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A randomized double-blind single center study of testosterone replacement therapy or placebo in testicular cancer survivors with mild Leydig cell insufficiency (Einstein-intervention)

Open AccessPublished:May 02, 2022DOI:https://doi.org/10.1016/j.clgc.2022.04.017

      Abstract

      Introduction

      Elevated luteinizing hormone (LH) in combination with low-normal testosterone (mild Leydig cell insufficiency) is common in testicular cancer (TC) survivors and is associated with impaired insulin sensitivity and metabolic syndrome. The aim was to evaluate if testosterone replacement therapy (TRT) improves metabolic health in this subgroup of TC survivors.

      Patients and Methods

      This was a single-center, double-blind, randomized, controlled trial. The main eligibility criterion was LH above the age-adjusted upper limit of normal in combination with free testosterone in the lower half of the age-adjusted normal range (mild Leydig cell insufficiency) >1 year after TC treatment. Eligible patients were randomly assigned (1:1) to 12 months transdermal TRT (Tostran, gel, 2%) or placebo with a maximum daily dose of 40 mg. The primary outcome was difference in Δ2 hour glucose measured with oral glucose tolerance test between groups assessed at 12 months. Outcomes were assessed after 6-, 12- and 3 months post-treatment. The study was registered at www.clinicaltrial.gov (NCT02991209) and ended June 2019.

      Results

      Between October 2016 and February 2018, 140 patients were screened for eligibility and 69 were randomized to testosterone (n = 35, 51%) or placebo (n = 34, 49%). TRT was not associated with a statistically significant difference in Δ2 hour glucose compared to placebo after 12 months of treatment (0.04 mmol/L (95% CI: -0.53, 0.60)). There was no statistically significant difference in Δ2 hour insulin between the groups after 12 months of treatment (28.23 pmol/L (95% CI: -34.40, 90.86)). Similarly, TRT was not associated with significant improvement in components of metabolic syndrome. TRT was associated with a decrease in fat mass after 12 months compared to placebo (-1.35 kg, (95% CI: -2.53, -0.18)).

      Conclusion

      In TC survivors with mild Leydig cell insufficiency, TRT was not associated with improvement of metabolic health. These findings do no not support routine use of TRT in these patients.

      Keywords

      Introduction

      Testicular cancer (TC) is the most common solid cancer in young men in Western Europe with an age-standardized incidence rate of 9.3 per 100.000 per year.
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      TC patients have increased risk of Leydig cell insufficiency already at the time of diagnosis suggested by elevated luteinizing hormone (LH) and lower testosterone levels than age-matched men.
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      Preorchiectomy leydig cell dysfunction in patients with testicular cancer.
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      The pituitary-Leydig cell axis before and after orchiectomy in patients with stage I testicular cancer.
      Around 98% of patients present with unilateral TC and are treated with unilateral orchiectomy.
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      In Denmark, disease spread is present in around 30% of patients requiring additional treatment with cisplatin-based chemotherapy or abdominal radiotherapy.
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      Prognostic factors and treatment results after bleomycin, etoposide, and cisplatin in germ cell cancer: A population-based study.
      After unilateral orchiectomy, TC patients are dependent on the function of the remaining testicle to ensure sufficient testosterone production. It is generally believed that an elevation of pituitary LH secretion can compensate for the orchiectomy and thus ensure enough testosterone in circulation. However, in around 30% of TC survivors serum testosterone is in the low-normal range despite elevated LH,
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      and it has been proposed that this condition represents an uncompensated state of mild Leydig cell insufficiency where testosterone replacement therapy (TRT) might be beneficial.
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      The metabolic syndrome and disturbances in hormone levels in long-term survivors of disseminated testicular cancer.
      This hypothesis is supported by the association between low serum testosterone concentrations and metabolic syndrome reported in studies of TC patients,
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      • et al.
      The metabolic syndrome and disturbances in hormone levels in long-term survivors of disseminated testicular cancer.
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      The metabolic syndrome in cancer survivors.
      as well as the increased long-term risk of vascular damage and cardiovascular disease among TC patients treated with cisplatin-based chemotherapy.
      • Lauritsen J
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      Cardiovascular risk factors and disease after male germ cell cancer.
      ,
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      • Jan de Jong I
      • Lefrandt JD
      • Gietema JA
      Vascular aging in long-term survivors of testicular cancer more than 20 years after treatment with cisplatin-based chemotherapy.
      A proposed mechanism has been Leydig cell insufficiency causing low grade inflammation, metabolic syndrome and increased risk of cardiovascular disease.
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      • Gietema JA.
      The metabolic syndrome in cancer survivors.
      A randomized study of male survivors of hematological malignancy with mild Leydig cell insufficiency did not find significant changes in body composition and quality of life after 6 months TRT.
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      Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy.
      However, these results might not be extrapolated to TC survivors where all patients are unilaterally orchiectomized.
      In the present study the primary objective was to investigate changes in insulin sensitivity after 12 months TRT in TC survivors with mild Leydig cell insufficiency. In addition, we investigated changes in components of the metabolic syndrome including body composition, inflammatory markers and adipocytokines.

      Patients and Methods

      Study design and participants

      This single-center, double-blind, randomized, controlled trial was conducted at Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
      Eligible TC patients were between 18 and 65 years of age without relapse ≥1 year after TC treatment. All patients were treated with unilateral orchiectomy and a contralateral biopsy was obtained in all patients at the time of diagnosis according to national Danish guidelines. Patients with clinical stage I were followed on a surveillance programme without adjuvant therapy. In case of relapse after clinical stage I or in primary disseminated disease, patients were treated with 3-4 courses of bleomycin, etoposide and cisplatin with the option of abdominal radiotherapy in patients with seminoma stage II. In case of contralateral germ cell neoplasia in situ, radiotherapy was applied to the remaining testicle with a radiation dose of 16-20 Gy. Serum concentrations of calculated free testosterone were compared to values from 304 healthy men aged 30-61 years participating in a population-based cross-sectional study (Health2008) from the Copenhagen area. Reference ranges were calculated using generalized additive model for location, scale and shape statistics allowing age-specific SD-calculations.
      • Damgaard–Olesen A
      • Johannsen TH
      • Holmboe SA
      • et al.
      Reference ranges of 17-hydroxyprogesterone, DHEA, DHEAS, androstenedione, total and free testosterone determined by TurboFlow-LC–MS/MS and associations to health markers in 304 men.
      Reference curves for serum LH vs. chronological age were constructed based on serum LH from 839 healthy men originated from 2 population-based studies.
      • Gyllenborg J
      • Rasmussen SL
      • Borch-Johnsen K
      • Heitmann BL
      • Skakkeb[aelig]k NE
      • Juul A
      Cardiovascular risk factors in men: The role of gonadal steroids and sex hormone-binding globulin.
      ,
      • Rasmussen SL
      • Torp-Pedersen C
      • Borch-Johnsen K
      • Ibsen H.
      Normal values for ambulatory blood pressure and differences between casual blood pressure and ambulatory blood pressure.
      Based on these men, reference curves for LH (mean, lower limit, upper limit) versus chronological age were constructed as described in.
      • Aksglaede L
      • Andersson AM
      • Jørgensen N
      • et al.
      Primary testicular failure in Klinefelter's syndrome: The use of bivariate luteinizing hormone-testosterone reference charts.
      Mild Leydig cell insufficiency was defined as serum concentration of free testosterone below the age-adjusted mean and above the age-adjusted lower limit of normal (LLN) (-2 standard deviations (SD)) in combination with serum LH above the age-adjusted upper limit of normal (ULN) (+2 SD). No universal definition of mild Leydig cell insufficiency exists, but we considered our definition to be appropriate and with the strength of taking age-related changes in LH and free testosterone into account. Presence of mild Leydig cell insufficiency was confirmed by a blood sample on a separate day.
      Due to slower recruitment than anticipated, a protocol amendment was decided on an investigator meeting, which was approved and implemented on November 10, 2017, as continuing with the same inclusion criteria would not make it possible to complete the study. The amendment allowed inclusion of patients with serum free testosterone below the age-adjusted ULN (+2 SD) and above -3 SD from the age-adjusted mean in combination with serum LH above +1 SD.
      Exclusion criteria were testosterone replacement therapy within 6 months prior to inclusion, contraindications to TRT, diabetes mellitus or paternity wish at time of inclusion. Additional details regarding inclusion and exclusion criteria are presented in the study protocol.
      • Bandak M
      • Jørgensen N
      • Juul A
      • et al.
      A randomized double-blind study of testosterone replacement therapy or placebo in testicular cancer survivors with mild Leydig cell insufficiency (Einstein-intervention).
      The study was approved by the Regional Ethics Committee for the Capital Region of Denmark (Protocol: H-15008901), the Data Protection Agency (Journal: 2012-58-0004) and the Danish Health Authorities (EudraCT: 2015-001452-30). All patients received oral and written information about the aims and concepts of the study and possible risks involved and gave written informed consent prior to inclusion.

      Randomization and masking

      Eligible patients were randomly assigned (1:1) to testosterone or placebo. Randomization was done on November 8, 2016 with the use of the web-based randomization tool (http://www.randomization.com) in 7 blocks of 10 (5 testosterone: 5 placebo). Double sealed opaque envelopes were prepared for all patients with a randomization number printed on the outside to be broken in case of suspected adverse events. The randomization sequence was done by an unblinded investigator, NJ, who also assigned patients to treatment. NJ and LA were unblinded investigators and evaluated safety issues. NJ and LA were not involved in any other trial related activities. Testosterone and placebo were supplied in identical canisters with identical labelling and a unique labelling number. Patients were assigned a trial number and a confirmatory email including the patient's trial number, name, date of birth and treatment kit numbers was sent to the investigators. Patients, investigators and study site staff were blinded to treatment assignment until the database was locked.

      Procedures

      Testosterone and LH are routinely assessed during surveillance for TC and eligible patients with mild Leydig cell insufficiency were invited for a screening visit where a blood sample was drawn to confirm mild Leydig cell insufficiency. Additional patients were included from a cross-sectional study of mild Leydig insufficiency and metabolic syndrome.
      • Bandak M
      • Jørgensen N
      • Juul A
      • et al.
      Leydig cell dysfunction, systemic inflammation and metabolic syndrome in long-term testicular cancer survivors.
      Patients fulfilling the inclusion criteria were randomly allocated to 12 months of TRT or placebo.
      Testosterone and placebo were supplied in canisters containing 60 grams of gel with a pumping mechanism delivering a fixed amount of gel. The gel was administered transdermally with a starting dose of 10 mg testosterone or placebo per day (1 depression of the pumping mechanism) applied on the stomach, thigh or shoulder. Dose titration was performed after 2 weeks to 20 mg (2 depressions of the pumping mechanism), 4 weeks to 30 mg, (3 depressions of the pumping mechanism) to a maximum dose of 40 mg (4 depressions of the pumping mechanism) after 8 weeks. At the time of study inclusion, patients were handed 5 containers while 6 containers were handed out at the 6 month's visit. Compliance was ensured by collecting empty containers and recording kit numbers. Safety parameters (serum levels of testosterone, LH, prostate specific antigen, erythrocyte volume fraction, alanine aminotransferase) were evaluated throughout the study period by the unblinded investigators NJ and LA and dose reductions were performed according to predefined criteria as shown in Supplementary Table 1. In case of dose reduction, an identical dose reduction was performed in a placebo-treated patient with age and body mass index (BMI) closest to the patient in order to keep the double-blind design (arranged by unblinded investigator NJ).
      The intervention was evaluated pre-treatment (baseline), and during treatment (6 months, 12 months), and 15 months (3 months post-treatment to further evaluate intervention, efficacy and safety). At each day of evaluation, blood samples were drawn between 07.00 AM and 11.00 AM after an overnight fast of minimum 8 hours, and an oral glucose tolerance test (OGTT) was performed where 75 grams of glucose was consumed. After 120 minutes, venous blood was drawn for determination of plasma insulin and plasma glucose. In addition, blood pressure, hip and waist circumference, height, and weight were measured. Adverse events were assessed by spontaneous reporting and systematic questioning.
      Blood samples were analyzed throughout the study period except for blood samples for inflammatory markers and adipocytokines, which were processed to plasma and stored at -80°C within 2 hours for analysis at the completion of the study in June 2019. Laboratory methodology for analysis of hormones, inflammatory markers, adipocytokines and other blood samples can be found in Supplementary Table 2.
      Dual-energy x-ray absorptiometry (DXA) scans were performed by a certified hospital technician at the Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, Copenhagen, Denmark. All patients were scanned on the same Lunar Prodigy Advance Scanner (GE Healthcare, Madison, WI, USA). The scanner was calibrated before use, using daily calibration procedures (Lunar, System Quality Assurance). Body composition, including visceral adipose tissue was calculated using the standard automatized software package (Encore v. 16, GE Healthcare).
      Patients reported outcome measures and changes in bone mineral density assessed with DXA will be included in a later publication. A detailed flowchart of study procedures is available in the study protocol.
      • Bandak M
      • Jørgensen N
      • Juul A
      • et al.
      A randomized double-blind study of testosterone replacement therapy or placebo in testicular cancer survivors with mild Leydig cell insufficiency (Einstein-intervention).

      Outcomes

      The primary outcome was change in Δ2 hour plasma glucose (mmol/L) after 12 months of treatment valuated by a 2 hour OGTT comparing the testosterone group with the placebo group.
      We decided to use the Δ2 hour plasma glucose as primary outcome as we considered it to be the most sensitive marker of insulin resistance which has been suggested as the core feature of metabolic syndrome.
      • de Haas EC
      • Oosting SF
      • Lefrandt JD
      • Wolffenbuttel BH
      • Sleijfer DT
      • Gietema JA.
      The metabolic syndrome in cancer survivors.
      ,
      • Reaven GM
      Banting Lecture 1988. Role of insulin resistance in human disease. 1988.
      A 12 months treatment duration was chosen to be sure to observe a possible effect on the outcomes as a 6 months treatment duration has been chosen in other studies,
      • Walsh JS
      • Marshall H
      • Smith IL
      • et al.
      Testosterone replacement in young male cancer survivors: A 6-month double-blind randomised placebo-controlled trial. Basu S, ed.
      and 12 months have shown to be a sufficient trial length in larger testosterone trials.
      • Snyder PJ
      • Ellenberg SS
      • Cunningham GR
      • et al.
      The Testosterone Trials: Seven coordinated trials of testosterone treatment in elderly men.
      Secondary outcomes included additional measures of insulin sensitivity; ∆2 hour insulin (pmol/L), 2 hour glucose (mmol/L) and 2 hour insulin (pmol/L) concentrations as well as the Gutt insulin sensitivity-index.
      • Gutt M
      • Davis CL
      • Spitzer SB
      • et al.
      Validation of the insulin sensitivity index (ISI0,120): Comparison with other measures.
      Additional secondary outcomes were metabolic syndrome components according to National Cholesterol Education Program (Adult Treatment Panel III) which included waist circumference (cm), triglycerides (mmol/L), HDL-cholesterol (mmol/L), fasting glucose (mmol/L), and systolic and diastolic blood pressure (mmHg).
      Expert Panel on Detection and Treatment of High Blood Cholesterol in Adults E
      Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III).
      DXA measurements related to metabolic syndrome
      • Foulkes SJ
      • Daly RM
      • Fraser SF.
      The clinical importance of quantifying body fat distribution during androgen deprivation therapy for prostate cancer.
      were evaluated and included total lean mass (kg), total lean mass divided by height^2 (kg/m2), total fat mass divided by height^2 (kg/m2), total fat mass (kg) and fraction (%), android and/or gynoid fat ratio and visceral fat mass (g).
      Adipocytokines leptin ((pg/ml) and adiponectin (ng/ml)) and inflammatory markers (tumor necrosis factor alpha (pg/ml), IL-1β (pg/ml), IL-6 (pg/ml), IL-8 (pg/ml), IL-10 (pg/ml) and high-sensitivity C-reactive protein (mg/L)) were also evaluated.
      Adverse events were reported by subjects and assessed by the investigators at all visits during the study period according to the common toxicity criteria (CTC) version 4.0. Participants were advised to contact the investigators immediately if severe adverse events were suspected. Assessments of adverse events were done before unblinding through the study. Once a year a report of the patient's safety was submitted to the Danish Health and Medicines Authority. Results were entered into EudraCT within 1 year after completion of the study.

      Sample size

      It was estimated that a minimum of 30 patients in each group was needed to detect a minimum difference of 0.5 mmol in Δ2 hour plasma glucose (mmol/L) between the intervention group and the placebo group with a statistical power of 80% and a 2 tailed significance level of 5%. The difference of 0.5 mmol was considered clinically meaningful by the investigators based on clinical experience as no minimal clinically important difference is universally accepted. The sample size calculation was based on an anticipated standard deviation of 0.83, which was based on clinical experience. Anticipating 10 dropouts, we aimed at including 35 patients in each group.

      Statistical analysis

      Initially, statistical analyses were planned to be done with independent t tests comparing baseline with measurements after 12 months.
      • Bandak M
      • Jørgensen N
      • Juul A
      • et al.
      A randomized double-blind study of testosterone replacement therapy or placebo in testicular cancer survivors with mild Leydig cell insufficiency (Einstein-intervention).
      But at a meeting (April 2019) before the study was completed and with no knowledge of data, it was decided to deviate from the original plan and use a statistical model which would take all outcome visits (baseline, 6, 12 and 15 months), individual patients and their correlation between visits into account. The decision was taken in collaboration with the study statistician (CD). Linear mixed effects models were used to compare longitudinal changes in primary and secondary outcomes between groups over time, according to the intention-to-treat principles as described by Twisk et al,
      • Twisk J
      • Bosman L
      • Hoekstra T
      • Rijnhart J
      • Welten M
      • Heymans M.
      Different ways to estimate treatment effects in randomised controlled trials.
      with adjustment for potential baseline differences. The models included fixed effects for visit number, treatment effects for visits at months 6, 12 and 15 and a random effect for each patient and for the correlation between visits for within patients we used an unstructured correlation structure, ie, not assuming a specific correlation structure. There was no allowance for multiplicity of secondary outcomes. The sample size was not re-evaluated after changing the statistical plan.
      All statistical analyses were done in R version 3.6.2 using package nlme. by R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.
      The study was independently monitored, and quality assured by the Unit for Good Clinical Practice (GCP) at the Copenhagen University Hospital, Bispebjerg, Denmark.
      The study was registered at www.clinicaltrial.gov (NCT02991209).

      Results

      Between October 2016 and February 2018, 140 patients were screened for eligibility and 69 were included and randomized to receive testosterone (n = 35) or placebo (n = 34), Figure 1. Five patients discontinued treatment between baseline and 6 months (2 in the testosterone group and 3 in the placebo group) and 6 patients discontinued treatment between 6 and 12 months (all in the testosterone group). Reasons for treatment discontinuation are shown in Figure 1.

      Baseline measures

      The median age at study inclusion was 42 years (interquartile range (IQR): 35-47) in patients treated with TRT and 44 years (IQR: 26-40) in patients treated with placebo. Median LH was 8.3 IU/L (IQR: 6.6-9.8) and 8.2 IU/L (IQR: 6.5-9.9) in the 2 groups respectively, while free testosterone was 285 pmol/L (IQR: 251-337) and 301 pmol/L (IQR: 249-361) in the 2 groups respectively. Further characteristics at baseline including TC treatment, metabolic syndrome components and self-reported lifestyle factors are presented in Table 1. The groups were well balanced apart from more patients treated with chemotherapy and/or abdominal radiotherapy in the TRT group (n = 23) compared to placebo (n = 12).
      Table 1Baseline Characteristics of 69 Included Testicular Cancer Survivors
      Testosterone (n = 35)Placebo (n = 34)
      Age at diagnosis
      Median (IQR)30 (25-38)33 (26-40)
      Age at study inclusion
      Median (IQR)42 (35-47)44 (37-49)
      Time since diagnosis, years
      Median (IQR)5 (3-10)5 (3-13)
      Ethnicity, No. (%)
      Caucasian35 (100)34 (100)
      Treatment, No. (%)
      Orchiectomy only10 (29)21 (62)
      Cisplatin-based chemotherapy13 (37)8 (24)
      Abdominal radiotherapy6 (17)2 (6)
      Chemotherapy and radiotherapy4 (11)2 (6)
      Orchiectomy and radiotherapy to the contralateral testicle due to germ cell neoplasia in situ2 (6)1 (3)
      Luteinizing hormone, IU/L
      Median (IQR)8.3 (6.6-9.8)8.2 (6.5-10.0)
      Free testosterone, pmol/L
      Median (IQR)285 (251-337)301 (249-361)
      Total testosterone, nmol/L
      Median (IQR)15.7 (12.9-17.8)14.5 (12.3-17.4)
      Body mass index, kg/m2
      Median (IQR)24.8 (23.3-27.6)27.0 (24.0-28.6)
      Waist circumference, cm
      Median (IQR)95 (89-101)98 (93-101)
      Triglycerides, mmol/L
      Median (IQR)1.2 (0.9-1.5)1.2 (1.0-1.9)
      HDL-Cholesterol, mmol/L
      Median (IQR)1.3 (1.1-1.5)1.4 (1.2-1.5)
      Fasting glucose, mmol/L
      Median (IQR)5.3 (5.0-5.7)5.2 (4.8-5.7)
      Systolic blood pressure, mmHg
      Median (IQR)132 (123-136)131 (123-141)
      Diastolic blood pressure, mmHg
      Median (IQR)79 (74-89)80 (78-87)
      Metabolic syndrome (NCEP-ATP-III), No. (%)
      Yes2 (6)4 (12)
      No33 (94)30 (88)
      Self-reported measures
      Smoking status, No. (%)
      Never13 (38)13 (38)
      Former8 (24)9 (26)
      Current13 (38)12 (35)
      Alcohol consumption, No. (%)
      0/week6 (18)6 (18)
      <7/week13 (38)16 (47)
      7-14/week10 (29)9 (26)
      14-21/week3 (9)3 (9)
      >21/week2 (6)0 (0)
      Physical activity, No. (%)
      Competitive sports1 (3)2 (6)
      Moderate ≥4h, week16 (47)13 (38)
      Modest <4h, week14 (41)16 (47)
      Sedentary lifestyle3 (9)3 (9)
      Prescription medication, No. (%)
      Lipid lowering drugs0 (0)2 (6)
      Antihypertensives1 (3)2 (6)
      Other
      Medication included: Beta2-agonists, antivirals, phosphodiesterase5 inhibitors, levothyroxine, anti-depressants, vitamin K antagonists.
      10 (29)13 (38)
      Abbreviations: IQR = Interquartile range; IDF = International Diabetes Federation; NCEP-ATPIII = National Cholesterol Education Program, Adult Treatment Panel.
      a Medication included: Beta2-agonists, antivirals, phosphodiesterase5 inhibitors, levothyroxine, anti-depressants, vitamin K antagonists.

      LH, free testosterone and dose titration

      A graphical overview of individual LH and free testosterone of included patients according to the original and amended inclusion criteria is presented in Supplementary Figure 1. Most included patients (61/69) had free testosterone levels below the age-adjusted mean and 6 patients had free testosterone levels less than -2SD. LH was above the age-adjusted upper limit (+ 2SD) in 40 of 69 patients.
      After dose titration, 26 patients were treated with 40 mg testosterone/day, 3 patients received 30 mg/day, 5 patients received 20 mg/day and 1 patient received 10 mg/day.
      Free testosterone and LH data at all patient visits are illustrated in Figure 2 while total testosterone data are illustrated in Supplemental Figure 2. After 12 months, median free testosterone was 542 pmol/L (IQR: 410-715), median total testosterone was 24.2 (IQR: 19.9-31.5) and median LH was 4.2 IU/L (IQR: 3.5-6.3) in the testosterone group while LH and free testosterone remained relatively unchanged in the placebo group, with median free testosterone 317 pmol/L (IQR: 274-347), median total testosterone 15.1 (IQR: 13.0-17.8) and median LH 7.6 IU/L (IQR: 6.3-8.7).
      Figure 2
      Figure 2Changes in free testosterone (A), luteinizing hormone (B), Δ2 hour glucose (C) and Δ2 hour insulin (D) during 12 months testosterone or placebo including evaluation 3 months post-treatment (15 months). Thin lines: Individual values. Thick lines: Mean values with 95% confidence bands. OGTT = Oral glucose tolerance test.

      Primary outcome

      Individual and mean values of Δ2 hour glucose and Δ2 hour insulin during the 12 months intervention including evaluation 3 months post-treatment (15 months) are illustrated in Figure 2, while the results from the linear mixed effects models are presented in Table 2.
      Table 2Difference in Outcomes Between Patients Treated With Testosterone and Patients Treated With Placebo During (6 Months, 12 Months) and 3 Months Post-Treatment (15 Months).
      6 months12 months15 months
      Primary outcome
      Δ2hour- glucose (mmol/L)-0.27 (-0.83, 0.30)0.04 (-0.53, 0.60)-0.46 (-1.09, 0.16)
      Insulin sensitivity
      Δ2hour- Insulin (pmol/L)27.16 (-29.19, 83.52)28.23 (-34.40, 90.86)-2.92 (-87.40, 81.56)
      Gutt-index-0.12 (-0.41, 0.17)0.02 (-0.25, 0.30)-0.19 (0.51, 0.13)
      2 hour glucose (mmol/L)-0.23 (-0.81, 0.34)-0.07 (-0.63, 0.48)-0.37 (-0.99, 0.25)
      2 hour insulin (pmol/L)18.19 (-39.11, 75.48)16.25 (-49.87, 82.36)6.39 (-80.04, 92.82)
      Metabolic syndrome components
      Waist circumference (cm)-1.22 (-3.00, 0.56)-0.62 (-2.38, 1.14)-1.18 (-3.43, 1.06)
      Triglycerides (mmol/L)-0.11 (-0.33, 0.12)-0.16 (-0.39, 0.06)-0.13 (-0.35, 0.09)
      HDL-Cholesterol (mmol/L)0.00 (-0.10, 0.10)0.04 (-0.07, 0.15)0.04 (-0.07, 0.14)
      Fasting glucose (mmol/L)0.05 (-0.18, 0.28)-0.13 (-0.31, 0.05)0.06 (-0.16, 0.28)
      Systolic blood pressure (mmHg)-0.26 (-5.31, 4.80)1.47 (-3.94, 6.88)-0.54 (-6.54, 5.45)
      Diastolic blood pressure (mmHg)0.67 (-2.25, 3.58)0.92 (-2.55, 4.39)0.28 (-3.15, 3.71)
      DXA
      Lean mass (kg)0.14 (-0.62, 0.91)0.08 (-0.72, 0.89)-0.23 (-1.26, 0.80)
      Lean mass index (kg/m2)0.05 (-0.17, 0.27)0.03 (-0.21, 0.27)-0.07 (-0.37, 0.23)
      Total fat mass (kg)-1.08 (-1.93, -0.220)-1.35 (-2.53, -0.177)-0.92 (-2.35, 0.52)
      Fat mass index (kg/m2)-0.32 (-0.58, -0.06)-0.42 (-0.78, -0.07)-0.29 (-0.72, 0.13)
      Total fat percent of total mass (%)-0.96 (-1.74, -0.18)-1.09 (-2.16, -0.02)-0.64 (-1.91, 0.63)
      Android-gynoid fat ratio0.00 (-0.05, 0.05)0.01 (-0.03, 0.04)-0.01 (-0.06, 0.03)
      Visceral fat mass (g)-12.94 (-152.95, 127.06)-50.07 (-175.80, 75.66)-49.12 (-204.12, 105.88)
      Adipocytokines
      Leptin (pg/ml)-36.21 (-1044.12, 971.70)-689.84 (-2255.58, 875.90)10.79 (-1483.27, 1504.85)
      Adiponectin (ng/ml)-291.93 (-1569.09, 985.23)44.45 (-1059.72, 1148.63)802.89 (-453.03, 2058.80)
      Inflammatory markers
      Tumour necrosis factor alpha (pg/ml)0.01 (-0.13, 0.16)-0.12 (-0.27, 0.03)0.01 (-0.15, 0.18)
      Interleukin 1β (pg/ml)-0.13 (-1.13, 0.87)0.40 (-0.69, 1.49)-0.99 (-2.07, 0.09)
      Interleukin 6 (pg/ml)0.08 (-0.21, 0.37)0.09 (-0.21, 0.40)0.07 (-0.25, 0.39)
      Interleukin 8 (pg/ml)0.12 (-0.09, 0.32)-0.06 (-0.28, 0.16)0.22 (0.00, 0.45)
      Interleukin 10 (pg/ml)0.12 (-0.16, 0.40)-0.21 (-0.50, 0.07)-0.08 (-0.37, 0.22)
      High-sensitivity C-reactive protein (mg/L)-0.04 (-0.36, 0.28)0.40 (-0.02, 0.81)-0.03 (-0.40, 0.34)
      The difference in changes between groups are reported with 95% confidence intervals and was estimated with a linear mixed effects model.
      The linear mixed effects model estimated that TRT was not associated with a significant difference in Δ2 hour glucose compared to placebo after 12 months of treatment (0.04 mmol/L (95% CI: -0.53, 0.60)).
      Furthermore, TRT was not associated with significant differences in Δ2 hour glucose compared to placebo after 6 months (-0.27 mmol/L (95% CI: -0.83, 0.30)), or 3 months post-treatment (-0.46 mmol (95% CI: -1.09, 0.16).

      Secondary outcomes

      There was no significant difference in Δ2 hour insulin between the groups after 6 months (27.16 pmol/L (95% CI: -29.19, 83.52)), 12 months of treatment (28.23 pmol/L (95% CI: -34.40, 90.86)) and 3 months post-treatment (-2.92 pmol/L (95% CI -87.40, 81.56)) (Figure 2 and Table 2).
      There were only minor, nonsignificant differences between groups in Gutt-index, 2 hour glucose and 2 hour insulin, Table 2.
      Individual and mean values of metabolic syndrome components are illustrated in Figure 3, while the results from the linear mixed effects models are shown in Table 2. TRT was not associated with significant differences in any metabolic syndrome components compared to placebo during treatment or 3 months post-treatment
      Figure 3
      Figure 3Changes in metabolic syndrome components during 12 months testosterone or placebo including evaluation 3 months post-treatment (15 months). Thin lines: Individual values. Thick lines: Mean values with 95% confidence bands.
      There were minor non-significant differences between groups in adipocytokines, inflammatory markers (Table 2).
      Individual and mean values of body composition evaluated by DXA-scan are illustrated in Supplemental Figure 3, while the results of a mixed linear model are shown in Table 2. TRT was associated with a statistically significant decrease in total fat mass compared to placebo at 6 months (-1.08 kg, (95% CI: -1.93, -0.22)), and 12 months (-1.35 kg, (95% CI: -2.53, -0.18)) and a non-significant difference 3 months post-treatment (15 months) (-0.92 kg, 95% CI: -2.35, 0.52). Similarly, TRT was associated with a statistically significant difference in fat mass index and total fat percent compared to placebo at 6 months and 12 months and a non-significant difference 3 months post-treatment.
      Finally, to account for a possible imbalance in treatment modalities between the 2 groups, we conducted an analysis adjusting for treatment modality (orchiectomy vs other treatment modalities) and this did not change any of the investigated outcomes. Data not shown.

      Safety

      Adverse events according to CTC are presented in Supplementary Table 3. Generally, the treatment was well tolerated, and no serious adverse events related to treatment were reported during the study period

      Discussion

      In this randomized double-blind study of 12 months TRT versus placebo in TC survivors with mild Leydig cell insufficiency, TRT was not associated with significant improvement in insulin sensitivity or improvement of components of the metabolic syndrome.
      As LH was effectively normalized while free testosterone was elevated to high-normal range, we are convinced that the testosterone dose used was adequate, and as less than 6 months of treatment has shown an effect on insulin sensitivity and metabolic syndrome in studies of non-cancerous men, a 12 months treatment duration should be adequate to observe an effect on these outcomes.
      • Kapoor D
      • Goodwin E
      • Channer KS
      • Jones TH.
      Testosterone replacement therapy improves insulin resistance, glycaemic control, visceral adiposity and hypercholesterolaemia in hypogonadal men with type 2 diabetes.
      To date, 2 randomized studies have investigated TRT in cancer survivors.
      • Howell SJ
      • Radford JA
      • Adams JE
      • Smets EMA
      • Warburton R
      • Shalet SM.
      Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy.
      ,
      • Walsh JS
      • Marshall H
      • Smith IL
      • et al.
      Testosterone replacement in young male cancer survivors: A 6-month double-blind randomised placebo-controlled trial. Basu S, ed.
      One study included 35 survivors of lymphoma and hematological malignancies after chemotherapy.
      • Howell SJ
      • Radford JA
      • Adams JE
      • Smets EMA
      • Warburton R
      • Shalet SM.
      Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy.
      Mild Leydig cell insufficiency was defined as LH ≥8 IU/I in combination with total testosterone in the lower half of the normal range (<20 nmol/L), which is comparable to the definition applied in the present study. Patients were randomized to 12 months testosterone 2.5 mg patches or placebo. Despite effectively normalizing testosterone and LH in the testosterone group (n = 16), no significant changes were observed in metabolic syndrome components or in fat and lean body mass assessed with DXA-scan.
      • Howell SJ
      • Radford JA
      • Adams JE
      • Smets EMA
      • Warburton R
      • Shalet SM.
      Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy.
      Similar findings were observed in the present study except for a significant decrease in fat mass. A more recent study by Walsh et al., included 136 cancer survivors (120 TC survivors) with morning total testosterone between 7 and 12 nmol/L.
      • Walsh JS
      • Marshall H
      • Smith IL
      • et al.
      Testosterone replacement in young male cancer survivors: A 6-month double-blind randomised placebo-controlled trial. Basu S, ed.
      Patients were randomized to 6 months of TRT or placebo. Testosterone replacement therapy was associated with decreased trunk fat mass, decreased whole-body fat mass and increased lean body mass assessed by DXA-scan, while there was no significant effect on metabolic syndrome components. Major contrasts to the present study were that elevated LH was not an inclusion criterion, a 6-fold higher starting dose of testosterone (60 mg vs. 10 mg) and shorter treatment duration (6 months vs. 12 months). In addition, patients appeared slightly more overweight (BMI at baseline 28 vs. 26 in the present study).
      In line with the guidelines of the Endocrine Society, we suggest that elevated LH needs to be considered to assess Leydig Cell insufficiency, as low-normal total testosterone in combination with normal or low LH is associated with obesity and not a sign of Leydig cell insufficiency.
      • Bhasin S
      • Cunningham GR
      • Hayes FJ
      • et al.
      Testosterone therapy in men with androgen deficiency syndromes: An endocrine society clinical practice guideline.
      In this group of patients, exercise and weight loss have been shown to elevate testosterone.
      • Kaukua J
      • Pekkarinen T
      • Sane T
      • Mustajoki P.
      Sex hormones and sexual function in obese men losing weight.
      Furthermore, decreased free testosterone rather than total testosterone is a better measure of Leydig cell insufficiency due to the inverse relation between SHBG and obesity.
      • Hautanen A.
      Synthesis and regulation of sex hormone-binding globulin in obesity.
      Future studies should aim to identify subgroups of TC survivors that might benefit from TRT. This could be TC survivors with mild Leydig Cell insufficiency in combination with impaired insulin sensitivity, metabolic syndrome, or sexual dysfunction. A Dutch study (NCT03339635) is currently investigating TRT in overweight (BMI>25) TC survivors with fat mass as the primary outcome. Furthermore, a simple evaluation of the pituitary-gonadal axis by assessment of LH and testosterone might not be sufficiently sensitive to evaluate Leydig cell insufficiency, and other biomarkers of Leydig cell capacity like insulin-like factor 3 (INSL3),
      • Toppari J.
      Insulin-like Factor 3 Emerges from the Shadow of Testosterone as a Leydig Cell Biomarker.
      as well as a human chorionic gonadotropin (hCG) stimulation test might be useful to identify patients with Leydig cell insufficiency with higher accuracy.
      Strengths of the present study included precise dose titration, high compliance, appropriate treatment duration, and that all outcome evaluations were done on the same equipment by the same operators.
      However, the study has several important limitations: (1) The inclusion criteria were adjusted after inclusion of 36 patients. Thus, although the study was adequately powered for the primary outcome, we cannot rule out that this influenced the analyses of secondary outcomes. We did not find it meaningful to perform a subgroup analysis of patients fulfilling the original inclusion criteria due to the small sample size, (2) There was an imbalance in treatment modalities between the 2 groups despite randomization. We tried to account for that by adjusting for treatment modality, which did not change the results, (3) The study was statistically powered to detect a 0.5 mmol difference in Δ2 hour-glucose between the groups. However, as the 95% CI of the difference in Δ2 hour-glucose presented in Table 2 includes 0.5 mmol at 12 months, it cannot be excluded that a statistically significant difference would have been found with a larger sample size, (4) The sample size calculation was based on an anticipated standard deviation in Δ2 hour-glucose of 0.83 mmol. However, the actual SD was 1.105 mmol at baseline (data not shown), which might indicate that the study was underpowered for the primary outcome, (5) The majority of included patients had limited indication of impaired insulin sensitivity at baseline suggested by the small change in ∆2 hour glucose. Thus, it could be hypothesized that it would not be possible to further improve insulin sensitivity in these patients with TRT. While this is a valid point, we would have expected to observe an improvement in ∆2 hour insulin, Gutt-index or 2 hour glucose and insulin if there was an effect of testosterone on insulin sensitivity, (6) It was a single center study which limits the generalizability of the findings.

      Conclusion

      Twelve months of TRT did not significantly change insulin sensitivity or metabolic health in TC survivors with mild Leydig cell insufficiency. Based on our present study, TRT should not be standard treatment in TCS with mild Leydig cell insufficiency. Future studies should investigate if subgroups of TC survivors, eg, TC survivors with mild Leydig cell insufficiency and metabolic syndrome or sexual dysfunction could benefit from testosterone substitution.

      Clinical Practice Points

      Testicular cancer (TC) is the most common solid cancer in young men and the high survival rate gives rise to a growing number of survivors who have required treatment related physical and psychological late effects. TC patients have increased risk of Leydig cell insufficiency already at the time of diagnosis suggested by elevated luteinizing hormone (LH) and lower testosterone levels than age-matched men. It is generally believed that an elevation of pituitary LH secretion can compensate for the orchiectomy and thus ensure enough testosterone in circulation. However, in around 30% of TC survivors serum testosterone is in the low-normal range despite elevated LH, and it has been proposed that this condition represents an uncompensated state of mild Leydig cell insufficiency where testosterone replacement therapy (TRT) might be beneficial.
      In this randomized double-blind study of TRT or placebo in 69 TC survivors with mild Leydig cell insufficiency we assessed the effect of TRT on insulin sensitivity and components of metabolic syndrome. Twelve months of treatment with TRT in testicular cancer survivors with mild Leydig cell insufficiency did not result in significantly changes in insulin sensitivity or components of metabolic syndrome. This study adds to the limited knowledge of the clinical handling of cancer survivors with low testosterone in combination with raised LH. TRT should not be standard treatment in cancer survivors with mild Leydig cell insufficiency. Future studies should focus on TRT in subgroups of cancer survivors e.g. cancer survivors with metabolic syndrome and Leydig cell insufficiency.

      Authors contribution

      Niels Jørgensen, Anders Juul, Mikkel Bandak, Jakob Lauritsen, Peter Oturai, Gedske Daugaard and Mikkel Bandak formulated the clinical question and designed the study. Michael Kreiberg, Niels Jørgensen, Anders Juul, Emma Grunwald, Lise Aksglaede, Peter Oturai, Gedske Daugaard and Mikkel Bandak were responsible for patient accrual, trial conduct, and obtaining the data. Jørn Wulff Helge, JesperFrank Christensen, Tim Schauer were responsible for analysis of biological samples. Michael Kreiberg, Christian Dehlendorff, Thomas Wagner, Josephine Rosenvilde and Mikkel Bandak analyzed the data and produced the results and figures.
      All authors participated in interpretation of the results and critically revised the manuscript, and all authors have approved the final version of the manuscript.

      Acknowledgments

      The Danish Cancer Society, The Danish Cancer Research Foundation and Rigshospitalet have supported the study. Kiowa Kirin International covered expenses for Tostran and placebo.

      Disclosure

      We declare no conflicts of interest.

      Data sharing statement

      • According to Danish law individual patient data that underlie the results reported in this paper cannot be shared. Anonymized access to data might be possible, please contact the corresponding author regarding this. Study protocol can be shared upon request.

      Appendix. Supplementary materials

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