P Gharagozloo1, A Gutiérrez-Adán2, A Champroux3, A Noblanc3, A Kocer3, A Calle2, S Pérez-Cerezales2, E Pericuesta2, A Polhemus4, A Moazamian4, J R Drevet3, R J Aitken5. 1. CellOxess LLC, 15 Roszel Road, Princeton, NJ 08540, USA parviz.gharagozloo@celloxess.com. 2. INIA, Animal Reproduction, Madrid 28040, Spain. 3. GReD Lab CNRS UMR6293-INSERM U1103 Université Blaise Pascal-Clermont II, Clermont-Ferrand 63001, France. 4. CellOxess LLC, 15 Roszel Road, Princeton, NJ 08540, USA. 5. Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
Abstract
STUDY QUESTION: Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? SUMMARY ANSWER: Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. WHAT IS KNOWN ALREADY: Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. STUDY DESIGN, SIZE, DURATION: The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. MAIN RESULTS AND ROLE OF CHANCE: Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). LIMITATIONS, REASONS FOR CAUTION: It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. WIDER IMPLICATIONS OF THE FINDINGS: Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.
STUDY QUESTION: Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? SUMMARY ANSWER: Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. WHAT IS KNOWN ALREADY: Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. STUDY DESIGN, SIZE, DURATION: The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. MAIN RESULTS AND ROLE OF CHANCE: Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). LIMITATIONS, REASONS FOR CAUTION: It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. WIDER IMPLICATIONS OF THE FINDINGS: Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.
Authors: Mojdeh Sabour; Arezoo Khoradmehr; Seyyed Mehdi Kalantar; Amir Hossein Danafar; Marjan Omidi; Iman Halvaei; Ali Nabi; Saeed Ghasemi-Esmailabad; Ali Reza Talebi Journal: Int J Reprod Biomed Date: 2017-03