Approximately 30% to 80% of infertile men having elevated seminal reactive oxygen species (ROS) levels suffer from a condition known as male oxidative stress infertility (MOSI) which impacts about 37 million men with idiopathic infertility around the world.1 Oral intake of individual antioxidants or a combination of several antioxidants such as L-carnitine, selenium, N-acetyl-cysteine, coenzyme Q10, ubiquinol, vitamin E, vitamin C, and lycopene have led to improvement in semen parameters and sperm DNA integrity of idiopathic infertile men.2-3 However, their role in modulating spermatozoa at a molecular level has not been reported thus far.
The objectives of our study was to 1) evaluate the sperm protein profile of idiopathic infertile men pre- and post-antioxidant supplementation and 2) to validate the proteins associated with the fertilization process using the conventional Western blot technique and correlate these findings with improved semen analysis post-treatment. Randomly selected idiopathic infertile men were provided with antioxidant capsules ((1000 mcg B12, 30mg zinc, 140mcg selenium, 350mg arginine, 2000mg, 200mg Co-Q10, 120mg vitamin C, 200IU vitamin E) (FH PRO for Men, Fairhaven Health, Bellingham, WA)) once daily for a period of 6 months.
No complications/side effects were reported in any of the participants.4 A shotgun proteomic approach using liquid chromatography and tandem mass spectrometry was adopted to unravel the mechanistic pathway responsible for improving the semen quality. Of the 379 differentially expressed proteins (DEPs) between pre- and post-antioxidant treatment patients, a majority (n=274) were overexpressed in men following antioxidant treatment. Bioinformatics analysis revealed the activation of oxidative phosphorylation pathway, and upregulation of key DEPs involved in spermatogenesis, sperm maturation, binding of sperm, fertilization and normal reproductive function. Also, the transcriptional factors associated with antioxidant defense system (PPARGC1A) and free radical scavenging system (NFE2L2) were predicted to be functionally activated in post-treatment group.
Key DEPs NDUFS1, CCT3 and PRKARA1 validated by western blot showed significant overexpression in post-treatment men. Our novel proteomic findings suggest that antioxidant supplementation in idiopathic infertile men has a beneficial effect on sperm function proteins associated with fertility at the molecular level particularly proteins involved in CREM signaling, mitochondrial function, and protein oxidation. Further, activation of TRiC complex helped in nuclear compaction, maintenance of telomere length, flagella function and expression of ZP receptors for sperm-oocyte interaction. Our proteomic findings suggest that sperm proteins NDUFS1, CCT3, PRKAR1A, CCT6B, and SPA17 can serve as potential biomarkers to monitor the efficacy of antioxidant therapy in men with idiopathic infertility.
Figure 1: Top ten enriched canonical pathways in idiopathic infertile men post-antioxidant treatment.
Figure 2: Activation of CREM signaling pathway in spermatozoa of idiopathic infertile patients post-antioxidant treatment
Figure 3: Proposed molecular mechanisms of action of combined antioxidant treatment on sperm function
Written by: Nicholas N. Tadros, MD, MCR,1 Ashok Agarwal, PhD,2 Manesh Kumar Panner Selvam, PhD,2 Rakesh Sharma, MD,2 Sarah C. Vij, MD,3 Neel Parekh, MD,3 Edmund Sabanegh, MD,3 Luna Samanta, MD,2 Mohamed Arafa, MD4
- Division of Urology, Southern Illinois University School of Medicine, Springfield, IL
- American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH
- Department of Urology, Cleveland Clinic, Cleveland, OH
- Department of Urology, Hamad Medical Corporation, Doha, Qatar
- Agarwal A. et al. Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility. World J Mens Health. 2019 Sep;37(3):296-312..
- Majzoub, A., A. Agarwal, and S.C. Esteves, Antioxidants for elevated sperm DNA fragmentation: a mini review. Transl Androl Urol, 2017. 6 (Suppl 4): p. S649-s653.
- Micic S, Lalic N, Djordjevic D, Bojanic N, Bogavac-Stanojevic N, Busetto GM, Micic S, Lalic N, Djordjevic D, Bojanic N, Bogavac-Stanojevic N, Busetto GM, Virmani A. Agarwal A. Double-blind, randomised, placebo-controlled trial on the effect of L-carnitine and L-acetylcarnitine on sperm parameters in men with idiopathic oligoasthenozoospermia. Andrologia, 2019. 51(6): e13267.
- Arafa, M., Agarwal A, Majzoub A, Khalafalla K, Alsaid S, Elbardisi H, Efficacy of antioxidant supplementation on conventional and advanced sperm function tests in patients with idiopathic male infertility. Fertility and Sterility, 2019.