european urology 53 (2008)

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1 european urology 53 (2008) available at journal homepage: Sexual Medicine An Evaluation of Semen Characteristics in Men 45 Years of Age or Older after Daily Dosing with Tadalafil 20 mg: Results of a Multicenter, Randomized, Double-Blind, Placebo-Controlled, 9-Month Study Wayne J.G. Hellstrom a, *, Marc Gittelman b, Jonathan Jarow c, Christopher Steidle d, James McMurray e, David Talley f, Steven Watts g, Carol L. Mitchell g, James M. McGill g a Department of Urology, Tulane University School of Medicine, New Orleans, LA, USA b South Florida Medical Research and Miami Center for Sexual Health, Miami, FL, USA c Brady Urological Institute, Johns Hopkins University, Baltimore, MD, USA d Northeast Indiana Research, Fort Wayne, IN, USA e Medical Affiliated Research Center, Huntsville, AL, USA f Urology San Antonio Research, San Antonio, TX, USA g Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Article info Article history: Accepted September 25, 2007 Published online ahead of print on October 5, 2007 Keywords: Tadalafil PDE5 inhibitor Spermatogenesis Testosterone Follicle-stimulating hormone Luteinizing hormone Semen parameters Abstract Objectives: Assess the effects on spermatogenesis of daily tadalafil 20 mg over three spermatogenesis cycles in men 45 yr. Methods: In this double-blind, placebo-controlled, noninferiority study, healthy men (or with mild erectile dysfunction) were randomized to receive tadalafil 20 mg (n = 125) or placebo (n = 128) for 9 mo followed by a 6-mo, treatment-free period. Semen and serum samples were provided at baseline and every wk. The primary outcome was the proportion of subjects with 50% reduction in sperm concentration at end point. Secondary outcomes included sperm concentration, number per ejaculate, motility and morphology; serum concentrations of testosterone, luteinizing and follicle-stimulating hormones; and tolerability. Results: Of 253 men enrolled, 191 (75%) completed treatment phase: 2 of 96 (2.1%, placebo) and 12 of 95 (12.6%, tadalafil) subjects had 50% reduction in sperm concentration. Tadalafil was noninferior to placebo because the upper 95% confidence interval for the difference in proportions of tadalafil and placebo subjects with a 50% reduction in sperm concentration was 17.5%, significantly less than the prespecified noninferiority margin of 20% ( p = 0.015). Ninety-four percent (179 of 191) of men completed the 6-mo, treatment-free period: Baseline sperm concentration levels were restored in 8 of 12 (tadalafil) and 1 of 2 (placebo) men. There were no significant differences between groups in secondary end points. Common treatment-emergent adverse events were headache, back pain, dyspepsia, gastroesophageal reflux disease, and myalgia. Twelve (9.6%) tadalafil and seven (5.5%) placebo subjects discontinued because of adverse events. Conclusions: This study demonstrated no deleterious effects of 9 mo of daily tadalafil 20 mg on spermatogenesis or hormones related to testicular function in men 45 yr. # 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Tulane University Medical Center, New Orleans, LA 70112, United States. Tel ; Fax: address: whellst@tulane.edu (W.J.G. Hellstrom) /$ see back matter # 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 european urology 53 (2008) Introduction Tadalafil is an inhibitor of phosphodiesterase (type) 5 (PDE5) used for treatment of erectile dysfunction (ED). Tadalafil 10 and 20 mg taken in anticipation of sexual activity have been shown to improve erectile function for up to 36 h [1,2]. Because of this extended period of efficacy, once-daily usage of tadalafil 2.5, 5, and 10 mg has been investigated in clinical trials and has been shown to be a well-tolerated and efficacious ED therapy [3,4]. The standard test for assessing potential adverse effects of a drug on human spermatogenesis is the analysis of semen parameters, with a focus on sperm concentration, total number per ejaculate, motility, and morphology [5,6]. In addition, the assessment of the reproductive hormones testosterone (total and free), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) are useful because there is less normal variability compared with sperm parameters, although these hormone levels can exhibit normal values despite significant abnormalities in spermatogenesis or sperm function [7]. The results of two previous studies evaluating the effects of tadalafil 10 and 20 mg taken daily on semen parameters and reproductive hormones have been published in a single report [8]. Each study lasted 6 mo, or at least two human sperm cycles. The two studies demonstrated no adverse effects of daily tadalafil on spermatogenesis or reproductive hormones. The objective of the current phase 4 study was to assess the effects of daily tadalafil 20 mg for 9 mo, or at least three human sperm cycles, on spermatogenesis. 2. Methods 2.1. Study population Healthy men or men with mild ED who were at least 45 yr of age were eligible to enroll if their semen samples at baseline met the following World Health Organization (WHO) 1987 criteria: sperm concentration of 20 million/ml (geometric mean of two samples), 50% motile sperm, 50% sperm with normal morphology, and semen volume 1.5 ml (arithmetic mean of two samples). Men were excluded for current use of nitrates, cancer chemotherapy, testosterone, or antiandrogens; or history of the following: moderate to severe ED, reproductive hormone levels outside of normal limits; infertility or vasectomy, testicular trauma or abnormality, known sperm defect, or retrograde ejaculation; sexually transmitted disease or relevant genitourinary infection; substance abuse; hyperprolactinemia, congenital adrenal hyperplasia, Cushing syndrome, hemochromatosis, panhypopituitarism, Klinefelter syndrome; active symptomatic hepatobiliary disease; significant renal insufficiency within the last 6 mo; chronic stable angina, unstable angina, or angina occurring during sexual intercourse in the last 6 mo; myocardial infarction or coronary intervention within the last 90 d; heart arrhythmia; sudden cardiac arrest, congestive heart failure (New York Heart Association class 2 or above); new, significant conduction defect within past 90 d; systolic blood pressure > 170 or < 90 mm Hg or diastolic blood pressure > 100 or < 50 mm Hg; significant central nervous system injuries, major surgery, severe burns, or thyrotoxicosis within the last 6 mo; current exposure to environmental toxins; or hemoglobin A1c > 13%. All patients signed an informed consent document before participating in the study. This study was conducted in accordance with the Declaration of Helsinki, and ethical review boards provided written approval of the study protocol and the informed consent document Study design This was a randomized, double-blind, placebo-controlled, multicenter, fixed-dose, parallel-design, noninferiority study. Subjects were randomized 1:1, via a central computerized voice response system, to receive tadalafil 20 mg or placebo once daily for 9 mo (40 wk). Subjects were stratified into three baseline sperm-concentration groups for randomization: (1) 20 to 50 million/ml; (2) greater than 50 but at least 80 million/ ml; (3) greater than 80 million/ml. The study consisted of a 4-wk screening phase, a 40-wk treatment phase, and a 26-wk treatment-free period (Fig. 1). Prior to semen sample collections, subjects were instructed to abstain from ejaculation for at least 48 h but not more than 5 d. Instructions regarding proper semen collection techniques were provided to subjects at screening and throughout treatment. For each visit, two semen samples were collected within a 1- to 2-wk period of each other. Blood samples for testosterone (total and free), LH, and FSH were collected at screening and weeks 40 (end of treatment phase), 53, and 66 (end of treatment-free period). All semen and blood sample parameters were analyzed at study sites with the exception of morphology, which was analyzed at a central laboratory. Free testosterone was measured by means of equilibrium dialysis. Standardized semen analysis techniques were taught to each technician at a central training facility at Tulane University, New Orleans, USA. Quality control and proficiency criteria were set for < 15% variation for certifying the technicians trained to do semen analysis for the study. Fig. 1 Study design.

3 1060 european urology 53 (2008) Primary and secondary end points The primary end point of the study was the proportion of subjects (P) who had a 50% or greater reduction in sperm concentration from baseline following 40 wk of treatment with tadalafil (P tadalafil ) or placebo (P placebo ). The 50% or greater reduction was chosen to allow for normal variations in sperm concentration within and between individuals [9 12]. Secondary end point measures included sperm concentration, total number per ejaculate, motility, and morphology; and serum concentrations of testosterone, LH, and FSH Post hoc analysis In addition to primary and secondary objectives, a post hoc analysis of ejaculatory frequency was done. Ejaculatory frequency was measured and calculated as the total number of ejaculations in the 4 wk prior to first sample collection for week 40. An increase in the number of ejaculations in the week prior to an abstinence period has been found to significantly decrease sperm concentration [13,14] Tolerability Adverse events reported by the patient were collected at each visit and coded to the Medical Dictionary for Regulatory Activities, version 8.0. An electrocardiogram was performed at screening, and standard laboratory tests were collected at screening and end point. Vital signs were collected at each visit Statistical analyses Three analysis populations were defined: randomized, treated, and completing. The randomized population consisted of all subjects randomized and grouped by assigned treatment. The treated population included subjects receiving at least one dose of study medication grouped by treatment received. For inclusion in a particular analysis, both a baseline and postbaseline value were required. The completing population consisted of subjects completing the study through week 40 (end point), for which this visit was at least 36 wk after baseline. The current study was requested by European regulators, which required calculation of geometric means. The previous two studies were conducted for the US Food and Drug Administration, which required arithmetic means. To make historical comparisons among the studies, we calculated both geometric and arithmetic means for the primary and secondary end points. The primary analysis of this noninferiority trial assessed the strength of evidence against the null hypothesis that tadalafil was inferior to placebo using a one-sided difference in binomial test [15] with a prespecified noninferiority margin of Proportions of subjects in the completing population with at least a 50% reduction from baseline to end point in sperm concentration were determined, and noninferiority was demonstrated if the upper limit of the 95% confidence interval (95%CI) for the difference between the two proportions (P tadalafil minus P placebo ) was less than 0.20 or 20%. Secondary analyses included mean sperm concentration, ejaculate sperm count (defined as the total sperm number per ejaculate), percentage normal motility, percentage normal morphology, and reproductive hormones, including testosterone, LH, and FSH. Each secondary variable was tested with an analysis of covariance (ANCOVA) model with effects for baseline, therapy, and investigative site (pooled according to number of patients and proximity to another site). The models for sperm parameters also included covariate terms for average number of ejaculations per week and mean hours from previous ejaculation and sample collection. For sperm concentration and total count, the data were subject to a natural logarithmic transformation prior to analyses, and point estimates were back-transformed estimates from the model. A post hoc test of significance using the Fisher exact test was performed for the proportion of subjects with a 50% or greater reduction from baseline in sperm concentration and was also calculated for total sperm count. When we analyzed data using an ANCOVA model, we determined the unadjusted p values and Bonferroni-adjusted p values [16] within each of the following sets of secondary analyses: sperm concentration, ejaculate sperm count, motility, and morphology at week 40; and serum FSH, serum LH, and serum testosterone at week 40. Within each of the above sets of variables, the adjusted p value was set to four times the unadjusted p value with a maximum allowed value of 1.0. Because of the number of statistical tests performed, unadjusted p values were more susceptible to type I error. Consequently, adjusted p values also were calculated. 3. Results 3.1. Patient demographics and disposition Of the 1306 men screened, 253 were enrolled and randomized, and 191 (75.5%) completed 40 wk of treatment (Fig. 2). Of these 191 men, 190 entered the subsequent 26-wk treatment-free period, and 179 of 190 (94.2%) completed this phase. Demographic characteristics at baseline were similar between the groups (Table 1). A large proportion (35%) of men failed screening either because their percentage of motile sperm was less than 50% (n = 243 of 1306, 18.6%) or their reproductive hormone levels were outside the normal range (n = 208 of 1306, 15.9%) Primary end point There were 2 of 96 (2.1%) men in the placebo group and 12 of 95 (12.6%) men in the tadalafil group with 50% or greater reduction from baseline in sperm concentration at end point. The difference between the tadalafil and placebo proportions was 10.5%. The upper limit of the two-sided 95%CI for the difference between the proportions was 17.5%, which was significantly less than the 20% prespecified

4 european urology 53 (2008) Fig. 2 Patient movement throughout the study. * One patient withdrew on the day of his last visit. noninferiority margin ( p = 0.015), establishing that tadalafil was noninferior to placebo. Among the 14 subjects with a 50% or greater decrease in sperm concentration at the end of treatment, 8 of 12 subjects taking tadalafil and 1 of 2 subjects taking placebo returned to near-baseline sperm concentrations by the end of the treatmentfree period. Overall, there were 6 of 86 (7.0%) men taking placebo and 8 of 88 (9.1%) men taking tadalafil with a 50% or greater reduction in sperm concentration at the end of the treatment-free period. The treatment difference between groups after the treatment-free period was 2.1% ( p < 0.001, tadalafil noninferior to placebo). Table 1 Baseline characteristics Parameter Placebo (n = 128) Tadalafil 20 mg (n = 125) Mean age (yr) Caucasian (%) Smokes (% yes) Consumes alcohol (% yes) Erectile dysfunction (% yes) * Baseline semen characteristics Concentration subgroups (M/ml) 31.3% 29.6% >50 80 (M/ml) 32.0% 35.2% >80 (M/ml) 36.7% 35.2% Sperm concentration, geometric mean (M/ml) Ejaculate volume, mean (ml) Ejaculate sperm count, geometric mean (million) Normal sperm morphology (%) Motile sperm (%) M/ml, million per milliliter. * Erectile dysfunction was determined through clinical assessment.

5 1062 european urology 53 (2008) Table 2 Total sperm concentration and total sperm count for patients completing the study Semen characteristic and week of study Placebo (n = 96) Geometric mean Tadalafil 20 mg (n = 95) p value * Placebo (n = 96) Arithmetic mean Tadalafil 20 mg (n = 95) p value * Sperm concentration (M/ml) 0 (baseline) (end of treatment) / (no treatment) Sperm count (million) 0 (baseline) (end of treatment) / (no treatment) / /1.00 M/ml, million per milliliter. * p values are expressed as unadjusted/adjusted. Table 3 Percent motile sperm and percent with normal sperm morphology for those subjects completing the study Sperm characteristic Placebo (n = 96) Tadalafil 20 mg (n = 95) p value * Motile sperm (%) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) Normal sperm morphology (%) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) / /1.00 * p values are expressed as unadjusted/adjusted Secondary outcomes There was a 9 million/ml decrease in the geometric least square mean sperm concentration in the tadalafil-treated group compared with the placebotreated group (Table 2; p = 0.02; p = 0.06 adjusted for multiplicity). There was a 7 million/ml decrease in the arithmetic mean sperm concentration in the tadalafil-treated group compared with the placebotreated group ( p =0.10;p = 0.39 adjusted). There was Table 4 Summary of reproductive hormones for subjects who completed the study Hormone Placebo (n = 96) Tadalafil 20 mg (n = 95) p value * Total testosterone (ng/dl) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) Free testosterone (pg/ml) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) FSH (IU/l) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) LH (IU/l) Week 0 (baseline) Week 40 (end of treatment) Week 66 (end of treatment-free period) / / / /0.21 FSH, follicle-stimulating hormone; LH, luteinizing hormone. * p values are expressed as unadjusted/adjusted.

6 european urology 53 (2008) no significant change in mean total sperm count observed when assessed by either geometric mean or arithmetic mean (Table 2). There were no significant differences between groups in percent of motile sperm or normal sperm morphology (Table 3). Among reproductive hormones tested, only total testosterone was significantly different between groups at end point. However, in light of the broad range of normal total testosterone values and the lack of effect in the bioactive free form, the difference was not considered clinically meaningful (Table 4) Post hoc analyses There was no significant difference between treatment groups in the mean ejaculatory frequency over the duration of the study. However, the ejaculatory frequency in the subjects who had a 50% or greater reduction in sperm concentration at the end of the treatment phase (5.41 ejaculations per week) was greater than in the subjects who did not have a 50% or greater reduction in sperm concentration (2.91 ejaculations per week, p < 0.001) Tolerability Treatment-emergent adverse events are summarized in Table 5. The majority of patients reported events to be mild or moderate in severity: 79% (33 of 42) of patients taking placebo and 81% (60 of 74) of patients taking tadalafil. Twelve (9.6%) tadalafil subjects and seven (5.5%) placebo subjects discontinued the study because of adverse events. There were no clinically significant changes in laboratory values or vital signs. Table 5 Treatment-emergent adverse events reported by at least 3% of patients during the 9-mo treatment phase Event Placebo (n = 128) Tadalafil 20 mg (n = 125) Headache 2 (2%) 20 (16%) Back pain 4 (3%) 14 (11%) Dyspepsia 1 (1%) 10 (8%) Gastroesophageal reflux disease 1 (1%) 10 (8%) Myalgia 1 (1%) 10 (8%) Pain 1 (1%) 5 (4%) Pain in extremity 0 5 (4%) Dizziness 1 (1%) 4 (3%) Influenza 1 (1%) 4 (3%) Nasopharyngitis 4 (3%) 4 (3%) Discontinued owing to adverse event 7 (5.5%) 12 (9.6%) 4. Discussion This randomized, double-blind, placebo-controlled study demonstrated that daily administration of tadalafil 20 mg for 9 mo, or approximately three human spermatogenesis cycles, had no significant adverse effects on sperm production or reproductive hormones in men at least 45 yr of age. Tadalafil was noninferior to placebo in the proportion of subjects with 50% or greater reduction in sperm concentration. A noninferiority trial seeks to determine whether a new treatment is no worse than a reference treatment. In this trial, tadalafil is the new treatment and placebo is the reference treatment. Thus, the design of the study seeks to determine whether tadalafil is no worse than placebo in terms of effects on spermatogenesis. Because actual proof of equivalence is not possible, a margin of noninferiority (in this study 20%) for the treatment effect is predefined [17]. Many factors have been shown to influence semen parameters, including body mass index, the duration of sexual arousal, seasonal variation, diurnal variation, heat and fever, and ejaculatory frequency [13,14,18 24]. To the best of our knowledge, there was no difference between groups in any of these parameters. However, the ejaculatory frequency in subjects who had a 50% or greater reduction in sperm concentration was higher than in the subjects who did not have a 50% or greater reduction in sperm concentration ( p < 0.001). An increase in ejaculatory frequency may be one possible factor contributing to a greater than 50% reduction in sperm concentration in these men. Results for total sperm concentration were equivocal. There was a significant decrease in total sperm concentration with tadalafil treatment in comparisons of geometric means but not arithmetic means. The clinical relevance of this finding is unclear, especially given that testicular toxins also typically decrease sperm count, motility, and morphology [5]. Thus, in the absence of any concurrent changes in total sperm count, motility, or morphology, the decrease in sperm concentration (geometric mean) seen in this study is not consistent with either impaired spermatogenesis or testicular toxicity [25,26]. These results are consistent with the previous report that found daily tadalafil 10 and 20 mg for 6 mo had no detrimental effect on spermatogenesis or reproductive hormones [8]. A different study has suggested that tadalafil may impair sperm function via inhibition of PDE11 [27]. Using PDE11 knockout mice, a surrogate for continuous, complete

7 1064 european urology 53 (2008) inhibition of PDE11 by tadalafil, Wayman showed that sperm from these knockout mice were more likely to undergo capacitance compared with wildtype mice (29% vs. 19%, respectively). Yet there was no difference in fertility rates between the two groups. Additionally, tadalafil has a 40-fold weaker potency for PDE11A4 compared with PDE5 and would be unlikely to exert a significant inhibitory effect upon PDE11A4 in patients taking the prescribed dosage [28,29]. One possible limitation of the current study is the entry criteria used to determine normal semen parameters. WHO reference values were used as inclusion/exclusion criteria for this study. Although these values are derived using samples from normal donors with a wide range of ages, they do not provide guidance on normal values for older men [30]. A large proportion of men in the current trial (557 of 1306, 43%) did not meet WHO standards. A recent analysis suggests that the high failure rate observed during the screening process of this study might be due to an overestimation of what is normal semen quality for men in their fourth and fifth decades [31]. Thus, WHO criteria used in this study may not be appropriate for the older population of men who were enrolled. Using agespecific semen and sperm reference values at enrollment may have made the conclusions more applicable to the older population of men who take tadalafil for ED. Another possible limitation of the study is the ability to extrapolate the results to younger men, men with more severe ED, and men with abnormal semen parameters. Although the results cannot be used to assess the effect of tadalafil on fertility, because pregnancy and live birth were not end points, there is no evidence from this study, nor the literature, nor reports in the spontaneous safety data base (collecting adverse events from more than 9 million men exposed to tadalafil) suggesting that tadalafil negatively impacts fertility [8,27]. effect on reproductive hormones. When taken in concert with previously published studies examining the effect of tadalafil on spermatogenesis, our findings show that sufficient evidence exists to demonstrate a lack of toxicity by tadalafil on spermatogenesis. Tadalafil 20 mg taken daily was well tolerated. Conflicts of interest Wayne J.G. Hellstrom: Advisory board member, speaker, consultant, and clinical study participant for Eli Lilly, Bayer and Pfizer. Marc Gittelman: Investigator, advisory board, speaker for Eli Lilly; Investigator, speaker for Pfizer; Investigator, advisory board, speaker for Bayer/ Schering/GSK. Jonathan P. Jarow: Speaker for Lilly and Pfizer. Christopher P. Steidle: Speaker for Lilly, GSK, Auxilium, Coloplast; Clinical investigator for Lilly, Pfizer, Auxilium, Schwarz-pharma, Falding. James G. McMurray: Speaker and investigator for Pfizer, Lilly, and GSK. David Talley: Paid investigator for Eli Lilly and Company. Steven Watts, Carol Mitchell, James M. McGill: Employees of Eli Lilly and Company. Acknowledgements The authors wish to thank Diane R. Stothard, PhD (Eli Lilly and Company) for writing assistance. Support for this study (H6D-MC-LVFE) was provided by Lilly ICOS LLC. Other investigators who participated in the study were Philip J. Aliotta, William B. Smith, Karen C. Evans, Bradley E. Davis, Eugene Dula, Robert A. Feldman, Joel M. Kaufman, Ira W. Klimberg, Andrew McCullough, Bruce Redmon, Samuel T. Thompson, Mitchell L. Wiatrak, and Jay M. Young. 5. Conclusions Results from this study support a lack of toxicity on spermatogenesis of tadalafil 20 mg compared with placebo after daily use for 9 mo. There were no statistically significant differences in the three sperm parameters predominantly determined by the testes: mean total sperm count, mean percent normal sperm motility, or mean percent normal sperm morphology. This lack of an effect on the testes is further supported by the lack of an adverse References [1] Carson CC, Rajfer J, Eardley I, et al. The efficacy and safety of tadalafil: an update. BJU Int 2004;93: [2] Young JM, Feldman RA, Auerbach SM, et al. Tadalafil improved erectile function at twenty-four and thirty-six hours after dosing in men with erectile dysfunction: US trial. J Androl 2005;26: [3] Porst H, Giuliano F, Glina S, et al. Evaluation of the efficacy and safety of once-a-day dosing of tadalafil 5 mg and 10 mg in the treatment of erectile dysfunction: results

8 european urology 53 (2008) of a multicenter, randomized, double-blind, placebo-controlled trial. Eur Urol 2006;50: [4] Rajfer J, Aliotta PJ, Steidle CP, Fitch III WP, Zhao Y, Yu A. Tadalafil dosed once a day in men with erectile dysfunction: a randomized, double-blind, placebo-controlled study in the US. Int J Impot Res 2007;19: [5] Guzick DS, Overstreet JW, Factor-Litvak P, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 2001;345: [6] Overstreet JW, Brazil C. Semen analysis. In: Lipschulz LI, Howards SS, editors. Infertility in the male. 3rd ed. St Louis; Mosby-Year Book 14; p [7] Sigman M, Jarow JP. Endocrine evaluation of infertile men. Urology 1997;50: [8] Hellstrom WJG, Overstreet JW, Yu A, et al. Tadalafil has no detrimental effect on human spermatogenesis or reproductive hormones. J Urol 2003;170: [9] Poland ML, Moghissi KS, Giblin PT, Ager JW, Olson JM. Variation of semen measures within normal men. Fertil Steril 1985;44: [10] Schrader SM, Turner TW, Simon SD. Longitudinal study of semen quality of unexposed workers. Sperm motility characteristics. J Androl 1991;12: [11] Tielemans E, Heederik D, Burdorf A, Loomis D, Habbema DF. Intraindividual variability and redundancy of semen parameters. Epidemiol 1997;8: [12] Auger J, Eustache F, Ducot B, et al. Intra- and inter-individual variability in human sperm concentration, motility and vitality assessment during a workshop involving ten laboratories. Hum Reprod 2000;15: [13] Carlsen E, Petersen JH, Andersson AM, Skakkebaek NE. Effects of ejaculatory frequency and season on variations in semen quality. Fertil Steril 2004;82: [14] Oldereid NB, Gordeladze JO, Kirkhus B, Purvis K. Human sperm characteristics during frequent ejaculation. J Reprod Fertil 1984;71: [15] Farrington CP, Manning G. Test statistics and sample size formulae for comparative binomial trials with null hypothesis of non-zero risk difference or non-unity relative risk. Stat Med 1990;9: [16] Sankoh AJ, Huque MF, Dubey SD. Some comments on frequently used multiple endpoint adjustment methods in clinical trials. Stat Med 1997;16: [17] Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJW. Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement. JAMA 2006;295: [18] Jensen TK, Andersson AM, Jorgensen N, et al. Body mass index in relation to semen quality and reproductive hormones among 1,558 Danish men. Fertil Steril 2004; [19] Pound N, Javed MH, Ruberto C, Shaikh MA, Del Valle AP. Duration of sexual arousal predicts semen parameters for masturbatory ejaculates. Physiol Behav 2002;76: [20] Centola GM, Eberly S. Seasonal variations and age-related changes in human sperm count, motility, motion parameters, morphology, and white blood cell concentration. Fertil Steril 1999;72: [21] Gyllenborg J, Skakkebaek NE, Nielsen NC, Keiding N, Giwercman A. Secular and seasonal changes in semen quality among young Danish men: a statistical analysis of semen samples from 1927 donor candidates during Int J Androl 1999;22: [22] Cagnacci A, Maxia N, Volpe A. Diurnal variation of semen quality in human males. Hum Reprod 1999;14: [23] Mieusset R, Grandjean H, Mansat A, Pontonnier F. Inhibiting effect of artificial cryptorchidism on spermatogenesis. Fertil Steril 1985;43: [24] Shafik A. The physiology of testicular thermoregulation in the light of new anatomical and pathological aspects. Adv Exp Med Biol 1991;286: [25] Nallella KP, Sharma RK, Aziz N, Agarwal A. Significance of sperm characteristics in the evaluation of male infertility. Fertil Steril 2006;85: [26] Agarwal A, Sharma RK, Nelson DR. New semen quality scores developed by principal component analysis of semen characteristics. J Androl 2003;24: [27] Wayman C, Phillips S, Lunny C, et al. Phosphodiesterase 11 (PDE11) regulation of spermatozoa physiology. Int J Impot Res 2005;17: [28] Saenz de Tejada I, Angulo J, Gadau M, Florio V. Comparative selectivity profiles of tadalafil, sildenafil, and vardenafil using an in vitro phosphodiesterase activity assay. Int J Impot Res 2002;14:S20. [29] Weeks JL, Zoraghi R, Beasley A, Sekhar KR, Francis SH, Corbin JD. High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodiesterase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients. Int J Impot Res 2005;17:5 9. [30] World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. World Health Organization; [31] Hellstrom WJ, Overstreet JW, Sikka SC, et al. Semen and sperm reference ranges for men 45 yr of age and older. J Androl 2006;27:421 8.