Supplementation for Male Subfertility

Study Overview
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Brief Summary
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Old age, physical inactivity, environmental factors and genetics may contribute negatively to fertility in both males and females. In males, specifically, certain supplements, such as single antioxidants and trace minerals, have previously been shown to improve sperm function marginally. One hypothesis is that sperm function can be improved even further by combining several different types of supplements (e.g., amino acids, energy carriers, vitamins, antioxidants, and trace minerals) to target several age-related cell pathways, for example, oxidative stress, mitochondrial dysfunction, inflammation and cell energetics. This 3-month placebo-controlled, randomized clinical trial, aims to test the effects of a novel multi-ingredient supplement (Fertility Enhancer) that targets several age-related cell pathways on sperm function in subfertile males.

Detailed Description
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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. 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 age-related pathways on World Health Organization (WHO) semen quality parameters in 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 with confirmed subfertility 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.

Official Title
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Nutraceutical Supplementation for Male Subfertility

Conditions
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Male Infertility

Intervention / Treatment
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* Dietary Supplement: Active multi-ingredient supplement (Fertility Enhancer, FE)
* Dietary Supplement: Inactive placebo (Placebo; PLA)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Males between the ages of 25-50 years diagnosed with subfertility through Ontario Networks of Experts in Fertility (ONE Fertility, Burlington, ON). For diagnosis of male subfertility, the 2010 and 2021 World Health Organization criteria will be used for sperm count, motility, morphology and vitality. Exclusion Criteria: Smoking, history and drug alcohol abuse, BMI > 30 kg/m2, genital disease (cryptorchidism, current genital inflammation, or varicocele), genital trauma or surgery to the male reproductive system, known Y chromosome microdeletions or karyotype abnormalities (if known prior), hepatobiliary disease, significant renal insufficiency, occupational exposures to reproductive toxins, endocrine abnormality, recent or current sexually transmitted infection, use of cytotoxic drugs, use of immunosuppressants, use of anticonvulsants, use of androgens or antiandrogens, history of central nervous system injury, neurological or psychiatric disease to potentially compromise study data collection, treatment of erectile dysfunction with any drugs during the past 4 weeks, history of cancer chemotherapy, current supplementation with ingredients being tested unless 1-month washout period

Ages Eligible for Study
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Minimum Age: 25 Years
Maximum Age: 50 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: This is a 3-month placebo-controlled, double-blinded, randomized clinical trial comparing an active supplement designed to enhance sperm function (Fertility Enhancer) to an inactive placebo (PLACEBO) in subfertile males.
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Active multi-ingredient supplement (Fertility Enhancer; FE)<br>Volunteers will be randomized in a double-blinded fashion into the experimental treatment group, which entails daily supplementation of an active multi-ingredient supplement designed to enhance fertility (Fertility Enhancer; FE) for 3 months. | Dietary Supplement: Active multi-ingredient supplement (Fertility Enhancer, FE)<br>* Consuming a multi-ingredient supplement targeting multiple cell pathways daily for 3 months.<br>|
| Placebo Comparator: Inactive placebo (Placebo; PLA)<br>Volunteers will be randomized in a double-blinded fashion into a placebo group, which entails daily supplementation of a calorie-matched, inactive placebo (Placebo; PLA) identical in flavor to the active supplement for 3 months. | Dietary Supplement: Inactive placebo (Placebo; PLA)<br>* Consuming an inactive placebo that is calorie-matched to the active supplement daily for 3 months.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Percent change in body composition index from baseline to 3 months | Lean mass/fat mass ratio by dual X-ray absorptiometry scan (body composition index; % change) | Baseline to 3 months | 
| Percent change in total fat mass from baseline to 3 months | Total fat mass by dual X-ray absorptiometry scan (kg; % change) | Baseline to 3 months | 
| Percent change in sperm count/concentration from baseline to 3 months | Sperm count/concentration (millions spermatozoa/mL semen) | Baseline to 3 months | 
| Percent change in sperm motility from baseline to 3 months | Proportion motile sperm (%) | Baseline to 3 months | 
| Percent change in sperm morphology from baseline to 3 months | Proportion normal sperm morphology (%) | Baseline to 3 months | 
| Percent change in sperm vitality from baseline to 3 months | Proportion viable sperm (vitality) (%) | Baseline to 3 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Percent change in sperm DNA fragmentation index from baseline to 3 months | Sperm DNA fragmentation index by flow cytometry (%) | Baseline to 3 months | 
| Percent change in sperm DNA 8-hydroxydeoxyguanosine from baseline to 3 months | Sperm DNA 8-hydroxydeoxyguanosine by ELISA (ng/mL; %) | Baseline to 3 months | 
| Percent change in sperm protein carbonyls from baseline to 3 months | Sperm protein carbonyls immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm lipid peroxidation (4-hydroxynonenal) from baseline to 3 months | Sperm 4-hydroxynonenal immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm antioxidant marker superoxide dismutase 1 from baseline to 3 months | Sperm superoxide dismutase 1 expression immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm antioxidant marker superoxide dismutase 2 from baseline to 3 months | Sperm superoxide dismutase 2 expression immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm apoptotic marker cleaved caspase 3 from baseline to 3 months | Sperm cleaved caspase 3 expression immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm apoptotic marker total caspase 3 from baseline to 3 months | Sperm total caspase 3 expression immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm mitochondrial OXPHOS from baseline to 3 months | Sperm mitochondrial OXPHOS expression immunoblot (optical density; %) | Baseline to 3 months | 
| Percent change in sperm cell cycle arrest marker p16 from baseline to 3 months | Sperm p16 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm cell cycle arrest marker p21 from baseline to 3 months | Sperm p21 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammatory marker interleukin 1 from baseline to 3 months | Sperm interleukin 1 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammatory marker TNF-alpha from baseline to 3 months | Sperm TNF-alpha messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammatory marker interleukin-6 from baseline to 3 months | Sperm interleukin-6 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammatory marker interleukin-8 from baseline to 3 months | Sperm interleukin-8 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammatory marker interleukin-18 from baseline to 3 months | Sperm interleukin-18 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm inflammasome marker caspase 1 from baseline to 3 months | Sperm caspase 1 messenger RNA levels by rtPCR (fold control/placebo; %) | Baseline to 3 months | 
| Percent change in sperm ATP levels from baseline to 3 months | Sperm ATP levels by ELISA (pM/100 mg protein; %) | Baseline to 3 months | 
| Percent change in sperm phosphocreatine levels from baseline to 3 months | Sperm phosphocreatine levels by ELISA (ng/100 mg protein; %) | Baseline to 3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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ATP, DNA, Sub-fertility, Infertility, Antioxidants, Supplements, Creatine, Arginine, Vitamins, Aging, Oxidative stress, Inflammation, Mitochondria, Fragmentation