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Accepted Preprint first posted on 1 June 2015 as Manuscript EJE-14-1061

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Original research

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Use of statins is associated with lower serum total and non-SHBG-bound testosterone

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levels in male participants of the Rotterdam Study

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Catherine E. de Keyser1,2*; Filipe Valerio de Lima1*; Frank H. de Jong3; Albert Hofman1;

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Yolanda B. de Rijke ; André G. Uitterlinden ; Loes E. Visser

3,4

1,3

1,3,5

; Bruno H. Stricker

1,2,3

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1

2

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Health Care Inspectorate, The Hague, the Netherlands; 3 Department of Internal Medicine,

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Erasmus Medical Center, Rotterdam, the Netherlands; 4 Department of Clinical Chemistry,

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Erasmus Medical Center, Rotterdam, the Netherlands; 5 Hospital Pharmacy The Hague

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Hospitals – HAGA, the Hague, the Netherlands. *Both authors contributed equally

Department of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands; The

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Running head: Statins lower total and bioactive testosterone

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Keywords: HMG-CoA reductase inhibitors; Statins; Total testosterone; Non-SHBG-bound

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testosterone; Cholesterol

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Word count main text:

4809

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Word count whole document:

6745

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Word count abstract:

250

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Number of references:

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Number of tables:

3

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Number of figures:

1

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Corresponding author:

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Bruno H. Stricker, PhD, MMed

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Department of Epidemiology, Erasmus MC, P.O. Box 2040, 3000CA, Rotterdam, the

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Netherlands. E: [email protected]; T: +31 10 7044958; F: +31 10 7044657

1 Copyright © 2015 European Society of Endocrinology.

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Submitting author (for correspondence during submission phase):

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Catherine E. de Keyser, MD

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Department of Epidemiology, Erasmus MC, P.O. Box 2040, 3000CA, Rotterdam, the

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Netherlands. E: [email protected]; T: +31 10 7044958; F: +3110 7044657

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ABSTRACT

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Objective: Statins or HMG-CoA reductase inhibitors decrease cholesterol production.

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Because cholesterol is a precursor of the testosterone biosynthesis pathway, there is some

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concern that statins might lower serum testosterone levels. Our objective was to investigate

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the association between use of statins and serum testosterone levels in men.

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Design: cross-sectional within the prospective population-based Rotterdam Study.

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Methods: We included 4166 men with total testosterone, non-SHBG-bound testosterone, and

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medication dispensing data available. Multivariable linear regression analysis was used to

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compare differences in serum testosterone levels (nmol/L) between current, past, and never

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statin users. We considered dose and duration of use. Analyses were adjusted for age, body

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mass index, cardiovascular disease history, diabetes mellitus, hypertension, and estradiol

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levels.

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Results: We identified 577 current (mean age 64.1 years), 148 past (mean age 64.6 years),

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and 3441 never (mean age 64.6 years) statin users. Adjusted for all covariables, current statin

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use 1-≤6 months and >6 months was significantly associated with lower total testosterone

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levels compared to non-users (β -1.24, 95%CI -2.17;-0.31 and β -1.14, 95%CI -2.07;-0.20

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respectively). Current use 1-≤6 months was also associated with significantly lower non-

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SHBG-bound testosterone levels (β -0.42, 95%CI -0.82;-0.02). There was a trend towards

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lower testosterone levels at higher statin doses, both for total (Ptrend 2.9x10-5) and non-SHBG-

56

bound testosterone (Ptrend 2.0x10-4). No association between past statin use and testosterone

57

levels was found.

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Conclusions: We showed that current use of statins was associated with significantly lower

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serum total and non-SHBG-bound testosterone levels. Clinical relevance of the association

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should be further investigated.

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INTRODUCTION

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Statins or HMG-CoA reductase inhibitors are widely used cholesterol lowering drugs which

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are effective in the primary and secondary prevention of cardiovascular disease (CVD).1-3

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They competitively inhibit HMG-CoA reductase, mainly in the liver, which is the rate-limiting

66

enzyme in the cholesterol biosynthesis pathway. By inhibiting this cholesterol biosynthesis,

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the number of low-density lipoprotein (LDL) receptors in the hepatic membrane is increased.

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This leads to increased serum uptake of LDL-cholesterol, and thus a decrease in serum

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cholesterol concentration.

4

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Testosterone is the main circulating androgenic hormone in men with important

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effects on libido, bone mass, fat distribution, muscle mass, strength and production of blood

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cells and sperm.5, 6 It is synthesized in the testes, and this process requires a continuous

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supply of cholesterol, which can be derived from plasma, mostly originating from the liver, or

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from de novo production within the gland.7 In contrast, women have in general 8 to 9 times

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lower levels of serum total testosterone; the hormone here also plays a role in sexual function

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and libido.8 Circulating testosterone in men is for 40-65% bound to sex hormone-binding

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globulin (SHBG), which regulates the serum concentration of testosterone and its transport to

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target tissues. SHBG has a high binding affinity for testosterone and the serum

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concentrations of total testosterone and SHBG are strongly correlated. In contrast, the non-

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SHBG-bound fraction of testosterone, which is considered to be bioactive, is barely

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associated with the serum SHBG concentration.9-11 This indicates that non-SHBG-bound

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testosterone, rather than total testosterone plays an important role in maintaining equilibrium

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in the negative feedback of the hypothalamo-pituitary-testicular axis and in other androgenic

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effects.

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Since statins decrease cholesterol biosynthesis, and cholesterol is the precursor of

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testosterone, there is some concern whether statins might impair testosterone production.

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Statins decrease serum availability of the substrate cholesterol, in vitro studies showed that

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statins decrease cholesterol production in testicular Leydig cells12, or inhibit enzymes within

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the testosterone biosynthesis pathway (e.g. 17β-hydroxysteroid dehydrogenase).

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testosterone level due to statins may be undesired in men with already low testosterone

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levels, since it may lead to symptoms such as a decrease in mood, libido, muscle strength, or

4

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A lower

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bone mineral density. The 2013 American guidelines for cardiovascular disease prevention

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lowered the threshold for treatment with statins and widened the target population.

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Furthermore, the prevalence of diseases such as type 2 diabetes mellitus (T2DM) and CVD is

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increasing.

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increase, and this might come along with an increase in non-beneficial effects of statins.17

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Consequently, it is important to elucidate whether statins decrease serum testosterone levels,

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and more specifically non-SHBG bound testosterone, as potential undesired effect of their

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use.

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15, 16

Therefore, the already substantial use of statins in clinical practice may further

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In this large population-based cohort study, our objective was to investigate whether

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the use of statins was associated with decreased serum levels of total and non-SHBG-bound

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testosterone in male persons aged 45 years or older.

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SUBJECTS and METHODS

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Setting and Study Population

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This research was conducted within the Rotterdam Study, a prospective population-based

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cohort study, which aims to examine the frequency and determinants of diseases in middle-

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aged and elderly people. The rationale and design of the Rotterdam Study have been

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described previously. 18, 19

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In short, at start, all 10,275 persons aged ≥55 years in the Ommoord district of

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Rotterdam, the Netherlands, were invited to participate. Of them, 7,983 (78%) were enrolled

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between 1990 and 1993 (RS-I). At baseline, participants underwent extensive clinical

114

examination at the research center. Participants have been followed during up to four follow-

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up rounds (1993-1995, 1997-1999, 2002-2004, 2009-2012). In 2000, an extended cohort was

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enrolled (RS-II), in which 3,011 inhabitants aged ≥55 years entered the study and are

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continuously followed since then. Furthermore, in 2006, a third cohort started (RS-III)

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including 3,932 inhabitants aged ≥45-54 years at enrollment.

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The Rotterdam Study has been approved by the medical ethics committee according

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to the Wet Bevolkingsonderzoek: ERGO (Population Study Act: Rotterdam Study), executed

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by the Ministry of Health, Welfare and Sports of the Netherlands. All participants gave

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informed consent to participate in the study and to obtain information from treating physicians

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and pharmacies, separately.

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All three cohorts of the Rotterdam study were considered in the current research,

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including a study population aged 45 years or over. The study population consisted of all male

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participants of the Rotterdam Study (n=4,255), for whom a serum total testosterone

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measurement was available, for whom a serum non-SHBG-bound testosterone concentration

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could be calculated, and for whom medication dispensing data were available. As most of the

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women in the Rotterdam Study are postmenopausal and have very low testosterone levels,

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we expected that any potential effect of statins could probably only be demonstrated in males

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or would not be clinically relevant in females. Therefore, we restricted our study population to

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only male participants.

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Outcome assessment

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Our outcomes of interest were the serum levels of total testosterone and non-SHBG bound

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testosterone. Serum hormone data were assessed from laboratory measurements during the

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third visit of the first cohort (RS-I-3, 1997-1999), first visit of the second cohort (RS-II-1, 2000-

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2001), and the first visit of the third cohort (RS-III-1, 2006-2008). Serum levels of non-SHBG-

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bound testosterone were used as a measure of bioactive testosterone.

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Exposure assessment

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The exposure of interest was the use of statin therapy. Medication dispensing data were

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obtained from all seven fully computerized linked pharmacies in the Ommoord district.

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Information on all filled prescriptions from January 1st 1991 until February 1st 2012 was

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available and included information on the product name of the drug, the Anatomical

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Therapeutical Chemical Code (ATC-code) , the amount dispensed, the prescribed dose

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regimen and the date of dispensing (http://www.whocc.no/atc_ddd_index; accessed October

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6 2014). For every dispensing of a statin, the duration of use (prescription episode) was

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calculated by dividing the number of dispensed tablets by the prescribed daily number.

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Repeated prescriptions, which were filled within 7 days after ending the previous filled

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prescription, were considered as continuous use.

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At the date of the testosterone measurement, every participant was classified into

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mutually exclusive categories: ‘Current use’ if the measurement occurred within a prescription

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episode, ‘Past use’ if the participant previously stopped using statins, or ‘Never use’ if the

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participant had not used statins during the study period. Current and past statin users were

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further stratified into 5 categories according to the duration of exposure to statins: current use

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≤1 month, current use >1 to ≤6 months, and current use >6 months; past use >6 months, and

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past use ≤6 months since the end of the last prescription episode. The 6-month cut-off was

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applied based on the median duration of current and past use in the population. To facilitate

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direct dose comparisons between drugs from the same therapeutic drug group, the daily dose

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of statin therapy was expressed in 'Defined Daily Doses' (DDD)

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(http://www.whocc.no/atc_ddd_index; accessed October 6 2014).

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Analytical determinations

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All steroids and SHBG were estimated in the same serum sample obtained from blood taken

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in the morning in the fasting state. Testosterone, dehydroepiandrosterone (DHEA) and DHEA

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sulphate (DHEAS) were measured simultaneously with a LC-MS/MS method using the

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CHS™ MSMS Steroids Kit (Perkin Elmer, Turku, Finland). The Steroids Kit uses a combined

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solvent extraction and protein precipitation method with acetonitrile containing the deuterated

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internal standards 2H5-testosterone, 2H6-DHEA and 2H6-DHEAS. The internal standard

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underwent processing identical to the analytes. The chromatographic separation was

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performed on a Waters® (Milford, MA, USA) Acquity™ UPLC HSS T3 1.8 µm column

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(diameter 1 mm, length 10 cm) and in-line filter frit 0.2 µm with an acetonitrile/MeOH gradient.

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A Waters XEVO-TQ-S system equipped with an ESI source operating in the electrospray

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positive mode was used for quantitation. The lower limits of quantitation for testosterone,

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DHEA and DHEAS were 0.07, 2.2 and 24.7 nmol/L, respectively. Non-SHBG-bound

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testosterone was calculated according to the method of Södergard et al.20, using previously

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described equations9 assuming a fixed albumin level of 40 g/L. SHBG, estradiol and insulin

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were measured using a Cobas 8000 Modular Analyzer (Roche Diagnostics GmbH,

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Mannheim, Germany). Fasting total cholesterol was measured on a cobas c702 system

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(Roche Diagnostics GmbH,Mannheim, DE).

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Covariables

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Variables were considered as potential confounders or effect modifiers influencing the

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association between the use of statins and serum testosterone levels, based on their clinical

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relevance or confounding effect in the analysis (a variable that changed the estimate >10%

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was considered as a confounder).21-23 Our selected confounders were: age, body mass index

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(BMI), a history of CVD, T2DM, hypertension, and serum estradiol levels. Testosterone is

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known to decrease with increasing age and BMI. CVD, hypertension and T2DM are

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conditions associated with lower testosterone levels. Estradiol changed the estimate >10%

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and is also correlated with testosterone and SHBG. Furthermore, analyses were adjusted for

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the prescribed DDD of statin therapy.

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In addition, we investigated whether additional adjustment for insulin, total

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cholesterol, DHEA, and DHEAS influenced the association between SHBG and testosterone,

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and investigated whether these variables were effect modifiers or intermediates. Insulin

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influences SHBG levels that are strongly correlated with total testosterone, total cholesterol

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and DHEA are part of the pathway from cholesterol to testosterone, and DHEAS is a

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metabolite of DHEA.

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BMI was calculated as the weight (in kg) divided by height-squared (in m2). CVD in

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history was defined as the occurrence of a myocardial infarction (MI), percutaneous

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transluminal coronary angioplasty (PTCA), coronary artery bypass grafting (CABG), heart

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failure, carotid desobstruction, cerebrovascular accident (CVA), or transient ischemic attack

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(TIA) before the date of testosterone measurement.24-26 T2DM was defined as a current

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prescription for an oral glucose-lowering drug or insulin (ATC-code A10). Hypertension was

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defined as blood pressure higher than 140/90 mmHg or a current prescription for an

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antihypertensive agent (ATC-code C02, C03, C07, C08 or C09)

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(http://www.whocc.no/atc_ddd_index; accessed October 6 2014).

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Statistical analysis

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Kolmogorov-Smirnov tests were used to test the normality of the distribution of the

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parameters. Correlations between variables were tested using Pearson’s correlation method

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for normally distributed data, and Spearman’s correlation method for non-normally distributed

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data. Differences in characteristics between current, past and never statin users were tested

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using unpaired two-sided Student’s T-test for normally distributed parameters and Mann-

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Whitney U-test for non-normally distributed parameters.

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In a cross-sectional design, we investigated the association between use of statins

217

and serum total and non-SHBG-bound testosterone levels, using multivariable linear

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regression analysis to adjust for confounders. Non-normally distributed data were log

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transformed in these analyses. First, we compared current and past use of statin therapy on

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serum testosterone levels, with never use as the reference category. In a second analysis, we

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considered duration of statin use (current use ≤1 month, >1-≤6 months and >6 months; past

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use ≤6 and >6 months), and investigated the effect of duration of use on serum testosterone

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levels, with never use as the reference category. Furthermore, we investigated whether

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additional adjustment for insulin, total cholesterol, DHEA, and DHEAS influenced this

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association, and investigated whether these variables were effect modifiers or intermediates.

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In an additional analysis, we investigated whether the association between statins

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and testosterone levels was dose-dependent. We stratified the DDD of statin therapy into

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tertiles, and compared current and past use of statins with never use in each stratum.

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A sensitivity analysis was performed in participants without CVD, to investigate

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whether associations are constant in men without CVD. Another sensitivity analysis was

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performed re-allocating most recent past users (within 14 days) from ≤6 months past use into

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the current use category. This was done because we believed that patients recently exposed

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to statins might still be affected by the drug (i.e. carry-over effect). In another sensitivity

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analysis, we adjusted the analyses on statins and total testosterone for serum SHBG levels,

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to investigate whether SHBG might play a role in the association between statins and total

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testosterone.

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Statistical analyses were performed using SPSS software (SPSS Inc., version 21.0,

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Chicago, Illinois, USA). All p-values are two-sided and were considered statistically significant

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if p1 to ≤6

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months current use and >6 months current use were associated with statistically significantly

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lower total testosterone levels, compared to never use (β -1.24, 95%CI -2.17 ; -0.31, P 0.009

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for >1 to ≤6 months current use; β -1.14, 95%CI -2.07 ; -0.20, P 0.017 for >6 months current

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use). For the past use categories and ≤1 month current use, no significant association with

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serum total testosterone levels was found (Table 2).

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In the multivariable analyses on non-SHBG-bound testosterone levels, current statin

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use was associated with lower non-SHBG-bound testosterone levels compared to never use

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(β -0.35, 95%CI -0.68 ; -0.01, P 0.042). Past statin use was not significantly associated with

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lower non-SHBG-bound testosterone levels compared to never use (β -0.26, 95% CI -0.65 ;

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0.13, P 0.191) (Table 2). When duration of use was considered, >1 to ≤6 months current use

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was associated with statistically significantly lower non-SHBG-bound testosterone levels with

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a beta of -0.42 (95%CI -0.82 ; -0.02, P 0.039). For the other past and current use categories,

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no significant association was found (Table 2).

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We investigated whether other hormones and total cholesterol were intermediates in

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the association between statins and testosterone levels. Additional adjustment for insulin and

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DHEAS did not change the results importantly. Additional adjustment for total cholesterol and

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DHEA showed the largest effects on the associations, and reduced the magnitude of the

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effect. Additional adjustment for total cholesterol showed a beta of -0.97 (95%CI -1.75 ; -0.18,

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P 0.015) for total testosterone and a beta of -0.25 (95%CI -0.58 ; 0.90, P 0.151) for non-

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SHBG-bound testosterone for current use, instead of the original betas of -1.18 and -0.35

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respectively. Additional adjustment for DHEA showed similar results as for total cholesterol

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adjustment. (Results not shown).

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Moreover, we stratified statin DDD in tertiles to investigate whether a higher statin

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dose was associated with a stronger testosterone lowering effect. As shown in Figure 1, there

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was a trend towards a stronger testosterone lowering effect at a higher statin dose, both for

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total testosterone (P for trend 2.9x10-5) and non-SHBG-bound testosterone (P for trend

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2.0x10-4). Current statin use in the mid and high dosage tertiles was associated with betas of -

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1.17 (95%CI -2.00 ; -0.35, P 0.005) and -1.89 (95%CI -2.75 ; -1.03, P 1.7x10-5) nmol/L of

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lower serum total testosterone, respectively, when compared to never users. For non-SHBG-

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bound testosterone, current statin use in the mid and high dosage tertiles was associated with

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betas of -0.44 (95%CI -0.79 ; -0.09, P 0.015) and -0.85 (95%CI -1.21 ; -0.48, P 6.0x10 )

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nmol/L, respectively, when compared to never users.

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In a sensitivity analysis, we excluded all participants with a history of CVD (8% of

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the population). Current users again showed significantly lower total testosterone levels (β -

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1.12, SE 0.43, P 0.048). The effect estimate for current users on lower non-SHBG-bound

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testosterone levels was similar to the original analysis, although non-significant (β -0.29, SE

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0.27, P 0.125). For past statin use, again no association was found.

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In an additional sensitivity analysis, we re-allocated 23 participants from ≤6 months

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past use to current use. These patients were the most recent past users (they had stopped

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statins ≤14 days before testosterone assessment). Results showed no significant association

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of past use with lower total and non-SHBG-bound testosterone levels. Current users again

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showed a stronger and more significant association with total testosterone levels (β -1.31,

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95%CI -2.06 ; -0.56, P 0.001) and non-SHBG-bound testosterone levels (β -0.42, 95%CI -

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0.74 ; -0.10, P 0.011), than never users (Table 3). When past users within 30 days were re-

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allocated to current use (40 participants re-allocated), the results were similar to the 14 days

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re-allocation (Results not shown).

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In an additional sensitivity analyses, we also adjusted the analyses on statins and

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total testosterone for serum SHBG levels. The association between current statin use and

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lower serum total testosterone levels was attenuated but remained statistically significant: β -

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0.65, 95%CI -1.27 ; -0.04, P 0.038 (original analysis); β -0.73, 95%CI -1.32 ; -0.14, P 0.015

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(sensitivity analysis with recent past users re-allocated).

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DISCUSSION

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In this cross-sectional population-based study, we showed that current use of statins was

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associated with statistically significantly lower levels of serum total and non-SHBG-bound

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testosterone in males. We demonstrated that the magnitude of the decrease in testosterone

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was directly proportional to the dosage of statin therapy. Considering duration of statin use,

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the association between current statin use and lower testosterone levels was present after at

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least 1 month current use. For past use, no association was found with testosterone levels.

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As far as we know, there are five cross-sectional studies in the literature that studied

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the association between statins and testosterone levels. Three studies are in line with our

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findings on total testosterone levels and showed that statins were associated with lower total

324

testosterone levels 27-29, while two showed no association at all 30, 31. The studies which

325

showed lower total testosterone levels demonstrated a difference of 1.5, 1.6 and 3.0 nmol/L,

326

respectively, while in our study mean total testosterone levels were 2.2 nmol/L lower in

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current users compared to never users. Three of these five studies also investigated free

328

testosterone, of which two did not find an association 28, 30 and one showed significantly lower

329

free and bioavailable testosterone levels

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lower in statin users than in non-users[30], while in our study mean non-SHBG-bound

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testosterone levels were 0.7 nmol/L lower in current users than in never users. Advantages

332

of our study compared with these studies are that one did not adjust for potential confounders

333

28

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these studies duration of therapy was analyzed. Moreover, one study was conducted only in

335

patients consulting for erectile dysfunction

336

selected

29

. Bioavailable testosterone levels were 1.0 nmol

, another had only 25 statin users 31, only one study had statin dosage 29, while in none of

337

28

29

, while in another exclusively T2DM patients were

.

Similarly, placebo controlled randomized trials studied total testosterone levels before

338

and after statin therapy. A recent meta-analysis including 5 such trials concluded that current

339

statin therapy (4 weeks - 3 months) induced a decrease in total testosterone of -0.66 nmol/L

340

in men. 32

341

Theoretically, there are several mechanisms by which statins may inhibit testosterone 12

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production. The main one is by inhibiting HMG-CoA reductase in the testis.

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the testis, statins consequently suppress de novo cholesterol production, impairing the

14

By operating in

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substrate source for the testosterone biosynthesis pathway. The second mechanism is by

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decreasing serum cholesterol concentrations, and therefore cholesterol uptake by the testis,

346

which could, again, limit its availability and impact on testosterone production. A third

347

mechanism is by directly inhibiting other enzymes in the testosterone biosynthesis pathway.

348

An alternative theory for the decrease in testosterone levels is that statin users have

349

increased insulin levels, because these patients often have metabolic syndrome, and statins

350

themselves may increase insulin levels.33 Insulin suppresses SHBG production in the liver34,

351

35

352

constant in response to the decrease in SHBG levels.9 This may be supported by the finding

353

that the association between statins and total testosterone is attenuated by additional

354

adjustment for SHBG in sensitivity analyses. However, no studies describe an effect of statin

355

use on SHBG levels, and sensitivity analyses in which we additionally adjusted for DHEA,

356

DHEAS, insulin, or cholesterol support that the association between statins and testosterone

357

is explained through cholesterol lowering by statins. Additional adjustment for total cholesterol

358

or DHEA attenuated the association between statins and lower testosterone levels, while

359

DHEAS and insulin showed no important effect. Both cholesterol and DHEA are substrates

360

for testosterone formation, and attenuation of the association through additional adjustment

361

supports the hypothesis that statins lower testosterone through reducing cholesterol. Also,

362

DHEA is a reflection of adrenal steroid production, and suppression of DHEA may suggest an

363

effect on adrenal steroidogenesis.36

13

, with consequently lower total testosterone levels, because free testosterone levels are kept

The impact of statins as testosterone lowering drugs has been criticized37 and several

364 365

arguments were used. First, a decrease of -0.66 nmol/L, as shown in the recent meta-

366

analysis

367

a poor correlation between the use of statins and symptoms such as decreased libido or

368

muscle strength, which questions the clinical relevance of this decrease. However, there is a

369

variability in response to statins and some patients might be more vulnerable to statins and

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will have a stronger decrease.38 A modest average decrease in a population might hide a

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substantial decrease in a handful of individuals and in those with an already low testosterone,

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it might be clinically meaningful. Moreover, even modest effects on a population-based scale

373

may gain more relevance now that statin therapy is increasingly used. For instance,

15

32

, is a small mean decrease in total testosterone level. Second, in literature there is

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application of the adapted American guidelines on male participants of the Rotterdam Study

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would imply that nearly all elderly men (i.e. 96.4%) should be prescribed statins.

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corroborates to the idea that adverse reactions to statins, e.g. a testosterone lowering effect,

377

deserve attention. Moreover, when the decrease in testosterone levels is clinically relevant,

378

statin users can become eligible for testosterone replacement therapy (TRT). Literature

379

discusses a possible association between TRT and an increase in cardiovascular events.

380

However, evidence is controversial, and most studies showed no increased cardiovascular

381

risk related to TRT

382

recently the U.S. Food and Drug Administration warned about the possible increased risk of

383

heart attack and stroke in the treatment of low testosterone levels due to ageing, and

384

confirmed cautions that TRT is approved only for men who have low testosterone levels

385

caused by certain medical conditions.45

40-43

39

This

44

, while only one study demonstrated a possible risk. Nevertheless,

386

Our study is cross-sectional, because regular assessment of serum testosterone

387

levels is very unusual in a population-based setting. Nevertheless, we were able to study

388

duration of use to establish a temporal relationship. Current use for >1 to ≤6 months and >6

389

months showed a significantly lower total testosterone level. The first month of therapy was

390

investigated separately to gain insight into how testosterone levels behave during the

391

beginning of statin use. During the first month, the testosterone lowering effect was non-

392

significant and less strong than for the other current use categories, suggesting that more

393

time is needed to induce a complete effect on testosterone levels. For >1 to ≤6 months of

394

statin use, non-SHBG-bound testosterone levels decreased in parallel with total testosterone.

395

However, for the > 6 months statin users, non-SHBG-bound testosterone levels also showed

396

a negative beta which was, however, not significant. As described in the introduction, non-

397

SHBG-bound testosterone is driving the negative feedback mechanism of the hypothalamo-

398

pituitary-gonadal axis to keep its concentration constant. Possibly, in long-term statin users,

399

the setpoint has adapted to the effect of statins on testosterone lowering, as a compensatory

400

effect to prevent the levels of bioactive non-SHBG-bound testosterone to fall. This might

401

explain why we could not demonstrate an effect of long-term statin use on the levels of

402

bioactive non-SHBG-bound testosterone.

16

Page 17 of 30

403

The magnitude of the association between current statin use and lower serum total

404

and non-SHBG-bound testosterone was stronger at a higher statin doses. This is in line with

405

what is expected, and strengthens our finding. Some misclassification of exposure may have

406

occurred in very recent past users who may actually be current users who took a drug holiday

407

or be subject to a carry-over effect after stopping the drug. In sensitivity analyses, the

408

association was strengthened for current users, which supports the possibility of some

409

misclassification of exposure.

410

Strengths and potential limitations should be considered. The Rotterdam Study is a

411

large prospective population-based cohort study with extensive data collection. For instance,

412

we were able to consider statin dose and were the first study that considered the effect of

413

duration of statin use. Furthermore, we accounted for potential confounding variables (e.g.

414

T2DM, hypertension and CVD). Compared to other cross-sectional studies, we managed not

415

only to overcome potential flaws, but also to further extend and strengthen the analysis. We

416

adjusted for confounding by indication which potentially plays a role since diseases such as

417

CVD and T2DM are independently associated with lower testosterone levels.46

418

A strength is that we measured total testosterone, DHEA and DHEAS by LC-MS/MS.

419

Although we measured estradiol by electrochemiluminescence immunoassay, the method

420

used has been standardized via ID-GC/MS. In the Rotterdam Study no direct measurement of

421

free testosterone by equilibrium dialysis was performed. Instead, we calculated non-SHBG-

422

bound testosterone using the formula by Sodergard et al.20 Results of both types of

423

estimations of free testosterone yielded highly correlated results.47 Another limitation is the

424

lack of data on gonadotropin levels in the Rotterdam Study. Ideally, the study should be

425

performed longitudinally with several testosterone assessments over time. However, this is

426

very unusual in a population-based setting. Unfortunately, we had too low numbers of current

427

statin users to investigate the association in users of the different types of statins separately.

428

However, we expected a class effect of statins on testosterone lowering. This study supports

429

the hypothesis that statins lower testosterone through cholesterol lowering, and thus all

430

statins will show this effect. Analyses of the association between current statin use and

431

testosterone levels showed a similar direction of the effect (negative beta) for all statins

17

Page 18 of 30

432

separately. However, the association only remained significant for the association of total

433

testosterone in simvastatin users, due to decreased power because of sample size.

434

In conclusion, our study showed that current use of statin therapy is associated with

435

significantly lower serum total and non-SHBG-bound testosterone levels after adjustment for

436

important confounders. Given the large number of statin-treated males and important

437

biological role of testosterone, the clinical relevance of this association should be further

438

investigated.

18

Page 19 of 30

439 440

DECLARATION of INTEREST

441

The authors declare that they have no conflict of interest

442 443

FUNDING

444

The Rotterdam Study is supported by the Erasmus Medical Center and Erasmus University

445

Rotterdam; the Netherlands Organization for Scientific Research (NWO); the Netherlands

446

Organization for Health Research and Development (ZonMW); the Research Institute for

447

Diseases in the Elderly (RIDE); the Netherlands Genomics Initiative (NGI); the Ministry of

448

Education, Culture and Science; the Ministry of Health, Welfare and Sport; the European

449

Commission (DG XII); and the Municipality of Rotterdam.

450 451

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

452 453

AUTHOR CONTRIBUTIONS

454

All authors contributed substantially to the manuscript, and have met the criteria for

455

authorship.

456 457

ACKNOWLEDGMENTS

458

The contributions of the study participants, general practitioners and pharmacists of the

459

Ommoord district to the Rotterdam Study are gratefully acknowledged.

19

Page 20 of 30

460 461

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Corona G, Boddi V, Balercia G, Rastrelli G, De Vita G, Sforza A, Forti G, Mannucci E & Maggi M. The effect of statin therapy on testosterone levels in subjects consulting for erectile dysfunction. J Sex Med 2010 7 15471556. Hall SA, Page ST, Travison TG, Montgomery RB, Link CL & McKinlay JB. Do statins affect androgen levels in men? Results from the Boston area community health survey. Cancer Epidemiol Biomarkers Prev 2007 16 1587-1594. Mondul AM, Selvin E, Rohrmann S, Menke A, Feinleib M, Kanarek N, Rifai N, Dobs AS & Platz EA. Association of serum cholesterol and cholesterollowering drug use with serum sex steroid hormones in men in NHANES III. Cancer Causes Control 2010 21 1575-1583. Schooling CM, Au Yeung SL, Freeman G & Cowling BJ. The effect of statins on testosterone in men and women, a systematic review and metaanalysis of randomized controlled trials. BMC Med 2013 11 57. Zafrir B & Jain M. Lipid-lowering Therapies, Glucose Control and Incident Diabetes: Evidence, Mechanisms and Clinical Implications. Cardiovasc Drugs Ther 2014 28 361-377. Plymate SR, Matej LA, Jones RE & Friedl KE. Inhibition of sex hormonebinding globulin production in the human hepatoma (Hep G2) cell line by insulin and prolactin. J Clin Endocrinol Metab 1988 67 460-464. Katsuki A, Sumida Y, Murashima S, Fujii M, Ito K, Tsuchihashi K, Murata K, Yano Y & Shima T. Acute and chronic regulation of serum sex hormonebinding globulin levels by plasma insulin concentrations in male noninsulin-dependent diabetes mellitus patients. J Clin Endocrinol Metab 1996 81 2515-2519. Goodarzi MO, Carmina E & Azziz R. DHEA, DHEAS and PCOS. J Steroid Biochem Mol Biol 2014. Sniderman AD & Thanassoulis G. Do statins lower testosterone and does it matter? BMC Med 2013 11 58. Thompson GR, O'Neill F & Seed M. Why some patients respond poorly to statins and how this might be remedied. Eur Heart J 2002 23 200-206. Kavousi M, Leening MJ, Nanchen D, Greenland P, Graham IM, Steyerberg EW, Ikram MA, Stricker BH, Hofman A & Franco OH. Comparison of Application of the ACC/AHA Guidelines, Adult Treatment Panel III Guidelines, and European Society of Cardiology Guidelines for Cardiovascular Disease Prevention in a European Cohort. JAMA 2014. Calof OM, Singh AB, Lee ML, Kenny AM, Urban RJ, Tenover JL & Bhasin S. Adverse events associated with testosterone replacement in middle-aged and older men: a meta-analysis of randomized, placebo-controlled trials. J Gerontol A Biol Sci Med Sci 2005 60 1451-1457. Haddad RM, Kennedy CC, Caples SM, Tracz MJ, Bolona ER, Sideras K, Uraga MV, Erwin PJ & Montori VM. Testosterone and cardiovascular risk in men: a systematic review and meta-analysis of randomized placebocontrolled trials. Mayo Clin Proc 2007 82 29-39. Fernandez-Balsells MM, Murad MH, Lane M, Lampropulos JF, Albuquerque F, Mullan RJ, Agrwal N, Elamin MB, Gallegos-Orozco JF, Wang AT, Erwin PJ, Bhasin S & Montori VM. Clinical review 1: Adverse

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Page 24 of 30

623 624

FIGURE LEGENDS

625 626 627

Figure 1 Multivariable linear regression of the association between tertiles of dosage of statin

628

therapy in all current users and serum total testosterone levels.

629 630

Legend Figure 1:

631

SHBG, sex-hormone binding globulin; CI, Confidence interval; DDD, defined daily doses.

632

A. Total Testosterone; B. Non-SHBG-bound Testosterone.

633

Tertiles of statin drug dosage compared to never use as the reference category. Analyses are

634

adjusted for age, body mass index, history of cardiovascular disease, diabetes mellitus,

635

hypertension, and estradiol level.

636

24

Page 25 of 30

TABLES Table 1 Characteristics of the study population of 4166 male participants.

Current Users

Past users

Never users

(N = 577)

(N = 148)

(N = 3441)

Age (mean ± SD, years)

64.1 ± 8.1

64.6 ± 7.9

64.6 ± 9.7

Body mass index (mean ± SD, kg/cm²)

28.1 ± 3.9#

28.0 ± 4.1*

26.8 ± 3.5

History of cardiovascular disease (n, %)

179 (31.0%)

#

39 (26.4%)*

115 (3.3%)

Type 2 diabetes mellitus (n, %)

100 (17.3%)#

24 (16.2%)*

185 (5.4%)

Hypertension (n, %)

298 (51.9%)

#

72 (49.0%)*

896 (26.0%)

Total T (median, IQR, nmol/L)

14.8 (11.5 – 18.8)

15.5 (12.0 – 19.9)*

17.0 (13.3 – 21.4)

Non-SHBG-bound T (median, IQR, nmol/L)

8.1 (6.6 – 9.9)#

8.2 (6.8 – 9.7)*

8.8 (7.2 – 10.6)

Insulin (median, IQR, pmol/L)

90 (63 – 136)

90 (62 – 137)*

71 (49 – 101)

Estradiol (median, IQR , pmol/L)

105 (79 – 132)#

97 (75 – 122)

99 (77 – 126)

SHBG (median, IQR, nmol/L)

40 (31 – 52)

42 (33 – 57)*

46 (35 – 58)

DHEA (median, IQR, nmol/L)

8 (6 – 14)#

8 (6 – 14)*

10 (6 – 15)

DHEAS (median, IQR, µmol/L)

2.44 (1.43 – 3.90)

2.48 (1.49 – 3.87)

2.81 (1.73 – 4.31)

Total cholesterol (median, IQR, mmol/L)

4.69 (4.14 – 5.32)#

5.47 (4.70 – 6.31)*

5.59 (4.96 – 6.20)

Duration of current use (mean ± SD, days)

190.8 ± 185.3

-

-

Statin Defined Daily Doses (mean ± SD)

1.6 ± 1.2

-

-

-

-

#

#

#

Type of statin

Simvastatin (C10AA01)

345 (59.8%)

Pravastatin (C10AA03)

68 (11.8%)

Fluvastatin (C10AA04)

34 (5.9%)

Atorvastatin (C10AA05)

112 (19.4%)

Rosuvastatin (C10AA07)

18 (3.1%)

#

Abbreviations: SD, standard deviation; T, testosterone; IQR, interquartile range; SHBG, sex hormonebinding globulin; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone

Page 26 of 30

sulphate.#current/*past users are statistically significantly different from never users (P1-≤6 Months current use

-1.24 (0.47)

-2.17 ; -0.31

0.009

288

>6 Months current use

-1.14 (0.48)

-2.07 ; -0.20

0.017

226

≤6 Months past use

-1.17 (0.62)

-2.39 ; 0.05

0.061

81

>6 Months past use

-0.35 (0.67)

-1.67 ; 0.97

0.600

67

(ref)

-

-

3441

Current use

-0.35 (0.17)

-0.68 ; -0.01

0.042

577

Past use

-0.26 (0.20)

-0.65 ; 0.13

0.191

148

(ref)

-

-

3441

≤1 Month current use

-0.29 (0.30)

-0.89 ; 0.31

.341

78

>1-≤6 Months current use

-0.42 (0.20)

-0.82 ; -0.02

.039

288

>6 Months current use

-0.29 (0.21)

-0.70 ; 0.11

.153

226

≤6 Months past use

-0.34 (0.27)

-0.87 ; 0.18

.200

81

>6 Months past use

-0.17 (0.29)

-0.73 ; 0.40

.563

67

Current and Past use categories

c

Never use

Non-SHBG-bound testosterone

Current – Past – Never useb

Never use

Current and Past use categoriesc

Never use

Abbreviations: β, beta; SE, standard error; CI, Confidence interval; SHBG, sex-hormone binding globulin. a Analyses are adjusted for age, body mass index, history of cardiovascular disease, diabetes mellitus, hypertension, estradiol level, and statin dose. b Current and past use of statin therapy compared to c never use as the reference category. Current and past use categories: the 1-month current use cutoff was applied to investigate whether statins exert an effect already after 1 month of therapy; the 6-

Page 28 of 30

month current and past use cut-off was applied based on the median duration of current and past use in the population. Bold values indicate a statistically significant association.

Page 29 of 30

1

Table 3 Sensitivity analysis: multivariable linear regression on the association between use of

2

statin therapy and serum total testosterone levels – recent past users reallocated.

β (SE)a

95% CI

P-value

N

Total testosterone

Current – Past – Never use

b

Never use

(ref)

-

-

3441

Current use

-1.31 (0.38)

-2.06 ; -0.56

0.001

600

Past Use

-0.49 (0.50)

-1.47 ; 0.49

0.331

125

(ref)

-

-

3441

Current use

-0.42 (0.16)

-0.74 ; -0.10

0.011

600

Past Use

-0.12 (0.21)

-0.54 ; 0.30

0.569

125

Non-SHBG-bound testosterone

Current – Past – Never useb

Never use

3

Abbreviations: β, beta; SE, standard error; CI, Confidence interval; SHBG, sex-hormone

4

binding globulin.

5

a

6

mellitus, hypertension, estradiol level, and statin dose. b Current and past use of statin therapy

7

compared to never use as the reference category.

8

Bold values indicate a statistically significant association.

Analyses are adjusted for age, body mass index, history of cardiovascular disease, diabetes

9 10

1

Page 30 of 30

140x76mm (300 x 300 DPI)