FEATURED NEW INVESTIGATOR

Testosterone, thrombophilia, thrombosis

JOEL FREEDMAN, CHARLES J. GLUECK, MARLOE PRINCE, RASHID RIAZ, and PING WANG

CINCINNATI, OHIO

Joel Freedman, MD, is a third yeat the Jewish Hospital Internal Marticle is based on a presentationMeeting of the Central SocietyResearch and the Midwestern SeMedical Research, held in ChicagFrom the Jewish Hospital Interna

Cincinnati, Ohio; Cholesterol, Met

Jewish Hospital of Cincinnati, Cinc

We screened previously undiagnosed thrombophilia (V Leiden–prothrombin muta-tions, Factors VIII and XI, homocysteine, and antiphospholipid antibody [APL]syndrome) in 15 men and 2 women with venous thromboembolism (VTE) orosteonecrosis 7 months (median) after starting testosterone therapy (TT), gel(30–50 mg/d), intramuscular (100–400 mg/wk), or human chorionic gonadotropin(HCG) (6000 IU/wk). Thrombophilia was studied in 2 healthy control groups withoutthrombosis (97 normal controls, 31 subjects on TT) and in a third control group(n 5 22) with VTE, not on TT. Of the 17 cases, 76% had $1 thrombophilia vs 19% of97 normal controls (P , 0.0001), vs 29% of 31 TT controls (P 5 0.002). Cases differedfrom normal controls by Factor V Leiden (12% vs 0%, P 5 0.021), by high Factor VIII(.150%) (24% vs 7%, P 5 0.058), by high homocysteine (29% vs 5%, P 5 0.007),and from both normal and TT controls for APL syndrome (18% vs 2%, P 5 0.023, vs0%, P 5 0.04). Despite adequate anticoagulation with TT continued after the firstdeep venous thrombosis–pulmonary embolus (DVT-PE), 1 man sustained 3 DVT-PEs5, 8, and 11 months later and a second man had 2 DVT-PEs 1 and 2 months later.Of the 10 cases with serum T measured on TT, 6 (60%) had supranormal T (.800 ng/dL) and of 9 with estradiol measured on TT, 7 (78%) had supranormal levels(.42.6 pg/mL). TT interacts with thrombophilia leading to thrombosis. TT continuationin thrombophilic men is contraindicated because of recurrent thrombi despite anti-coagulation. Screening for thrombophilia before starting TT should identify subjects athigh risk for VTE with an adverse the risk to benefit ratio for TT. (Translational Research2015;165:537–548)

Abbreviations:APL¼antiphospholipid antibody; DVT¼deep venous thrombosis; E2¼ estradiol;FV ¼ Factor V; HRT ¼ hormone replacement therapy; ON ¼ osteonecrosis; PE ¼ pulmonaryembolus; PTG ¼ prothrombin gene mutation; T ¼ testosterone; TT ¼ testosterone therapy; VTE¼ venous thromboembolism

INTRODUCTION

A ndrogen use in men aged $40 has increasedmore than 3-fold from 0.81% in 2001 to2.91% in 2011.1 The broad use of testosterone

therapy (TT) may have major public health ramifica-

ar resident in Internal Medicineedicine Residency Program. Hisgiven at the Combined Annualfor Clinical and Translationalction American Federation foro, Ill, on April 2014.l Medicine Residency Program,

abolism, and Thrombosis Center,

innati, Ohio.

tions, given recent reports of thrombotic2-7 andcardiovascular disease (CVD) events8-10 associatedwith TT. Despite lessons learned about venousthromboembolism (VTE) and CVD associated withsex-hormone therapy in postmenopausal women from

Submitted for publication September 30, 2014; revision submitted

December 16, 2014; accepted for publication December 17, 2014.

Reprint requests: Charles J. Glueck, Cholesterol, Metabolism, and

Thrombosis Center, Suite 430, 2135 Dana Avenue, Cincinnati, OH

45207; e-mail: sonar16@gmail.com.

1931-5244/$ - see front matter

� 2015 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.trsl.2014.12.003

537

AT A GLANCE COMMENTARY

Freedman J, et al.

Background

When testosterone therapy (TT) is given to men

and women, thrombotic events occur, reflecting

thrombophilic interactions between TT and famil-

ial and acquired thrombophilia-hypofibrinolysis.

Translational Significance

TT appears to interact with thrombophilia leading

to thrombosis. TT continuation in thrombophilic

men is contraindicated because of recurrent

thrombi despite adequate anticoagulation.

Screening for thrombophilia (PCR measures of

the Factor V Leiden, and prothrombin gene muta-

tions, Factors VIII and XI, homocysteine, and

antiphospholipid antibodies) before starting TT

should identify subjects at high risk for venous

thromboembolism with an adverse risk/benefit ra-

tio for TT.

Translational Research538 Freedman et al May 2015

the Women’s Health Initiative,11,12 TT is oftenindiscriminately prescribed to middle-age, obese menwithout understanding of its long-term risks.13 Manyof these men do not meet highly specific criteria fordiagnosis and therapy of hypogonadism.14 Testosterone(T) levels fall with increasing age,15,16 with chronicdisease17,18 and obesity,17 and rise with smoking.19,20

Part of the problem of increased TT use lies indetermination of an age-specific lower normal rangefor T, because most normal ranges come from healthyyounger men.21 There are also differences in T assaymethods22,23 and recognition that adverse musclesymptoms occur at different T levels in differentsubjects.24

Previously, in aggregate,2-7 we have described VTEdeveloping after 5 months (median) in 42 patients onTT, 38 men and 4 women, including 27 with deepvenous thrombosis–pulmonary embolism (DVT-PE),12 with osteonecrosis (ON),4,25,26 1 with centralretinal vein occlusion (CRVO), 1 with amaurosisfugax, and 1 with spinal cord infarction. None of the42 cases in our previous studies2-7 had increasedhemoglobin or uncontrolled hypertension during TT,which might have contributed to their thrombi. Ofthe 40 cases having studies of thrombophilia, 39 werefound to have previously undiagnosed thrombophilia-hypofibrinolysis, including 28% heterozygous for theFactor V (FV) Leiden mutation, 28% with high Factor

VIII, and 15% with high Factor XI.7 In 8 men whoseTT was continued, second thrombotic event occurreddespite adequate anticoagulation with warfarin.7

Beyond interacting with familial and acquired throm-bophilia,2-7 TT is associated with physiological changesthat predispose to clotting and thrombosis includinghypertension,27 increased hemoglobin,28 low high-density lipoprotein cholesterol,27,28 hyperviscosity,and platelet aggregation.28-30 Dihydrotestosteroneenhances monocyte activation,31 further promotingacute coronary events.32 TT also increases circulatingestrogens33 that subsequently play a role in thromboticevents.7 Oral contraceptives and hormone replacementtherapy (HRT)34,35 have been identified as risk factorsfor VTE in women. Given that T is converted byaromatization to estradiol (E2),36 it may be prothrom-botic by the same mechanism as reported in women,where HRT interacts with the FV Leiden mutation to in-crease risk of VTE.37

In June 2014, on the basis of postmarketing surveil-lance reports including our studies,2-7 both the USFood and Drug Administration (FDA)38 and CanadaHealth39 added a warning regarding the risks of VTEto the label of all T products. VTE, particularly PE, isassociated with significant mortality risk.40 The FDAhad previously warned about a TT-associated increasein VTE in men with a diagnosis of increased hemoglo-bin.28 However, the June 2014 FDA warning38 wasbased on reports of VTE in men without increased he-moglobin.In the present report, our specific aim was to further

focus on thrombotic events in 17 newly reported pa-tients on TT (1 receiving HCG injections as a form ofTT41), subsequently found to have previously undiag-nosed familial and acquired thrombophilia. Our secondspecific aim was to assess thrombophilia in our totalcohort of 57 patients who sustained VTE on TT, 17newly reported and 40 previously reported.7 Our thirdspecific aim was to compare thrombophilia in 57 caseswho developed VTE on TT, in 97 healthy normal con-trols, in 31 men receiving TT without VTE, and in 22cases with VTE, not taking TT.

MATERIALS AND METHODS

Patients. The procedures followed were in accordancewith the ethical standards of the institutional reviewboard of the Jewish Hospital, Cincinnati, Ohio, whichapproved the research protocol. The protocol was car-ried out with the understanding, and signed informedconsent was taken from each participant.We excluded patients and controls whose VTE was

associated with cancer, polycythemia vera, recent soft

Fig 1. Percent of subjects with no thrombophilia or $1 abnormality

of thrombophilia (V Leiden mutation, prothrombin gene mutation,

high Factors VIII, XI, homocysteine, and antiphospholipid antibody

[APL] syndrome), in 17 cases with venous thromboembolism on

testosterone therapy (TT-VTE), 97 healthy normal controls, 31 con-

trols on TT without VTE (TT controls), and 22 controls with VTE

not on TT (NTT-VTE). Comparisons were made by Fisher’s exact

test (significance level, P , 0.05).

Fig 2. Differences in thrombophilias between 17 venous thromboem-

bolism on testosterone therapy (TT-VTE) cases and 97 normal

controls, 31 TT controls, and 22 controls with VTE not on TT

(NTT-VTE). Comparisons were made by Fisher’s exact test (signifi-

cance level, P , 0.05).

Translational ResearchVolume 165, Number 5 Freedman et al 539

tissue trauma, bone fracture, hip-knee-foot surgery,airline flights .8 hours, or extensive immobilization.Fifteen men and 2 women were studied in their serial

order of referral in the last 14 months in our ThrombosisCenter by their family physicians-urologists because ofthrombotic events. TT had been prescribed by the refer-ring family physicians-urologists, and, if not alreadystopped, it was discontinued by us before our assessmentof thrombophilia and hypofibrinolysis. Measurement ofthrombophilia-hypofibrinolysis was carried out at least2 months after the initial thrombotic event (Figs 1and 2). We focused on 6 thrombophilias previouslyimplicated7 in a TT-thrombophilia interaction for VTE(FV Leiden25 and prothrombin gene mutations[PTGs], high Factors VIII and XI, high homocysteine,and the antiphospholipid antibody [APL] syndrome).The 17 newly acquired cases of the present study were

added to 40 previously reported cases7 to provide 57cases for comparison with controls (Figs 3 and 4).At study entry, a detailed history42-44 for VTE, DVT,

PE, recurrent miscarriage, pre-eclampsia, eclampsia,and hemolysis-elevated liver enzymes-low plateletcount syndrome was taken for all the cases and theirfirst-degree relatives, along with a history of cigarettesmoking.In the 17 cases and 31 controls receiving TT but

without VTE, total serum T and E2 were measured inblood drawn in the morning.

Controls. Six tests for thrombophilia (FV Leiden,PTG, Factors VIII and XI, homocysteine, and APL syn-drome) were carried out in 110 healthy controls (46 men

and 64 women), free of VTE, not on TT, and in a secondcontrol group, 56 healthy subjects (52 men and 4women) who took T (TT controls), with neither controlgroup having DVT-PE or ON. A third control groupincluded 22 cases referred to us for evaluation ofVTE, not taking TT (NTT-VTE controls).In 31 TT controls and the 22 NTT-VTE controls, at

study entry, a detailed history42-44 for VTE, DVT, PE,recurrent miscarriage, pre-eclampsia, eclampsia, andhemolysis-elevated liver enzymes-low platelet countsyndrome was taken for all probands and their first-degree relatives, along with a history of cigarette smok-ing.In our analysis to optimize comparability of cases and

controls, we included cohort of only those cases andcontrols having complete data for the 6 tests of throm-bophilia (FV, PTG, Factors VIII and XI, homocysteine,and APL syndrome), including 97 of the 110 normalcontrols, 31 of the 56 TT controls, and all 22 of theVTE controls (Table I).

Laboratory assessment of thrombophilia. Polymerasechain reaction assays. Polymerase chain reaction(PCR) measures of thrombophilia (G1691A FV Leidenand G20210A prothrombin) were performed in all casesand controls using previously published methods25,26 bylaboratory staff blinded to the participants’ status(diagnosis and severity of disease).

Serologic measures of thrombophilia. Serologic mea-sures of thrombophilia included Factors VIII and XI,homocysteine, and the APL syndrome (anticardiolipinantibodies [IgG, IgM], the lupus anticoagulant, andanti-beta2-glycoprotein). Previously published estab-lished methods were used.25 High homocysteine was

Fig 3. Percent of subjects with no thrombophilia or$1 thrombophilia

(V Leiden mutation, prothrombin gene mutation, high Factors VIII,

XI, homocysteine, and antiphospholipid antibody [APL] syndrome)

in 57 cases with venous thromboembolism on testosterone therapy

(TT-VTE), 97 normal controls, 31 controls on TT without VTE (TT

controls), and 22 controls with VTE not on TT (NTT-VTE). Compar-

isons were made by Fisher’s exact test (significance level, P , 0.05).

Fig 4. Difference in thrombophilias between 57 venous thromboem-

bolism on testosterone therapy (TT-VTE) cases and 97 normal

controls, 31 TT controls, and 22 controls with VTE not on TT

(NTT-VTE). Comparisons were made by Fisher’s exact test (signifi-

cance level, P , 0.05).

Translational Research540 Freedman et al May 2015

identified by levels .the 95th percentile45 for controls.The 3 components of the APL syndromewere measuredby enzyme-linked immunosorbent assay using previ-ously published methods.46-48

Measures of T and E2. Total T, E2, and free E2 weremeasured by LabCorp in a single laboratory. Total Tand E2 were measured by electrochemiluminescenceimmunoassay.49

Statistical methods. All statistical analyses were doneusing Statistical Analysis System version 9.4. Wilcoxonnonparametric tests were used for comparisons ofcontinuous variables. For categorical variables, Fisher’sexact test was used. Analysis of variance was used aftercovariance adjusting for age to compare homocysteinelevels in patients vs normal controls.

RESULTS

Patients and controls. By selection, none of the newlyreported 17 cases, the 40 previously reported cases,7 orthe 22 patients with NTT-VTE control group hadpathoetiologic causes for DVT-PE (cancer, increasedhemoglobin, antecedent trauma or surgery, recentairline travel longer than 8 hours, or extendedimmobilization).Because we did not initiate TT in either the TT-treated

patients with VTE or the TT controls without VTE, wedo not know the etiology of the hypogonadism in the 2TT-treated groups.Fifteenmen and2womenwere allCaucasianwith ages

ranging from 23 to 80, mean 546 16, median 55 years,

12 of whom were aged #60 (Table II). None of the pa-tients had sustained thrombotic events before TT.The 17 cases were older than the 97 normal controls

(43 6 12, median 42, P 5 0.006), but their age didnot differ from the 31 TT controls (52 6 12, median52) or from the 22 NTT-VTE controls (596 17, median63), P . 0.05 for both.Of the 17 cases 6 (35%) had normal body mass index

(BMI) (18.5 to ,25 kg/m2), 6 (35%) were overweight(25 to ,30), and 5 (30%) were obese (BMI $ 30)(Table II). Of the 97 normal controls, 40% had normalBMI, 28% were overweight, and 32% were obese. Ofthe 31 TT controls, 13% had normal BMI, 48% wereoverweight, and 39% were obese. Of the 22 VTE con-trols, 10% had normal BMI, 14% were overweight,and 76% were obese. BMI in the 17 cases did not differfrom normal or TT controls (P . 0.2 for both), but the22 VTE controls were heavier than the 17 cases (median33.1 vs 27.1 kg/m2, P 5 0.01, Wilcoxon).Of the 17 cases, 10 had measures of serum Twhen on

TT, and serum T was supranormal ($800 ng/dL) in 6(60%) (Table II), compared with 8 of 29 (28%) TT con-trols (P5 0.12). Median T in the cases was higher thanin the TT controls (1198 vs 460 ng/dL, P 5 0.018). Ofthe 17 cases, 9 had measures of E2 when on TT, and ofthese 9, 7 (78%) had high serum E2 $42.6 pg/mL(Table II), vs 11 of 28 (39%) TT controls (P 5 0.06).Median E2 in cases differed from TT controls (52.5 vs35.1 pg/mL, P 5 0.053).Family history of VTE was positive for 1 of the 17

cases (6%), not different (P. 0.05) from the 31 TT con-trols (0%) or the 22 VTE controls (10%).

Table

I.Comparisonofthrombophiliain

the97norm

alc

ontrols,31controlsontestosteronetherapy(TT),and22controlswithVTE

notta

kingTT

Norm

al

FactorV

PTG

FactorVIII

FactorXI

Homocysteine*

APLsy

ndrome

$1of6thrombophiliaabnorm

alities

CC

CC

#150%

#150%

Umol/L

Negativ

e

Percentageof97norm

alcontrolswith

thrombophilia

0/97(0%)

3/97(3%)

7/97(7%)

3/97(3%)

5/97(5%)

2/97(2%)

18/97(19%)

Percentageof31TTcontrolswith

outVTE

vs97norm

alcontrols

1/31(3%)

NS

1/31(3%)

NS

4/31(13%)

NS

1/31(3%)

NS

3/31(10%)

NS

0/31(0%)

NS

9/31(29%)

NS

Percentageof22VTEcontrolswith

outTT

vs97norm

alcontrols

3/22(14%)

P5

0.0056

1/22(5%)

NS

11/22(50%)

P,

0.0001

1/22(5%)

NS

5/22(23%)

P5

0.019

1/22(5%)

NS

12/22(55%)

P5

0.0018

Percentageof31TTcontrolswith

outVTE

vs22controlswith

VTEwith

outTT

NS

NS

P5

0.005

NS

NS

NS

P5

0.089

Abbreviations:APL,antiphosp

holip

idantibody;C

C,w

ild-typenorm

al;NS,

nonsignificant;PTG

,prothrombin

genemuta

tion;TC,h

eterozygote

muta

nt;TT,h

omozygousmuta

nt;VTE,v

enousthrom-

boembolism.

Bold

signifiesstatisticalsignificancese

parate

from

thePvalueitse

lf.*H

omocysteinedatednorm

alrange(,

laboratory

95th

percentile):,13.5

mmol/L(b

efore

March21,2

005),,12.0

(March21,2

005to

March27,2

006),,10.4

(March28,2

006to

April14,2

008),,11.4

(April15,2008to

November14,2008),and,15(sinceNovember15,2008).

Translational ResearchVolume 165, Number 5 Freedman et al 541

Of the 17 cases, 1 smoked (6%), not different (P. 0.05)from 0 of 31 TT controls (0%) or 3 of 22 NTT-VTE con-trols (14%).

Thrombotic events in cases. Of the 15 men and 2women, 6 (case IDs 1, 3, 5, 10, 11, and 12, Table II)had DVT alone, 4 (case IDs 2, 6, 8, and 9) had DVT-PE, 1 (case ID 4) had PE alone, 1 (case ID 7) hadCRVO and DVT, 2 (case ID14 and 15) had CRVOalone, and 3 (case ID 13, 16, and 17) had ON, In 15men and 2 women, thrombotic events occurred7 months (median) after starting TT, with 9 eventsfrom 1 to 7 months after starting TT and 8 eventsfrom 12 to 36 months (Table II).After the initial thrombotic event, TTwas continued in

2 men (case IDs 2 and 9), with concurrent anticoagula-tion (Table II). In case ID 2, despite adequate anticoagu-lation with warfarin (international normalizedratio $ 3.0), DVT-PE occurred a second, third, andfourth time 5, 8, and 11 months after the initial event(Table II). In case ID 9, after an initial right arm and sub-clavian thrombus and PE, second and third DVT-PEoccurred 1 and 2 months after the initial event, despitefull dose Xarelto (20 mg/d) (Table II).

Thrombophilia. Of the 17 cases, 13 (76%) had $1thrombophilia vs 19% in 97 normal controls(P , 0.0001), vs 29% in 31 TT controls (P 5 0.002),vs 55% in the 22 VTE controls (P 5 0.2) (Fig 1).Healthy normal and TT controls did not differ(P 5 0.2) in having $1 thrombophilia 19% vs 29%(Fig 1, Table I). VTE controls were more likely thanhealthy normal controls to have $1 thrombophilia55% vs 19% (P 5 0.002), but did not differ(P 5 0.09) from TT controls 29% (Fig 1, Table I).The 17 cases differed from normal controls by FV

Leiden (12% vs 0%, P 5 0.021), by high Factor VIII(.150%) (24% vs 7%, P 5 0.058) and by high homo-cysteine (29% vs 5% P 5 0.007) (Fig 2, Table III).After covariance adjusting for age, least squaresmean mean homocysteine in the 17 cases(11.5 6 0.9 mmol/L) was higher than in the normalcontrols (8.2 6 0.4), P 5 0.001. The 17 cases differedfrom both normal and TT controls for the APL syn-drome (18% vs 2% in normal controls, P 5 0.023; vs0% TT controls, P 5 0.039) (Fig 2, Table III). The17 cases did not differ (P . 0.05) from the 22 VTEcontrols for Factor VIII (24% vs 50%), for high homo-cysteine (29% vs 23%), or for the APL syndrome (18%vs 5%) (Fig 2, Table III).Adding coagulation measures from 40 recently pub-

lished cases7 to the current 17 cases allowed comparisonof 57 patients (53 men, 4 women with VTE on TT) with97 normal controls, 31 TT controls, and 22 VTE con-trols. As displayed in Fig 3 and Table IV, 57 caseswere much more likely to have $1 of the 6 major

Table II. Deep venous thrombosis (DVT)-pulmonary embolus (PE)-central retinal vein occlusion (CRVO)-osteonecrosis (ON), which developed in 17

patients receiving testosterone therapy (TT) (1 receiving HCG injections as a form of TT)

Case ID Gender AgeBodymass

index Testosterone

Event no., time between TT startand development of

Serum testosteronenormal 280–800 ng/dL

Serum estradiolnormal #42.6 pg/mL

DVT PE On Pre-Rx On Rx Pre-Rx On Rx

1 M 46 27.3 230 IM/wk 12 mo no PE 258 — 5.1 50.92 M 42 30.0 50 mg gel 1, 1 mo 1, 1 mo 120 — — 50.0

50 mg gel, warfarin 2, 6 mo 2, 6 mo50 mg gel, warfarin 3, 9 mo 3, 9 mo50 mg gel, warfarin 4, 12 mo 4, 12 mo

3 M 46 31.1 50 mg gel 3 mo no PE 192 120 42.7 —4 M 23 20.1 50 mg gel no DVT 7 mo 165 — — —5 M 75 26.7 100 mg IM 13 mo no PE 3.5 — — —6 M 80 23.5 30 mg gel 15 mo 15 mo — 750 — —7 M 72 53.0 200 IM 2/wk DVT 1 CRVO 3 mo no PE — 1500 — 1448 M 62 32.8 50 mg gel 36 mo 36 mo — 342 — 739 M 46 27.9 200 mg IM 2/wk Right arm subclavian PE

1, 14 mo 1, 14 mo 150 2000 5.9 —100 mg IM 1/wk

(Xarelto)2, 15 mo 2, 15 mo

100 mg IM 1/wk(Xarelto)

3, 16 mo 3, 16 mo

10 M 68 21.8 200 mg IM/wk 1 mo No PE 329 1500 21 7111 M 60 38.1 200 mg IM/wk 12 mo No PE 228 1198 — 5312 M 41 27.1 50 mg gel 36 mo No PE — 1467 — 5313 M 60 22.1 50 mg IM/wk No DVT No PE ON 12 mo — 1197 — 33.2, free E2 25.2

(UNL 18 pg/mL)14 M 32 22.7 HCG (6000 IU/wk)

anastrozole 1 mg/dCRVO 4 mo No DVT, no PE — 777 — 28.4

15 M 50 25.0 50 mg gel CRVO 3 mo No DVT, no PE 286 — — —16 F 40 28.0 50 mg gel 1 mo, shoulder

and knee— — — —

17 F 58 27.8 50 mg gel 6 mo, knee — — — —

Abbreviations: IM, intramuscular; UNL, upper normal limit.The bold values represent supranormal serum T or E2.

Time between TT start to development of event 10.5 6 10.8 months, median 5 7, range [1, 36].

Transla

tionalR

ese

arch

542

Freedmanetal

May2015

Table III. Coagulation disorders in 17 cases with deep venous thrombosis, pulmonary embolism, central retinal vein occlusion, osteonecrosis on

testosterone therapy (TT) compared with 97 normal controls, to 31 controls on TT without VTE, and to 22 controls with VTE, not receiving TT

Case ID, Age (y) Factor V PTG Factor VIII Factor XI Homocysteine* APL syndrome$1 of 6 thrombophilia

abnormalitiesNormal CC CC #150% #150% Umol/L Negative

1, 46 CC CC 131 94 12.9 1 Y2, 42 CC CC 234 108 9.1 2 Y3, 46 CC CC 161 133 16.5 2 Y4, 23 CC TC 110 126 8.0 2 Y5, 75 CC CC 137 72 18.6 2 Y6, 80 CC CC 115 125 19.0 2 Y7, 72 CC TC 186 145 14.1 2 Y8, 62 CC CC 76 129 17.3 2 Y9, 46 CC CC 110 105 9.3 1 Y10, 68 CC CC 238 105 8.0 2 Y11, 60 CC CC 129 125 9.2 2 No12, 41 CC CC 130 135 8.0 1 Y13, 60 CC CC 71 113 8.0 2 No14, 32 TC CC 27 135 10.0 2 Y15, 50 CC CC 55 108 11.3 2 No16, 40 TC CC 121 N 10.0 2 Y17, 58 CC CC 145 112 9.4 2 NoPercentage of 17 cases

with thrombophilia2/17 (12%) 2/17 (12%) 4/17 (24%) 0/17 (0%) 5/17 (29%) 3/17 (18%) 13/17 (76%)

Percentage of 97 normalcontrols withthrombophilia vscases

0/97 (0%)P 5 0.021

3/97 (3%)NS

7/97 (7%)P 5 0.058

3/97 (3%)NS

5/97 (5%)P 5 0.007

2/97 (2%)P 5 0.023

18/97 (19%)P , 0.0001

Percentage of 31 TTcontrols without VTEvs cases

1/31 (3%)NS

1/31 (3%)NS

4/31 (13%)NS

1/31 (3%)NS

3/31 (10%)NS

0/31 (0%)P 5 0.04

9/31 (29%)P 5 0.002

Percentage of 22 VTEcontrols without TT vscases

3/22 (14%)NS

1/22 (5%)NS

11/22 (50%)NS

1/22 (5%)NS

5/22 (23%)NS

1/22 (5%)NS

12/22 (55%)NS

Abbreviations: APL, antiphospholipid antibody;CC, wild-type normal;NS, nonsignificant; PTG, prothrombin genemutation; TC, heterozygotemutant; TT, homozygous mutant; VTE, venous throm-boembolism.Abnormal represent by bold.*Homocysteine dated normal range (,laboratory 95th percentile):,13.5 mmol/L (beforeMarch 21, 2005),,12.0 (March 21, 2005 toMarch 27, 2006),,10.4 (March 28, 2006 to April 14, 2008),,11.4

(April 15, 2008 to November 14, 2008), and ,15 (since November 15, 2008).

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tionalR

ese

arch

Volume165,Number5

Freedmanetal

543

Table

IV.Coagulationdisordersin

57case

swhohaddeepvenousthrombosis,pulm

onary

embolism,c

entralretinalvein

occlusion,o

rosteonecrosison

testosteronetherapy(TT)

comparedwith97norm

alc

ontrols,to

31controlsontestosteronetherapy(TT)

withoutVTE,andto

22controlswithVTE,not

receivingTT

Norm

al

FactorV

PTG

FactorVIII

FactorXI

Homocysteine*

APLsy

ndrome

$1of6thrombophiliaabnorm

alities

CC

CC

#150%

#150%

Umol/L

Negativ

e

Percentageof5

7caseswith

thrombophilia

13/57(23%)

4/57(7%)

15/57(26%)

6/57(11%)

10/57(18%)

5/57(9%)

43/57(75%)

Percentageof97norm

alcontrolswith

thrombophiliavs

cases

0/97(0%)

P,

0.0001

3/97(3%)

NS

7/97(7%)

P5

0.0017

3/97(3%)

P5

0.077

5/97(5%)

P5

0.022

2/97(2%)

P5

0.10

18/97(19%)

P,

0.0001

Percentageof31TTcontrolswith

outVTE

vscases

1/31(3%)

P5

0.016

1/31(3%)

NS

4/31(13%)

NS

1/31(3%)

NS

3/31(10%)

NS

0/31(0%)

NS

9/31(29%)

P,

0.0001

Percentageof22VTEcontrolswith

outTT

vscases

3/22(14%)

NS

1/22(5%)

NS

11/22(50%)

P5

0.062

1/22(5%)

NS

5/22(23%)

NS

1/22(5%)

NS

12/22(55%)

P5

0.10

Abbreviations:APL,antiphosp

holip

idantibody;C

C,w

ild-typenorm

al;NS,

nonsignificant;PTG

,prothrombin

genemuta

tion;TC,h

eterozygote

muta

nt;TT,h

omozygousmuta

nt;VTE,v

enousthrom-

boembolism.

Bold

signifiesstatisticalsignificancese

parate

from

thePvalueitse

lf.

*Homocysteinedatednorm

alrange(,

laboratory

95th

percentile):,13.5

mmol/L(b

efore

March21,2

005),,12.0

(March21,2

005to

March27,2

006),,10.4

(March28,2

006to

April14,2

008),,11.4

(April15,2008to

November14,2008),and,15(sinceNovember15,2008).

Translational Research544 Freedman et al May 2015

thrombophilias (75%) than either normal controls(19%, P , 0.0001) or TT controls (29%, P , 0.0001).As displayed in Fig 4 and Table IV, 57 cases were

much more likely than normal controls to have FV Lei-den heterozygosity, high Factor VIII, and high homo-cysteine, and were also more likely to have V Leidenheterozygosity than the TT controls. VTE controls didnot differ significantly from the 57 TT-VTE casesfor FV 14% vs 23%, high Factor VIII 50% vs 26%(P 5 0.06), and high homocysteine 23% vs 18%(Fig 4, Table IV).Fifty-seven cases with VTE on TT did not differ

(P. 0.05) from the 31 TT controls or the 22 VTE con-trols for a positive family history for VTE 4% vs 0% vs10%, and did not differ for smoking 11% vs 0% vs 14%(P . 0.05 for all).

Testosterone and estradiol. Of the 57 cases with VTEon TT, 14 had measures of E2 when taking TT (1 takingHCG). E2 or free E2 was high (.42.6, .18 pg/mL) inthe 5 cases receiving intramuscular T, and E2 high in 7of 8 on T gel (P5 1.0). Of the 57 cases with VTE on TT,16 had measures of serum T when taking TT, 10 on Tgel, and 5 on intramuscular T, and 1 on HCG. SerumT was high (.800 ng/dL) in 5 of 5 cases on intramus-cular T and 2 of 10 cases on T gel, Fisher’s P 5 0.007.

DISCUSSION

As shown in the present report of 15 men and 2women who sustained thrombotic events on TT and inour previous report,7 TT appears to interact with throm-bophilia leading to thrombosis. Of the 17 cases, 76%had $1 abnormality of the 6 measured thrombophilias,higher than in 97 healthy controls (19%, P , 0.0001)and higher than in 31 TT controls (29%, P 5 0.0024).In the present study, FV Leiden (12%), high FactorVIII (24%), high homocysteine (26%), and the APLsyndrome (18%) were more common in the 17 caseswho sustained VTE on TT than in healthy normal con-trols, and cases also were more likely to have the APLsyndrome than in TT controls free of thrombotic events(18% vs 0%).In the elderly, mild reduction of renal function is

accompanied by increased serum homocysteine,50 andof the 5 cases with serum homocysteine .normals’95th percentile, the second highest levels were in menaged 75 and 80 years, whereas the remaining 3 subjectswith hyperhomocysteinemia were 46, 62, and 72 yearsold. However, after adjusting for age, mean homocyste-ine in the 17 cases differed from the 97 normal controls(11.5 vs 8.2 mmol/L, P 5 0.001).Adding the 17 new cases of the present report to those

40 previously reported,7 the 57 cases were more likelyto have $1 thrombophilia than 97 normal controls

Translational ResearchVolume 165, Number 5 Freedman et al 545

(75% vs 19%), or 31 TT controls (75% vs 29%). FV Lei-den heterozygosity (23%), high Factor VIII (26%), andhigh homocysteine (18%) were more common in 57cases than in 97 healthy normal controls (0%, 7%, and5%). FV Leiden heterozygosity was also more commonin the 57 cases than in TT controls (23% vs 3%).We do not believe that the high percentage of our pa-

tients with high Factor VIII (26%) in our study wasdriven by TT, because the percentage of subjects withhigh Factor VIII in our TT controls (13%) did not differ(P5 0.46) from that in 97 normal controls not receivingTT (7%). TT has not been reported to raise FactorVIII,51 and in a study of risk of thrombosis associatedwith high Factor VIII,52 TT was not recognized as acause of high Factor VIII.Mild-moderate homocysteinemia was much more

common in our 57 cases than in the 97 normal controls(18% vs 5%, P5 0.022). Mild-moderate homocysteine-mia in the range of our patients is a recognized risk fac-tor for venous thrombosis45,53-55 and particularly forocular vascular thrombosis.56

The percentage of our 57 patients who developedVTE after starting TT with $1 thrombophilia (75%)was higher than in the 97 normal controls (19%) orthe 31 TT controls (29%). Moreover, the percentageof our 57 patients with familial and acquired thrombo-philia (23%VLeiden heterozygotes, 7% PTG heterozy-gotes, 26% high Factor VIII, 11% high Factor XI, 18%high homocysteine, and 9% APL syndrome) was com-parable with or higher than reported studies of patientswith VTE not taking TT.57-61 Turan et al61 observed FVLeiden heterozygosity in 19% of patients with PE. In astudy of 3005 patients with VTE, Gadelha et al57 re-ported that 345 (11.4%) had the FV Leiden mutationand 8.7% had the PTG. Ivanov et al58 reported thatthe V Leiden mutation was present in 23.5% of patientswith PE and the PTG in 5.9%. In 44 patients with DVT,Caprini et al59 found 23% with the V Leiden mutation.In 318 patients with DVT, Ben Salah et al60 reported that22.6% had hereditary thrombophilia and 19.1% had theAPL syndrome.We speculate that TT is likely to result in VTE among

men or women3 with underlying familial or acquiredthrombophilia. Similarly, in women on HRT, the pres-ence of the FV Leiden mutation or high Factor VIII in-creases the risk of DVT 17-fold compared with womenon HRT without these thrombophilias.62 Moreover, thetime course between starting TT and VTE, 5 months(median) in our first 42 patients7 and 7 months (median)in the current 17 patients, is comparable with VTE inwomen receiving HRT, where VTE rates are highestin the first year of treatment and are much more pro-nounced in women at higher risk for VTE by virtue ofthrombophilia.35

In our study, family history of VTE was low, being6% in the 17 cases who developed VTE on TT, 0% inthe 31 TT controls, and 10% in the 22 VTE controls.These findings were congruent with previousstudies,42-44 emphasizing that family history is a weakpredictor of VTE. Cigarette smoking (6%) also didnot differ between our 17 patients with VTE on TT vsour 31 TT controls (0%) or our 22 VTE controls (14%).In men, most E2 comes from aromatization of T.63We

speculate that when exogenous T is aromatized to E2and E2-associated thrombophilia is superimposed on fa-milial and acquired thrombophilia, as in our present andprevious7 reports, thrombosis occurs. We have previ-ously reported that 45% of men receiving conventionalgel TT (50 mg/d) have high serum E2 ($42.6 pg/mL).4

In the present study, of the 9 men having measures of E2on TT, 7 (78%) had high E2. The high E2 may alsoreflect overtreatment with TT. Of the 10 men havingmeasures of T during TT, 6 (60%) had supranormalvalues (.800 ng/dL), and 5 of these 6 men also hadhigh E2 or free E2. We speculate that in the 17 caseswith VTE in the present study, high T and E2 on TTcontributed to their VTE, because median T in the 17cases was higher than in the 31 TT controls (1198 vs460 ng/dL, P 5 0.018). Median E2 in the 17 caseswas higher than in the 31 TT controls (52.5 vs35.1 pg/mL, P 5 0.053).After a thrombotic event, if TT is continued, concom-

itant and adequate anticoagulation does not appear toprevent recurrent thrombotic events.7 In the presentstudy, when TTwas continued along with optimal anti-coagulation with warfarin (international normalizedratio $ 3) or (Rivaroxaban 20 mg/d) after an initialthrombotic event, 1 patient sustained 3 subsequentDVT-PEs 5, 8, and 11 months after his first DVT-PEand a second patient had 2 DVT-PEs 1 and 2 months af-ter his first DVT-PE.There are similar time intervals between starting TT

and thrombotic events (median 5 months7) or cardiovas-cular events (�3 months8-10), and we speculate that theshort duration between starting TT and thrombotic andcardiovascular events may indicate a sharedthrombotic pathophysiology, because CVD events�3 months after starting TT cannot reflect aconventional arterial atherosclerotic event.Before starting TT, to facilitate prevention of VTE,

we recommend PCR measures of the FV Leiden andPTGs, as well as measures of Factors VIII and XI, ho-mocysteine, and the APL syndrome. In a parallelfashion, the issue of pretreatment thrombophiliascreening before giving exogenous estrogens has alsobeen raised in women.62,64-66

Our findings may have important clinical implica-tions, because VTE risk is an important determinant

Translational Research546 Freedman et al May 2015

of the benefit to risk ratio of TT. In women, for example,PE accounts for about one third of the incidence ofpotentially fatal VTE events associated with HRT,67

and HRT increases the risk of VTE by 2- to 3-fold.35

We studied only morbid VTE in the present study, alimitation, because mortal VTE might be associatedwith a larger percentage of subjects with thrombophilia.Because we did not initiate TT in either the TT-treatedpatients with VTE or TT controls without VTE, we donot know the etiology of the hypogonadism in the 2TT-treated groups. We had only limited serum T andE2 data in the 57 patients when on TT, with T high in7 of the 16 cases and E2 high in 12 of 14, and not enoughdata to determine whether intramuscular T led to higherT and E2 levels than T gel.An optimal study, paralleling the Women’s Health

Initiative studies in women11,12 would be prospective,placebo-controlled, and double-blind, with thromboticand CVD end points, and evaluation of increased hemo-globin, hypertension, prostate cancer, and osteoporosis.Alternatively outcomes of TT therapy could be studiedretrospectively using administrative health data fromhealth insurance sources provided that laboratory re-sults for thrombophilia measures were consistentlycoded. This study might allow an examination of aninteraction term (TT 3 thrombophilia) revealing, spec-ulatively, that TT is more likely to result in VTE amongthe subgroup of men with underlying thrombophilia.This study would also allow assessment of whethermen with low (,280 ng/dL) or normal serum T levels(280–800 ng/dL) before TT and those who reach supra-physiological levels of T (.800 ng/dL) during TT andthose have high E2 on TT (.42.6 pg/mL) are atincreased risk for thrombotic and CVD events with orwithout underlying thrombophilias.

ACKNOWLEDGMENTS

Conflicts of Interest: All authors have read the jour-nal’s policy on disclosure of potential conflicts of inter-est and have none to declare.This work was supported by the Lipoprotein Research

Fund of the Jewish Hospital of Cincinnati.All authors have read the journal’s authorship agree-

ment and the manuscript has been reviewed andapproved by all named authors.There were no external sources of editorial support

for preparation of the manuscript.

Supplementary data

Supplementary data related to this article can befound at http://dx.doi.org/10.1016/j.trsl.2014.12.003.

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