Supplementation for Male Subfertility

BACKGROUND: Infertility is characterized by the failure to become pregnant after one year of regular intercourse without the use of contraceptives and impacts 10-15% of couples worldwide. Both male and female partners contribute to a couple's reproductive health, with approximately one third of infertility cases caused by male factors, one third by female factors, and the remaining by either a combination of both or unknown causes. The prevalence of infertility is a growing concern in Canada, as is seen in an increased use of assisted reproductive technology (ART), which may be both invasive and expensive. Cost-effective, safe, and accessible alternatives to ART are therefore needed. The most common cause of subfertility is 'biological aging', characterized by the hallmarks of aging, such as mitochondrial dysfunction, oxidative damage and inflammation. Another common cause of male and female subfertility is obesity, which is associated with multisystemic oxidative damage and inflammation.

PURPOSE: The aim of this placebo-controlled, double-blind randomized clinical trial is to test the effects of a multi-ingredient supplement (Fertility Enhancer) designed to target several aging- and obesity-related pathways on World Health Organization (WHO) semen quality parameters in overweight and obese and subfertile males (sperm count, motility, morphology and vitality).

SAMPLE-SIZE ESTIMATE AND DESIGN: Sperm count/concentration is strongly correlated to all World Health Organization semen quality parameters. With significance set at 0.05 (Z = 1.96) and power to 0.8 (Z = 0.84), a sample-size of 17-32 per group is sufficient to detect an increase of 10 x 10\^6 spermatozoa/mL with a standard deviation of 15 to 20 x 10\^6 spermatozoa/mL. Thus, sixty-four (n = 64) males between 25 and 50 years of age that are confirmed overweight or obese and subfertile will be randomized into age-matched Placebo (PLA, n = 32) vs Fertility Enhancer (FE, n = 32) groups and undergo daily supplementation for 3 months.

SUPPLEMENTS: The FE supplement contains energy carriers (creatine), conditionally essential amino acids (arginine), Omega 3 fatty acids (DHA and EPA), vitamins (B9, B12, E, and D3), antioxidants (CoQ10 and alpha lipoic acid), trace minerals (selenium, iron, zinc, and copper), and plant extracts (beet root, green tea, and green coffee bean). The isocaloric and inactive placebo contains safflower oil, microcrystalline cellulose and sugar and is identical in flavor to FE.

CO-PRIMARY OUTCOMES: All outcomes will be measured at baseline and post intervention for assessing % pre-to-post changes. Co-primary outcomes are body composition by dual x-ray absorptiometry, including lean mass to fat mass ratio (body composition index; BCI) and total fat mass, and the WHO semen quality parameters; specifically, % improvements in sperm count, motility, morphology, and vitality.

SECONDARY OUTCOMES: Secondary outcomes are % improvements in sperm DNA fragmentation (flow cytometry-assessed) and markers of oxidative damage (protein carbonyls, lipid peroxidation, 8-hydroxydeoxyguanosine)), inflammation (interleukin-1, tumor necrosis factor-alpha, interleukin-6), apoptosis (total and cleaved caspase 3), cell cycle arrest (p16 and p21), mitochondrial biogenesis (complexes I-V), antioxidant status (superoxide dismutases 1 and 2), and energy state (ATP and phosphocreatine).

OTHER: Additional outcomes are body morphology (bodyweight, waist/height ratio, and body mass index), other body composition outcomes (lean mass and appendicular skeletal muscle mass index), and blood markers of oxidative damage (malondialdehyde), inflammation (c-reactive protein, interleukin-1, tumor necrosis factor-alpha, interleukin-6), antioxidant status (ORAC, TEAC), liver enzymes (alanine aminotransferase, aspartate aminotransferase, and creatinine) and energy state (ATP \& phosphocreatine levels).

HYPOTHESIS: The main hypothesis of the current trial is that co-primary body composition outcomes and the World Health Organization (WHO) semen quality parameters (count, motility, morphology, and/or vitality) will be significantly improved following FE supplementation and superior to PLA.

STATISTICS: A standard omnibus one-way repeated measures ANOVA F-test followed by Duncan post hoc analyses will be used for all parametric data analyses. Non-parametric equivalents will be used for non-normally distributed data with significance set at p = 0.05. Delta pre-post changes (% improvements) for all outcomes within and between groups are biologically relevant and planned a priori comparisons.

• Percent change in sperm DNA fragmentation index from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm DNA 8-hydroxydeoxyguanosine from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm protein carbonyls from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm lipid peroxidation (4-hydroxynonenal) from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm antioxidant marker superoxide dismutase 1 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm antioxidant marker superoxide dismutase 2 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm apoptotic marker cleaved caspase 3 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm apoptotic marker total caspase 3 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm mitochondrial OXPHOS from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm cell cycle arrest marker p16 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm cell cycle arrest marker p21 from baseline to 3 months [Time frame: Baseline to 3 months]
• Percent change in sperm inflammatory marker interleukin 1 from baseline to 3 months [Time frame: Baseline to 3 months]