ray absorptiometry ............................................................................................................. 11 
13 
Description of procedures .............................................................................................................. 11 
14 
Clinical examination ...................................................................................................................... 11 
15 
Office blood pressure (OBP)...................................................................................................... 11 
16 
Blood and urine sampling .............................................................................................................. 11 
17 
Prespecified routine blood analyses ........................................................................................... 11 
18 
Whole-body dual x-ray absorptiometry ......................................................................................... 12 
19 
Biobank ...................................................................................................................................... 12 
20 
Biomarker analyses by international associates ......................................................................... 12 
21 
Registry-based follow-up ............................................................................................................... 12 
22 
Extended study programmes (3 sub studies) ...................................................................................... 15 
23 
Semen samples (Extended study program 1) ................................................................................. 16 
24 

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Study visits ......................................................................................................................................... 17 
1 
Core program ................................................................................................................................. 17 
2 
Extended program 1 ....................................................................................................................... 17 
3 
Extended program 2 ....................................................................................................................... 17 
4 
Extended program 3 ....................................................................................................................... 17 
5 
Statistical considerations .................................................................................................................... 17 
6 
Sample size .................................................................................................................................... 17 
7 
Data analyses.................................................................................................................................. 19 
8 
Time schedule .................................................................................................................................... 19 
9 
Feasibility ........................................................................................................................................... 20 
10 
Data extraction from participant files and hospital records ............................................................... 20 
11 
Registry follow-up ......................................................................................................................... 21 
12 
Data protection of Personal Information in the Study ....................................................................... 21 
13 
Economy, Funding and Participant Insurance ................................................................................... 21 
14 
Recruitment of Participants and Informed Consent ........................................................................... 22 
15 
Initial contact.................................................................................................................................. 22 
16 
Scheduled meeting ......................................................................................................................... 22 
17 
Acquisition of informed consent .................................................................................................... 23 
18 
Informed consent............................................................................................................................ 23 
19 
Consent regarding core program, registry-based follow-up and research biobank ................... 23 
20 
Consent regarding extended program ........................................................................................ 23 
21 
Consent regarding establishment of a biobank of future research ............................................. 23 
22 
Risks and discomforts ........................................................................................................................ 23 
23 
Ethical considerations ........................................................................................................................ 24 
24 

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Dissemination of Results ................................................................................................................... 24 
1 
Future perspectives ............................................................................................................................ 25 
2 
Overview over appendices ........................................................... Fejl! Bogmærke er ikke defineret. 
3 
 
 
4 

VEK nr. H-21035759 
Protocol version 3, 19-05-22 
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Background  
1 
Epidemiology of illicit use of anabolic androgenic steroids  
2 
Anabolic androgenic steroids (AAS) are synthetic derivates of the primary male reproductive 
3 
hormone, testosterone, but have been chemically modified and thereby featuring a higher 
4 
anabolic/androgenic ratio, longer plasma half-life and stronger affinity for the androgen receptor [1]. 
5 
During the 1980’s, illicit AAS use was limited to elite athletes and bodybuilder communities, thereby 
6 
restricting the incidence of complications of AAS use to a small, highly selected group. But within 
7 
recent years studies suggest illicit AAS use has moved to the broader population and is now 
8 
widespread among young men and should therefore be considered as a public health concern [1]. A 
9 
meta-analysis from 2014 based on all accessible studies on illicit AAS use worldwide, estimated that 
10 
almost 20% of male recreational athletes and approximately 6% of all men worldwide have 
11 
experience with illicit AAS use [2]. Data on the prevalence of AAS use among women is virtually 
12 
non-existent.  
13 
 
14 
Health-related complications of illicit AAS use 
15 
A recent Danish register-based study noted a factor three increased risk of all-cause mortality and 
16 
hospital outpatient contacts among men who had tested positive for AAS as compared with a 
17 
randomly selected age- and sex-matched cohort [3]. Within recent years, we and others have reported 
18 
several adverse effects of AAS abuse among men that is supportive of long-term AAS complications 
19 
years after AAS cessation in terms of long-lasting male hypogonadism, impaired fertility, decreased 
20 
cardiac function as well as impaired lipid and glucose metabolism, and incipient atherosclerosis [4–
21 
9]. Furthermore, sustained AAS use has been associated with adverse behavioral consequences, 
22 
including impulsivity, aggression, and violence. AAS users may also develop a condition of aberrant 
23 
body image, often referred to as muscle dysmorphia or bigorexia. A condition defined by a warped 
24 
perception of one’s body size being smaller than what can objectively be measured. Loss of muscle 
25 
triggers anxiety and contributes to AAS dependence and relapse.  
26 
In summary, current information on adverse effects related to illicit AAS use is based on cross-
27 
sectional studies demonstrating only momentary health status among AAS users and therefore not 
28 
the true impact of illicit AAS use on health during following many years. Furthermore, studies based 
29 
on registers only provide even less valuable information since absolutely no conclusions on causality 
30 
can be drawn. Therefore, it would be of high interest to examine a large cohort of current and former 
31 
AAS users for detailed information with regular follow-up the following years to assess their health 
32 
status and potentially find screening biomarkers which could suggest development of severe health 
33 
adverse effects.  
34 
 
35 
AAS-induced male hypogonadism 
36 
Male hypogonadism is not frequently observed among younger men and has traditionally been 
37 
attributed congenital conditions such as Klinefelter and Kallmann’s syndrome or cryptorchidism 
38 
[10,11]. Interestingly, a recent US study reported approximately 55% of younger men referred to 
39 
urologic outpatient clinics with profound male hypogonadotropic hypogonadism (plasma total 
40 

VEK nr. H-21035759 
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testosterone < 1.7 nmol/L) were related to prior AAS use [11]. These findings are corroborated by a 
1 
recent Danish register-based study reporting substantially increased risk of male hypogonadism 
2 
among AAS users: 22-fold increased risk of testicular dysfunction, 2.5-fold increased risk of male 
3 
infertility, 15-fold more likely of being treated with testosterone supplementation [3]. The 
4 
mechanisms behind AAS-induced male hypogonadism relates to the hypothalamic-pituitary-gonadal 
5 
(HPG)-axis suppression after supraphysiologic plasma androgen concentrations and leads to a rapid 
6 
decline in plasma testosterone following AAS cessation [9,12] with various concomitant hypogonadal 
7 
symptoms, such as erectile dysfunction, decreased sexual desire, fatigue, depression even after 2-3 
8 
years since AAS discontinuation [9]. Indeed, from our own and others experience, many of these 
9 
patients will resume their AAS abuse due to these severe symptoms which lead to AAS-dependence 
10 
[13].  The current knowledge on impaired fertility after AAS cessation is sparse being primarily from 
11 
case reports, [14–19] although we reported higher prevalence of previous AAS abusers (median of 
12 
2.6 years since AAS cessation) with plasma inhibin B, a fertility marker, below the lower cut off level 
13 
abusers compared with non-AAS users [9]. Thus, suggesting that oligo- or azoospermia may become 
14 
permanent or long-lasting. New state-of-art methods of investigating fertility among men, such as 
15 
DNA fragmenting and acrosome reaction, could be of high interest in men with previous abuse of 
16 
AAS [20,21]. Although testosterone is presently the best biomarker for the symptoms following AAS 
17 
cessation, regarding the pleiotropic adverse effect following AAS use, and psychological well-being 
18 
especially, testosterone alone may be limited in its ability to capture important characteristics needed 
19 
to guide clinical decision-making, why further studies using a broader approach are warranted to 
20 
investigate the clinical, biomedical and physical well-being in these young individuals. 
21 
 
22 
Illicit use of AAS among women 
23 
Information on AAS use among women is very sparse. Information on prevalence, patterns of AAS 
24 
use, AAS compounds used have not been reported from systematic studies in the literature. We now 
25 
from case-reports, and from Anti Doping Denmark that illicit AAS use seems to have a masculinizing 
26 
effect in women and therefore give rise to several adverse effects such as: hirsutism, acne, irreversible 
27 
deepening of voice, absent or irregular menstrual cycle and clitoris enlargement. Nevertheless, 
28 
scientific literature on the subject is extremely limited. However, a recent minor qualitative study in 
29 
female AAS users confirmed several of the proposed side-effects. (Havnes et el. International journal 
30 
of drug policy, 2021). Thus, it would be of high interest to perform a cohort study including both 
31 
current and former female AAS users to gain information on illicit AAS use among women to develop 
32 
prevention strategies and therapy options. 
33 
 
34 
Cardiovascular disease among AAS users 
35 
An increased risk of cardiovascular disease (CVD) and impaired cardiac function has been suggested 
36 
among former male AAS abusers7,10. The precise mechanisms linking AAS with myocardial 
37 
dysfunction are unknown, however, a direct toxic effect of supraphysiologic levels of androgens 
38 
could be implicated 9.  We have previously reported in a Danish community-based cross-sectional 
39 
study that past illicit male AAS use is associated impaired left ventricular (LV) systolic function 
40 
expressed as impaired global longitudinal strain (GLS) 9. Impaired LV GLS independently predicted 
41 

VEK nr. H-21035759 
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adverse cardiovascular outcomes, including heart failure in  a low risk general population 27. By using 
1 
coronary computed tomography angiography, a US study, demonstrated higher coronary artery 
2 
plaque volume and reduced LV systolic function in AAS users than nonusers28. Interestingly, both 
3 
current and former AAS abusers exhibited higher aortic stiffness 18.  
4 
 
5 
Notably, AAS may have even worse adverse effects on the heart and metabolism among women, 
6 
since they by biology only have very low testosterone levels, and increasing knowledge from the 
7 
condition polycystic ovary syndrome, which is characterized by increased testosterone levels, we 
8 
know that these patients have a markedly increased risk of type 2 diabetes and CVD. Therefore, it 
9 
could be hypothesized that myocardial blood flow reserve will be impaired among former male and 
10 
female AAS abusers. 
11 
 
12 
Early signs of AAS-induced cardiomyopathy, such as microvascular dysfunction with reduced 
13 
myocardial perfusion can be measured as myocardial blood flow reserve (MFR) quantitatively as ml 
14 
per gram per minute, and noninvasively by cardiac positron emission tomography (PET) with 
15 
Rubidium-82 (Rb-82). Several advantages using PET with Rb-82 should be addressed, such as higher 
16 
diagnostic accuracy, improved image quality, less radiation dose to patient and staff, and especially 
17 
rapid examinations time 29, and the Rb-82 PET CT method has not previously been used among AAS 
18 
users. Therefore, it will be relevant to use this highly sensitive Rb-82 PET CT to further examine the 
19 
mechanism behind cardiovascular disease in former AAS abusers.  
20 
 
21 
Dysmetabolism among AAS users  
22 
We have previously noted a dysmetabolic state in both current and former male AAS users compared 
23 
with age-matched healthy controls (9). Interestingly, both current and former AAS users showed 
24 
increased volume of visceral adipose tissue (VAT) and impaired insulin sensitivity compared with 
25 
healthy controls [6]. Increased VAT among AAS users was associated with decreased insulin 
26 
sensitivity in a multivariate linear regression [6]. Further, a few case reports noted debut of diabetes 
27 
among current AAS users. Thus, it would be of interest to perform a dual X-ray absorption (DXA) 
28 
scan, which is a low dose x ray scan, to assess body composition, especially VAT, and to assess a 
29 
potential link with later development of fulminant metabolic syndrome including diabetes in male 
30 
and female AAS abusers.  
31 
 
32 
Objective 
33 
The overarching aim of this study is to characterize a large cohort of former and current male and 
34 
female AAS users and to identify whether certain characteristics such as duration or intensity of illicit 
35 
AAS use could play a role for development of cardiovascular disease, diabetes, male hypogonadism, 
36 
female infertility, and psychiatric disease during the following years. Furthermore, we will pay 
37 
attention to the prevalence of subclinical disease including impaired gonadal function and fertility, 
38 
psychological well-being and cardiovascular disease and the predictive value of markers subclinical 
39 
disease in terms of developing overt disease requiring therapy during the following years.  
40 

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1 
The study will especially focus on the following among current and former male and fremale illicit 
2 
AAS users:  
3 
- 
Long-term complications and outcomes related to: cardiovascular disease, diabetes, gonadal 
4 
disease (women and men) and psychiatric disease using data from Danish registries including 
5 
addressing central questions such as whether the following characteristics play a role for 
6 
development of overt disease: duration of and intensity of AAS use, increased risk of certain 
7 
AAS compounds, age when starting AAS use and other characteristics. 
8 
- 
Characterization of illicit AAS use among women: duration and intensity of AAS use, AAS 
9 
compounds used, apparent side-effects including reproductive health and hirsutism. 
10 
- 
Current male reproductive health: reproductive serum hormone levels, semen status and 
11 
erectile dysfunction and gynecomastia  
12 
- 
Current psychological well-being, aggressive tendencies, cognitive function and quality of 
13 
life 
14 
- 
Current cardiovascular and metabolic status: blood pressure, serum levels of lipids, insulin 
15 
resistance, cardiac function, body composition  
16 
 
17 
Study Methodology 
18 
The study design is a combined cross-sectional and prospective cohort study including male and 
19 
female participants with an ongoing or former illicit AAS use. The study will establish a cohort with 
20 
participants of current and former users of AAS containing clinical data, and a biobank, consisting of 
21 
blood and urine samples. Participants in the AAS cohort are to be followed-up every third year from 
22 
inclusion of the last study participants for 15 years with regard to diagnoses related to gonadal 
23 
function, mental health, cardiovascular disease and diabetes mellitus using national registry data. The 
24 
study aims at including a total of 800 participants during an inclusion period of three to four years.  
25 
To ensure a contemporary cohort of comparison (control), a concurrent, smaller cohort, including 100 
26 
healthy athletes (80 men and 20 women) without current or former AAS use, will constitute a control 
27 
cohort for in-depth analysis of clinical characteristics. Further, a larger age- and sex-matched cohort 
28 
based on national registry-data (n = 8000) will be used to assess the clinical implication of clinical 
29 
characteristics in prospective follow-up.  
30 
Participants 
31 
Participants will be recruited from the outpatient clinic at the Department of Endocrinology and from 
32 
the local community through advertisement in fitness- and weightlifting gyms and the internet: 
33 
(Social 
media 
such 
as 
:Facebook, 
Instagram, 
homepage 
of 
AntiDoping 
Denmark, 
34 
forsoegspersonen.dk). 
35 
Inclusion criteria (AAS Cohort) 
36 
 Male and female recreational athletes (≥18 years of age) with current or former illicit use (> 
37 
3 months since AAS cessation) of PIEDs (e.g. AAS) for a minimum four weeks. 
38 

VEK nr. H-21035759 
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Inclusion criteria (Control cohort) 
1 
 Male or female recreational athletes (≥18 years of age) with NO current or former illicit use 
2 
of AAS. 
3 
Exclusion criteria 
4 
 Severe psychiatric or somatic diseases which makes it impossible to give informed consent or 
5 
comply with the investigatory program. 
6 
 Known pregnancy. 
7 
Procedures 
8 
Core study programme  
9 
Participants are to undergo a semi-structured medical interview, a clinical examination, fill out 
10 
questionnaires, and be submitted to blood and urine sampling (Table 1). This core study program will 
11 
be performed during a single clinical visit. Furthermore, the participants will be presented to 
12 
participate in the extended research programs (Table 3). 
13 
Table 1: Core study program
Procedures
                                 Outcome
Semi-structured interview 
- 
Medical history, including concomitant chronic disease, medication 
and dietary supplements. 
- 
PIED history  
- 
Alcohol, tobacco and substance abuse 
- 
Fertility (characteristics of menstrual cycle) and sexual complaints 
- 
Diet and physical activity 
- 
Socioeconomic status 
Clinical examination
- 
Anthropomorphic measures (Age, height, weight, waist and hip 
circumference, body mass index (BMI, kg/m2))  
- 
Dermatology Score (Acne) 
- 
Alopecia and face and body hair (Ferriman-Gallway Score); 
(Women) 
- 
Office blood pressure (OBP) 
- 
Orchidometer to determine testicular size (Men) 
- 
Gynecomastia (Tanner-staging; Men) 
Patient reported outcome
 
- 
”Dit helbred og velbefindende” (SF-36®; Quality of life)  
- 
Inventory of Interpersonal problems ® (Interpersonal relations) 
- 
Body Q (Bodily perception) 
- 
Buss-Perry Aggression Questionnaire (BPA) (Aggressive 
tendencies) 
- 
General Anxiety Disorder – 7 (GAD 7) (Anxiety and depression) 
- 
Major Depression Index (MDI) (Depression score) 
- 
Cognitive complaints in bipolar disorder rating (COBRA) 
(Cognition)  

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- 
Sexual function (IIEF5) (Sexual function) 
- 
Inventory of interpersonal problems (IIP32) 
- 
Testosterone deficiency (ADAM) 
- 
Physical activity and sports injury questionnaire 
Blood and urine samples
(please see Table 2 for details) 
Routine biochemical panel
Biomarkers of: 
- 
Organ function (TAME biomarkers) 
- 
Hormone function (Hormones and their binding proteins) 
- 
Adipose tissue function (derived hormones and inflammation 
markers) 
- 
Endothelial function 
- 
Extracellular matrix 
- 
Skeletal muscles growth 
- 
Hemostatic system 
- 
Circulating micro-RNA 
- 
AAS derivatives in urine 
Extra biological material acquired will constitute a research biobank of 
future research. (See Consent regarding biobank of future research) 
Dual x-ray absorptiometry 
 
- 
Body composition including distribution of VAT 
Description of procedures 
1 
Clinical examination 
2 
The following anthropomorphic measures will be acquired for every participant: Age, height, weight, 
3 
waist and hip circumference, body mass index (BMI, kg/m2). Acne is estimated by dermatology 
4 
score. Testicular size is measured using an orchidometer and gynecomastia is estimated using the 
5 
Tanner score (men). Alopecia and face and body hair are scored using the Ferriman-Gallway Score 
6 
(women). 
7 
Office blood pressure (OBP) 
8 
An automatic, oscillometric apparatus is used to measure blood pressure on the non-dominant arm of 
9 
the participant after a predefined period of rest (above 5 min). The measurement will be repeated two 
10 
times divided by a minute of additional rest. 
11 
Blood and urine sampling 
12 
Venous blood will be drawn through a cannula in antecubital vein. Urine sampling will be acquired 
13 
by participants providing a fresh urine-sample performed on-site supervised by one of the 
14 
investigators. A research biobank will be established for blood and urine for analysis not performed 
15 
immediately. A total of 300 ml blood and 200 mL urine will be sampled from the participants in the 
16 
study. 
17 
Prespecified routine blood analyses 
18 
The routine blood panel analyses are performed at the local hospital laboratory immediately after 
19 
acquisition (Table 2). Surplus biological material from samples sent for the routine analyses will be 
20 
destroyed following analyses.  
21 

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Whole-body dual x-ray absorptiometry 
1 
Full body dual x-ray absorptiometry (DXA - lumbar spine, hip, and whole body) is used to measure 
2 
body composition including VAT and bone density. Radiation amounts to 0.004 mSv (See: Ethical 
3 
considerations). The estimated examination time is 5 minutes. 
4 
 
5 
Biobank  
6 
As part of the protocol a subset of samples are frozen as part of a biobank, where extra material, if 
7 
any, acquired will constitute a biobank of future research. The biobank will be used to perform 
8 
measurements according to the prespecified list of biomarkers from the protocol. The biobank of 
9 
future research will consist of the extra material acquired at the scheduled visit (See Consent 
10 
regarding biobank of future research).  
11 
Blood and urine samples will be frozen and stored in a secure freezer at Rigshospitalet, University 
12 
Hospital. Samples will be stored in aliquots at -80°C as part of the abovementioned biobank, 
13 
established at the Department of Endocrinology, Rigshospitalet according to specifications in this 
14 
protocol. Samples will be stored as pseudo-anonymized form until use. The purpose of the  biobank 
15 
is to be able to perform analyses in one assay batch, whereby assay variation is minimized and a more 
16 
exact analysis is achieved.  
17 
After the prespecified analyses in the protocol, any material remaining in the  biobank will stored in 
18 
a biobank for future research until the completion of register-based follow-up of specified in the study 
19 
and then destroyed, however, the samples will at most be stored for 15 years. If biomarkers not pre-
20 
specified are to be analysed, the investigators will apply VEK for permission to perform these 
21 
laboratories measurements without obtaining novel informed consent from the participants.  
22 
The biobank will be registered at the National Biobank Register and will comply with the legislation 
23 
of the Danish Data Protection Agency. No analyses, besides the prespecified in this protocol, will be 
24 
performed without a new assessment and subsequent approval from the regional ethics committee on 
25 
health research. 
26 
Biomarker analyses by international associates 
27 
Regarding the urine samples, samples to be used in AAS analyses will be sent to Centre for 
28 
Preventative Doping Research at the German Sports University Cologne, Germany (B), as specified 
29 
in the protocol. The urine samples will subject to a specialized analysis for traces of AAS and PIEDs 
30 
(B). The urine samples will be pseudo-anonymized prior to shipment. The legislation and regulations 
31 
of the Danish Data Protection Agency will apply to these samples, including the Data Protection Act, 
32 
chapter V (C).  The transfer and handling of samples will follow the above stated legislation and 
33 
regulations. 
34 
Registry-based follow-up 
35 
All citizens of Denmark are given a permanent unique civil registry number allowing us to perform 
36 
individual-level linkage in nationwide registries. Data for these studies will be obtained from: 1) the 
37 
Danish Civil Registration System registry (sex, date of birth, immigration, emigration and vital 
38 
status), 2) the Danish National Patient Registry (discharge diagnoses coded according to the ICD-10 
39 
system since 1994), 3) the Danish National Prescription Registry (claimed prescriptions), 4) the 
40 
Danish National Cause of Death Registry (primary and underlying causes of death from death 
41 

VEK nr. H-21035759 
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certificates), 5) the Population’s Education Register (sociodemographic characteristics and education 
1 
level), and 6) the Danish Psychiatric Center Research Register (depression) 7) participants  hospital 
2 
records. It is essential to gain access to the participants’ hospital records since information such as 
3 
examination results (CT scans, echocardiography, ultrasound, blood samples) van verify diagnoses 
4 
given to a participant. Furthermore, examination results in the hospital record may reveal diagnoses 
5 
the participant have not formally been given but fulfill criteria for. We will seek information on 
6 
previous relevant conditions (from the 4 conditions of interest) from the hospital records as from a 
7 
maximum of 10 years before the participant entered the study.  
8 
 
9 
Registry-based prospective follow-up will be performed every third year until 15 years past initial 
10 
establishment of the cohort. This follow-up will be centered on the 4 primary conditions of interest: 
11 
reproductive health, type 2 diabetes, cardiovascular complications and mental health (depression, 
12 
anxiety, psychotic episodes, schizophrenia) which leads to subsequent hospitalization or outpatient 
13 
visits in the tertiary hospital sector, for the duration of the follow-up. Moreover, date of death and 
14 
cause will be obtained. Data on prescriptions and ICD10-diagnoses will internally validate data 
15 
registration and determine severity of underlying disease (e.g. the more severe disease, the more 
16 
aggressive the treatment is). Data extracts will be obtained for both the AAS cohort, baseline controls 
17 
and the excerpt of the general population. 
18 
Data will be kept until the end of the trial. The duration of the trial is 15 years. 
19 
The following ICD codes will be applied in the Danish National Patient registry every third 
20 
year: 
21 
Gonadal diseases: Testicular hypofunction (men) (ICD-10: DE291-C); erectile dysfunction of any 
22 
kind (men): ICD-10 codes: DN484, DF522-A; Female infertility (women) (DN970-9); Hirsutism 
23 
(women) (ICD-10: DL680) 
24 
Cardiovascular diseases: cardiac diseases including arrythmias (ICD-10: DI44- 49), ischemic 
25 
heart disease/nonfatal myocardial infarction (ICD-10: I20-I25), heart failure (ICD-10: DI110, 
26 
DI500-501, DI509, DI420-422, DI426, DI429, DJ819) and valve diseases (ICD-10: DI05, DI06, 
27 
DI34-36, DZ952); nonfatal hemorrhagic or ischemic stroke (ICD-10: DI60-64); venous 
28 
thromboembolism (ICD-10: DI26, DI74, DI81, DI646, DI801-803, DI822- 823, DI828-829); 
29 
Hypertension (ICD-10: DI10-109) 
30 
Diabetes mellitus: established diabetes of any kind including previous gestational diabetes (ICD-
31 
10: DE10, DE11, DE13, DE14, and DO244). 
32 
Mental health: depression (DF33-DF339); Psychotic episodes of any kind (DF22-DF28); Anxiety 
33 
of any kind (DF40-DF419); Schizophrenia (ICD-10 codes: DF20-DF209) 
 
34 

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Table 2: Prespecified biomarker analyses 
1 
 
Blood 
Urine 
Local routine lab, 
immediate analysis 
Hormones  
LH 
FSH 
Total T 
Free T  
SHBG 
Estradiol, 
Prolactin  TSH 
ACTH Cortisol 
Aldosterone, 
FGF23, 
Ghrelin  
TT3 
TT4 
T3R 
17OH 
Progesterone 
 
Metabolism and 
inflammation  
Lipids 
p-glucose, HbA1c 
(TAME) 
25-OHD  
NT-proBNP (TAME) 
TNT, TNI 
 
Other  
Hemoglobin Hematocrit  
Creatinine Sodium, 
Potassium, ALAT,  
GGT,  
BASF 
Ionized-Ca 
NA 
Frozen for later 
analysis in batches 
 
 
C-peptide  
Adiponectin, leptin 
sCD36 
SuPAR, IGF-II, 
IGFBPs, bioactive 
IGF- I,  
IGF activating 
enzymes and 
regulators (PAPP-A 
and PAPP-A2) 
INSL-3 
Stanniocalcins 
YKL40, 
IL6, TNFα 
Ferritin, PTH 
Inhibin B, AMH, 
TAME markers: IL-6, 
TNFα-receptor I or II, 
CRP, GDF15, fasting 
insulin, IGF1, 
cystatin C, NT-
Autoantibodies 
Myostatin, Follistatin 
Fibulin 1 
CK 
Hemostatic tests 
ACE  
White cell count, 
Albumin 
Urea 
Endocrine disruptors 
(PFAS, phthalate and 
metabolites) 
Metabolomics 
endocrine 
disruptors 
(PFAS, phthalate 
and metabolites), 
suPAR, markers 
of oxidative 
stress 
 

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proBNP, and 
hemoglobin A1c.  
Amount of 
biological material 
300 ml blood 
200 ml urine 
Extended study programmes (3 sub studies) 
1 
1) Semen sample (in total 200 current and former male AAS users and all male controls (n 
2 
= 80)) 
3 
2) GnRH and hCG stimulation tests (30 former male AAS users and 30 male controls) 
4 
3) Rubidium PET CT scan (90 men: 30 current and 30 former AAS users and 30 controls 
5 
and 60 women: 20 current, 20 former and 20 controls). 
6 
All participants in the core study program are eligible for participation the extended study 
7 
programmes. Recruitment for the extended study programmes will be in a consecutive manner, thus 
8 
all who consent to participate in the core study programme will be offered participation in the 
9 
extended study programmes until recruitment for these sub studies is complete. Participants who 
10 
choose to participate in the extended study programs will be asked to sign an additional informed 
11 
consents for each sub study Procedures related to the extended study programmes will, if possible, be 
12 
performed during the same day as the core study programme or within two weeks.  
13 
Table 3: Extended study program 1 (semen analyses)
Algorithm 
Investigation 
Outcome  
N = 280 (200 men from the 
AAS cohort included 
+ all controls) 
Semen analyses 
morphology, motility, 
volume, pH, acrosome 
reaction, DNA 
fragmentation  
Table 4: Extended study program 2 (hormonal gonadal axis)
Algorithm 
Investigation 
Outcome  
N = 60 (30 former male AAS 
users and all  
+ 30 male controls) 
GnRH and hCG stimulation tests
Serum gonadotropins and 
serum total testosterone, 
respectively   
 
 
 
 
 
Table 5: Extended study program 3 (cardiac microcirculation) 
Algorithm 
Investigation 
Outcome  

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N = 90 males (30 former and 
30 current male AAS users 
+ 30 male controls) 
 
N = 60 women (20 former 
and 20 current female AAS 
users and 20 controls 
 
 
Rubidium PET CT 
Myocardial flow reserve 
and myocardial 
microcirculation  
 
1 
 
2 
Semen samples (Extended study program 1) 
3 
Participants will provide a semen sample by masturbation in a private room at the laboratory at 
4 
Rigshospitalet following at least 48 hours of sexual abstinence. The samples will be analyzed 
5 
immediately for: sperm count, morphology, vitality, motility, pH, acrosome reaction and DNA 
6 
fragmenting. 
7 
GnRH stimulation test (Extended study program 2) 
8 
Intravenous injection with 100 mikrogram of GnRH will administered through a cannula in an 
9 
antecubital vein. Blood for analyses of FSH, LH and total testosterone (freezed for later analyses in 
10 
batches using LC-MS) will be drawn at time 0 min and +30 min. 
11 
 
12 
hCG stimulation test (Extended Study program 2) 
13 
Following GnRH test, at the same visit, an intramuscular injection with 5000 IE of hCG will be 
14 
administered intramuscularly. 72 hours after the injection, blood for analyses of serum total 
15 
testosterone (freezed for later analyzes in batches using LC-MS) will be drawn in an overnight fasting 
16 
state during morning hours. 
17 
 
18 
Ultrasound of testes (Extended Study Program 2) 
19 
Ultrasound of testes will be performed to measure size and morphology. Examination time: 5 minutes 
20 
 
21 
Rubidium-82 PET cardiac scan (Extended Study Program 3) 
22 
Rubidium-82 is a potassium analogue with a short half-life, which permits quantitative 
23 
measurements of myocardial perfusion in absolute values (mL/g/min) during stress and rest, as the 
24 
myocardial flow reserve (MFR) [37]. PET scans will be performed using a Siemens Biograph 
25 
mCT/PET 64-slice scanner (Siemens Medical Solutions, Knoxville, USA). Serial rest followed by 
26 
stress imaging with Rb-82 from a CardioGen-82 Sr-82/Rb-82 generator (Bracco Diagnostics, Inc., 
27 
Princeton, New Jersey, USA). A standard clinical protocol will be used. Stress is induced with 
28 
adenosine infusion of 140 μg/kg/min for 6 min. Myocardial blood flow quantification is performed 
29 
ECG gating under pharmacological stress will be used for measurement of LVEF and wall motion 
30 
in relation to peak hyperemia. Rubidium-PET procedures will be performed at the Department of 
31 
Nuclear Physiology, Rigshospitalet.  
32 

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1 
Study visits  
2 
Participants eligible for inclusion in the study will participate either solely in the core study program 
3 
(1 visit) or in both the core program as well as the extended study programs. 
4 
Core program 
5 
Each participant enters the study facility (Total examination time 2 h) 
6 
Table 5: 1 Visit 
Investigations
Time/hours
- 
Written informed consent 
- 
Semi-structured medical interview 
- 
Physical examination 
- 
Acquisition of blood and urine sample 
- 
DXA scan 
 
2 hours in total
Extended program 1 
7 
Table 6: 1 Visit 
Investigations
Time/hours
- 
Semen sample 
10 minutes
 
8 
Extended program 2 
9 
Table 7: 2 Visits 
Investigations
Time/hours
Visit 1 
- 
GnRH test 
- 
hCG test (injection) 
Visit 2 (72 hours later) 
- 
hCG test (blood sample)  
 
40 minutes 
10 min 
 
5 min 
 
10 
Extended program 3 
11 
Table 8: 1 Visit 
Investigations
Time/hours
- 
Rubidium PET CT 
35 minutes
 
12 
Statistical considerations 
13 
Sample size 
14 
In general, exact sample size calculation for observational studies is not unproblematic and is 
15 
primarily used for randomized controlled trials, but to do the best to ensure an appropriate sample 
16 

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size, we have calculated sample sizes for the prospective cohort study with registry follow-up and the 
1 
three sub studies.  
2 
 
3 
For the prospective cohort study with registry-based follow-up, a sample size of 800 AAS users is 
4 
appropriate based on the following calculations: 
5 
A major interest in the present study is risk of CVD among AAS users and therefore chosen as 
6 
primary endpoint for the prospective cohort study. A previous registry-based study noted a 3% 
7 
incidence of “other forms of heart diseases” during a mean follow-up of seven years among 545 
8 
AAS users and that the risk was 3-fold lower among healthy age- and sex-matched controls than 
9 
AAS users [3].  
10 
Using a reduced two-sided alpha (significance) level of 0.01 in our cohort study, due to expected 
11 
multiple comparisons, and furthermore, using a factor 10 larger sample size of age- and sex 
12 
matched registry-based control group (n = 8000) we find a power of 90% of detecting a 3-fold 
13 
difference in heart diseases between the AAS group (n =800) and registry-based control group (n = 
14 
8000). With these assumptions and calculations, the risk of statistical type I and II errors both seem 
15 
to be appropriately low in the prospective cohort study.  
16 
 
17 
The sample sizes in the extended programs are based on a number of recent cross-sectional 
18 
studies [8-9, 35-36]. 
19 
 
20 
For the extended sub study 1 sample size is based on the following considerations: 
21 
Data from a recent study demonstrated that the total motile sperm count decreased among 
22 
recreational athletes after one cycle of illicit AAS use and was persistently low one year following 
23 
AAS cessation, decreased by 14 x 106 compared with prior to the AAS cycle [36]. We expect a 
24 
major decrease in the total motile sperm count among current AAS users whereas a large variation 
25 
could occur among former AAS users due to expected variation in elapsed duration since AAS 
26 
cessation. Including a total of 200 AAS (approximately 100 current and 100 former AAS users) and 
27 
80 non-AAS users as control participants and using a 90% power and a reduced two-sided alpha 
28 
level of 0.01 (due to three study groups and multiple comparisons in the study), we will be able to 
29 
detect a difference in total motile sperm count of 5 x 106 (SD = 9) between the three study groups. 
30 
We find this difference clinically relevant. 
31 
 
32 
For the extended sub study 2 sample size is based on the following considerations: 
33 
Data from a prior study demonstrated lower mean serum total testosterone of 10.2 nmol/L in 
34 
persistently hypogonadal former AAS users following a hCG stimulation test compared with non-
35 
AAS users who were hypogonadal due to unknown reasons [35]. We expect a lesser group difference 
36 
in mean serum total testosterone following the hCG stimulation test in our study as the participants 
37 
are not all hypogonadal, although a large individual variation in serum total testosterone could occur 
38 
as we expect a more heterogenic group of participants. Including 30 participants in each of the two 
39 
study groups (former AAS users and controls) and using a 90% power and a reduced two-sided alpha 
40 
level of 0.01 (due to multiple comparisons in the study), we will be able to detect a difference in 
41 

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serum total testosterone of 5 nmol/L (SD = 5) between the two study groups following the hCG 
1 
stimulation test. We find this difference in serum total testosterone clinically relevant during a hCG 
2 
stimulation test.  
3 
 
4 
For the extended sub study 3 sample size is based on the following considerations: 
5 
 
6 
A difference in Myocardial Blood Flow of 0.5 ml/g/min is considered clinically relevant. With a SD 
7 
of measurements of global MBF by Rb-82 PET of 0.5, based on recent studies in the department; a 
8 
sample size of 30 participants is required is each of the three male study groups with a power of 90 
9 
% and a reduced two-sided alpha level of 0.015 (three study groups and multiple comparisons in the 
10 
study) to detect a difference of 0.5 ml/g/min.  
11 
 
12 
Data analyses 
13 
Analyses of basic anthropometric data, clinical characteristics and the measured biomarkers will be 
14 
performed to elucidate the underlying phenotypic characteristics on the AAS cohort, with the healthy 
15 
athlete cohort acting as controls. Cross-sectional data will be analyzed using conventional parametric 
16 
and non-parametric analyses as appropriate and with post-hoc adjustment for multiple comparisons.  
17 
Prospective follow-up will rely on registry-based data from national registries as described above and 
18 
is planned to consist of data on the three cohorts. 
19 
Planned prospective analyses include survival analyses of time-to-event data testing the impact of 
20 
comorbidities and baseline characteristics distinguishing the cohort from controls. Competing risks 
21 
analysis or extending analyses to adjust for recurrent events will be performed if deemed necessary. 
22 
Though no machine learning-based statistical methods are planned, future exploratory analyses 
23 
investigating the predictive power of the found clinical characteristics will adhere to the following: 
24 
- 
In case the statistical analyses need a random seed, the seed chosen will be ‘343635’ (FIDO-
25 
DK) to ensure integrity of approach and reproducibility of results. 
26 
- 
The statistical models are to be subject to cross-validation, using an 80/20-split to train the 
27 
model and subsequently test the predictive power of the models, respectively. Further, a 5-
28 
fold cross-validation will be used unless suggestions from an independent statistical advisor 
29 
provides evidence of a different approach providing a more robust estimate.  
30 
- 
The final model will use all data and be the product of the best performing test. 
31 
Time schedule 
32 
 September 2020– April 2022: Application for funding and approval from local ethical 
33 
committee on health research  
34 
 June 2022: First participant first visit 
35 
 June 2025: Last participant last visit 
36 
 July 2025 – June 2026: Lab and statistical analyses  
37 
 July 2026 – November 2027: writing of manuscripts and publications 
38 
 Data extraction recurring every three years counting from date of last participant visit +/- 3 
39 
months until 15 years of follow-up (i.e. approximately July 2040) 
40 

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1 
Feasibility 
2 
Conducting the study is deemed highly feasible. All equipment and personnel with the necessary 
3 
experience and expertise for the practical conduction of the study are employed at Rigshospitalet in 
4 
Copenhagen. Principal investigator, Professor Caroline Kistorp and co-investigator post.doc Jon 
5 
Rasmussen have previously conducted community-based clinical research among current and former 
6 
AAS abusers and published several papers on the subject in well-recognized international peer-
7 
reviewed journals [4–7,9]. Our experience from previous studies in illicit AAS users is that 
8 
recruitment of several hundreds of AAS as many of these are highly motivated to participate and, 
9 
furthermore, in- and exclusion criteria are few which increases the probability of recruiting a study 
10 
cohort which is representative of illicit AAS users in the community.  The current study is an apparent 
11 
continuation of our previous research in the field. Furthermore, the research group has previously 
12 
been involved in the conduction of several clinical trials including other patient groups. Jon 
13 
Rasmussen is employed as post-doctoral scientist, 20% of full-time for six years, and work on the 
14 
study during this time and a PhD student (Yeliz Bulut) will be involved with the study full-time.  In 
15 
addition, a collaboration has been established with Professor and Head of Department, Anders Juul 
16 
and Consultant, Niels Jørgensen, Department of Growth and Reproduction, Rigshospitalet, Associate 
17 
Professor Philip Hasbak, Department of Nuclear Physiology, Professor, Finn Gustafsson, Department 
18 
of Cardiology, Rigshospitalet and Professor Lars Kessing, Department of Psychiatry, Rigshospitalet 
19 
and Associate Professor, Morten Schou, Department of Cardiology, Herlev-Gentofte Hospital, 
20 
Professor Marianne Andersen and Professor Jan Frystyk, Department of Endocrinology, Odense 
21 
University Hospital, Associate Professor, Ebbe Eldrup, Herlev and Gentofte University Hospital, and 
22 
Professor, Michael Kjær, Bispebjerg and Frederiksberg University Hospital. 
23 
Data extraction from participant files and hospital records 
24 
Source data will be composed of the participants’ hospital records, files and charts (including 
25 
electronic charts), and laboratory/scan reports up to 15 years after participation in the study. Direct 
26 
access to source data is allowed during audit and inspection from national or local ethics committee 
27 
on biomedical research, the Danish Data Protection Agency and other relevant health authorities. 
28 
Direct access to source data is allowed for the primary investigator, sponsor and the sponsor’s 
29 
representatives. This must be accepted by participants and is explicitly stated in the informed written 
30 
consent. All legislations, regulations and laws of the Danish Data Protection Agency will be complied 
31 
with.  
32 
The participants consent to investigators reading and the transfering information from the electronical 
33 
hospital records to investigators and REDCap. The transfered data include medical history, blood and 
34 
urine test results, previous DXA scan results, echocardiographic and CT scans, and prescribed as well 
35 
as over-the-counter medication. Consent includes giving permission for the study group to see all 
36 
blood test results as AAS use may be associated with damage or illness in every organ system. 
37 
Furthermore, consent includes permission to retrieve information on prescribed and over-the-counter 
38 

VEK nr. H-21035759 
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medication, as it may influence the risk and progression of disease. hence all information on 
1 
medication is relevant. 
2 
In the initial assessment of the eligibility of participants, who have contacted the investigators, will 
3 
give oral consent of access to their hospital records to allow the investigators the ability to assess 
4 
whether the individual characteristics is in line with the stated inclusion and exclusion criteria. 
5 
Investigators will only tranfer source data after the acquisition of oral and written informed consent. 
6 
Registry follow-up 
7 
To avoid bias due to inevitable data-loss, we want to compare participants who drop out or who are 
8 
lost-to-follow-up to those continuing in the study. Therefore, the present application from this study 
9 
group applies for permission to collect registry data after anonymization regarding ICD10 diagnostic 
10 
codes, medical treatment, socioeconomic status, mortality and morbidity in all participants, including 
11 
those who drop out or are lost to follow-up. 
12 
The data will be kept in encrypted format for the duration of the trial. The duration of the trial is 15 
13 
years. 
14 
Data protection of Personal Information in the Study 
15 
All legislations, regulations and laws of the Danish Data Protection Agency will be complied with. 
16 
Permission to handle personal data will be sought from the Danish Data Protection Agency. All data 
17 
are stored pseudo-anonymized and analyzed electronically and no unauthorized access to data is 
18 
allowed. Original data is filed according to a unique participant number. REDCap, hosted by OPEN 
19 
(Open Patient data Explorative Network) will be used for registration of clinical data. REDCap meets 
20 
the safety requirements set by the Danish Data Protection Agency for storage of person-sensitive 
21 
data; prescribed medication, medical history, height, weight are examples of such data. Data will be 
22 
encrypted and stored for 15 years in accordance with recommendations on data storage from the 
23 
Danish Data Protection Agency and thereafter transferred to the Danish Data Archives. 
24 
The study is reported to the Danish Data Protection Agency Pactius in Region H and will be handled 
25 
according to the regulations of the General Data Protection Regulation: GDPR: REGULATION (EU) 
26 
2016/679 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 27th of April 2016 on 
27 
the protection of natural persons with regard to the processing of personal data and on the free 
28 
movement of such data, and repealing Directive 95/46/EC and the Act on Processing of Personal 
29 
Data. Data is stored for 15 years. 
30 
Economy, Funding and Participant Insurance 
31 
The initiator of the study Caroline Kistorp, a professor and researcher at the Department of Medical 
32 
Endocrinology at Rigshospitalet, University Hospital, Denmark. The study is solely funded by 
33 
external private and public foundations. Any funding is deposited in a research account administered 
34 
at Rigshospitalet, University Hospital, Denmark. None of the investigators or departments will have 
35 
any financial gain from conducting the study. The participants will not receive payment, but 
36 
transportation cost will be covered according to the guidelines of the Capital Region of Denmark.  
37 
 The investigators have received an unrestricted grant from the Novo Nordisk Foundation of 
38 
approximately 10 million Dkr for the interdisciplinary project: “Fitness Doping in Denmark (FIDO) 
39 

VEK nr. H-21035759 
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novel treatment strategies and somatic and mental health complications with anabolic androgenic 
1 
steroids’, grant number: 0065138, which comprises several studies including the present trial. 
2 
Furthermore, Jon Rasmussen, post.doc, MD, PhD has received 590.000 Dkr from ”Region 
3 
Hovedstadens Forskningsfond til Sundhedsforskning” to be used for conducting the present trial. 
4 
Funding ensuring the completion of the trial is therefore in place.  
5 
Participants in the trial are covered by the existing patient insurance ”Patienterstatningsordningen og 
6 
ordningen om erstatning for lægemiddelskader”. Healthy athletes participating as controls are 
7 
covered by the existing insurance “Arbejdsskadeforsinkringen”. 
8 
Recruitment of Participants and Informed Consent 
9 
Initial contact 
10 
Patients from the endocrine outpatient clinic at the Department of Medical Endocrinology, Center of 
11 
Cancer and Organ Disease at Rigshospitalet, who are eligible to participate in the study, will be 
12 
informed of the possibility to participate in study by a physician when they attend the outpatient clinic 
13 
for a scheduled visit. If the patient is interested in further information on the study, contact 
14 
information on the investigators will be given to the patient. If the patient favors contact being 
15 
initiated by an investigator from the study instead and provides oral consent, the patients contact 
16 
information will be forwarded to an investigator. Posters informing of the study will be displayed in 
17 
the hospital departments and information about the study will be advertised on the facebook, and on 
18 
varies homepages (sundhed.dk, antidoping.dk and forsoegspersonen.dk), where contact information 
19 
on investigators can be found. Over the years we have gained much experience in recruiting AAS 
20 
users for research studies in a discrete manner without incrimination using social media and we find 
21 
that this way of communicating with potential participants works for both parties. Potential 
22 
participants can contact the Departments of Endocrinology, Rigshospitalet by email or telephone. 
23 
Regarding advertisements on Facebook, any advertisement/post will have its ability to be shared or 
24 
write a comment to the advertisement/post disabled. 
25 
Following contact with one of the investigators if the individual is interested further written and oral 
26 
information on the study will be given to the patient during an initial meeting or sent by mail. In 
27 
addition to the written information pertaining to study, it will be accompanied by the brochure: 
28 
“Forsøgspersoners rettigheder i et sundhedsvidenskabeligt forskningsprojekt” (Your rights as a 
29 
participant in medical research). If the individual accepts further information on the study, a meeting 
30 
in a quiet, undisturbed room will be scheduled with one of the investigators, and the person will be 
31 
informed of the right to have a bystander present at that moment.  
32 
Scheduled meeting 
33 
At the scheduled meeting, the individual will receive additional oral information, including the 
34 
pamphlet “Dit væv, din ret”, from an investigator, and it will be clarified whether the potential 
35 
participant fulfill the general participation criteria. The potential participant will be informed about 
36 
the terms and restrictions of the form of ‘informed written consent’ and is informed of 24 hours 
37 
deciding time. The investigator will ensure that the patient is adequately informed about the study 
38 

VEK nr. H-21035759 
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background and design both orally and in writing. It will be made clear that the patient can withdraw 
1 
from the study at any time.  
2 
Acquisition of informed consent 
3 
If the person is still interested in participating in the study after the initial information meeting and 
4 
after the allotted deciding time, a new meeting will be scheduled where oral and written informed 
5 
consent will be obtained. After consent to participate in the study has been obtained, the visit (Core 
6 
program: visit 1) will be scheduled. 
7 
No study-related examinations will be conducted prior the acquisition of informed consent. 
8 
Investigators will only access and transfer electronic hospital records after acquisition of written 
9 
informed consent.  
10 
Informed consent 
11 
The participant will be required to sign the individual forms of informed consent applicable. 
12 
Consent regarding core program, registry-based follow-up and research biobank 
13 
The participant’s consent includes the right to read and transfer information from electronical hospital 
14 
records to RED-Cap by investigators. The data transferred include information such as medical 
15 
history, blood and urine test results, prescribed and over-the-counter medication. Further, the 
16 
informed consent includes permission to establish a research biobank regarding the analyses of 
17 
biomarkers specified in the present protocol. The informed written consent grants permission to use 
18 
registry data prospectively after anonymization, including information on ICD10 diagnostic codes, 
19 
medical treatment, socioeconomic status, morbidity and mortality.  
20 
The informed consent will be valid for the duration of the trial. The duration of the trial is 15 years. 
21 
Consent regarding extended program 
22 
Participants undergoing additional procedures will be required to sign additional forms of informed 
23 
consent for each extended program. Data will be stored in REDcap as previously described and kept 
24 
in encrypted form for the duration of the trial. 
25 
The informed consent will be valid for the duration of the trial. The duration of the trial is 15 years. 
26 
Consent regarding establishment of a biobank of future research 
27 
A separate form of informed consent gives permission of the establishment of a biobank of future 
28 
research consisting of extra material, if any, acquired at the initial visit of the core program. The aim 
29 
of the future research biobank is to ensure the ability of the cohort to answer contemporary research 
30 
questions in a rapidly evolving field. Future analyses may therefore include, but not be limited to 
31 
analyses of circulating levels of hormones, proteins, DNA-excerpts, not specified in the current 
32 
protocol. Future analyses will be required to be approved by the regional ethics committee prior to 
33 
analysis. 
34 
The informed consent will be valid for the duration of the trial. The duration of the trial is 15 years. 
35 
Risks and discomforts  
36 
Phlebotomy to acquire blood can be uncomfortable, but is considered safe. In comparison to blood 
37 
donation, where 500 mL is donated, the current study will require a sample of 300 mL, which may 
38 
induce slight lightheadedness, but is otherwise not associated with any known risk. No genome-
39 

VEK nr. H-21035759 
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mapping is planned in the current protocol. In the event the routine blood panel gives indication of 
1 
organ damage, which requires immediate action or a referral for further analysis, the attending 
2 
physician is obliged to do so. 
3 
The radiation dose delivered by the DXA scans (0.001-0.004 mSV per scan) during the entire trial is 
4 
comparable to less than or equal to half a day of radiation when compared to the natural background 
5 
radiation (0.008 mSV/day) and hence does not form any known health risk [34]. Radiation has a 
6 
stochastic risk of inducing spontaneous mutation, why pregnant women are ineligible to participate 
7 
in the trial. The Rb-82 PET scan delivers radiation much less than a single photon emission 
8 
tomography (SPECT) scan which is the current noninvasive clinical standard of diagnosing coronary 
9 
artery disease. Radiation doses of Rb-82 PET scans (Participants in the extended Study program 3) 
10 
are approximately 4 mSv per investigation including rest and stress.. In general, Danish citizens’ 
11 
lifetime risk of cancer is approximately 25.00%. Participating in this extended study program 3 
12 
increases the risk of cancer to approximately 25,02% (risk category: IIb) (International Commission 
13 
on Radiation Protection (ICRP) and Europe Commission).  
14 
 
15 
Ethical considerations  
16 
The study is in accordance with the Helsinki II declaration and the regulations of the General Data 
17 
Protection Regulation. The study will be approved by the Danish Data Protection Agency and the 
18 
Regional Ethics Committee of Copenhagen Denmark.  
19 
Our study involves the recruitment of AAS users, who have self-administered AAS for long periods 
20 
and may continue to do so. All study participants will receive information material of the hazardous 
21 
effects of AAS created in collaboration with AntiDoping Denmark. Further, to protect the participants 
22 
from future stigma or potential prosecution, information specific to the use of PIEDs by each 
23 
individual will only be registered in the database and will not appear in the medical journal. If we 
24 
encounter persons, we find are in need of acute medical or psychiatric therapy (including suicidal 
25 
risk) we will refer or admit the participant as appropriate to the relevant specialty.  
26 
In summary, illicit AAS misuse is associated numerous adverse effects on health related to almost 
27 
every organ system. Furthermore, previous AAS users often suffer from prolonged male 
28 
hypogonadism, increased cardiovascular risk, increased risk of infertility and a range of psychological 
29 
symptoms related to this condition. We find that the potential therapeutic gain and benefits for the 
30 
participants and future patients outweigh the risks and discomforts of participating in the study and 
31 
therefore, we believe conducting the study is justified. 
32 
 
33 
Dissemination of Results 
34 
The investigators oblige themselves to publish all clinically relevant findings in peer-reviewed 
35 
journals irrespective of their ability to achieve statistical significance. Positive, inconclusive and 
36 
negative results will be presented. Results will be published following the International Committee 
37 
of Medical Journal Editors (ICMJE) recommendations. Further, the findings will be presented at 
38 
national and international conferences. 
39 

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1 
Future perspectives 
2 
To the best of our knowledge, no trials have previously been conducted among men with AAS-
3 
induced hypogonadism and no consensus on therapy exists to mitigate the risks following the 
4 
condition in Denmark or abroad. This study will provide incremental value to the evidence-based risk 
5 
assessment of men and women current and former, which will be crucial in maintaining a normal 
6 
health condition or mitigating the adverse effects of continued AAS use.  
7 
 
 
8 

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