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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 INTEREST(S) The study was funded by the Clermont Universite 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.;A.P. are employees of CellOxess, LLC. J.R.D.;A.G.-A.;R.J.A. are honorary members of the CellOxess advisory board. © 2016 The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction a d Embryology. All rights reserved.

Intracellular lipids accumulated in embryos produced invitro have been linked to reductions in both quality and post-cryopreservation viability. Diacylglycerol O-acyltransferase 1 (DGAT1) is an enzyme that catalyzes the final step in triglyceride synthesis, which is a major component of the lipid droplets in embryos. Thus, the aim of this study was to evaluate the development and quality, in regard to changes in the expression of candidate genes of lipid metabolism, of bovine blastocysts cultured invitro with DGAT1 inhibitor (A922500 Sigma-Aldrich). Zygotes were cultured in synthetic oviduct fluid supplemented with 5% FCS (TC) or in TC supplemented with 10 or 50 µM DGAT1 inhibitor (T10 and T50, respectively) or TC with 0.1% dimethyl sulfoxide (TDMSO: vehicle for DGAT1 dilution) from 54h post-insemination (hpi; major embryonic genome activation, EGA) until Day 8. Cleavage rate (48 hpi) and blastocyst yield (Day 7-8) were evaluated. Day 7 blastocysts were snap-frozen in LN2 for gene expression analysis. The mRNA abundance of candidate genes related to lipid metabolism (DGAT1, PLIN2, GLUT5, GLUT1, GPX1, PPAR1b, and G6PD) was measured by quantitative PCR. The H2AFZ and ACTB genes were used as housekeeping genes. Statistical analysis was assessed by one-way analysis of variance. No differences were found in cleavage rate, whereas blastocyst yield at Day 8 was higher (P

Embryonic genome activation (EGA) is a critical event in early embryonic development and occurs in 8-16-cell stage embryos in bovine. Invitro embryo production increases reactive oxygen species (ROS), leading to low yield and cell death. Nobiletin is an antioxidant that inhibits ROS production and affects cell cycle regulation. The aim of this study was to evaluate the effect of nobiletin supplementation in two key periods of early embryo development on blastocyst yield and expression of candidate genes of the Akt pathway. Invitro-produced zygotes were cultured in synthetic oviductal fluid supplemented with 5% FCS (control; C); C with 5 or 10 µM nobiletin (MedChemExpress; N5, N10) or C with 0.03% dimethyl sulfoxide (CD vehicle for nobiletin dilution) during the minor (2-8-cell stage; MNEGA) or major (8-16-cell stage; MJEGA) phase of EGA, considered as two separate experiments. For all groups, the speed of development was considered, and normally developing embryos that reach ≥8 cells at 54h post-insemination and ≥16 cells at 96h post-insemination were selected and cultured in control medium until Day 8, respectively. Embryos at ≥8-cell stage (N5/N10 MNEGA), 16-cell stage (N5/N10MJEGA), and Day 7 blastocysts of both periods were snap-frozen in LN2 for gene expression analysis. Cleavage rate and blastocyst yield (Day 7-8) were evaluated. The mRNA abundance of candidate genes related to the Akt pathway (CDK2, PGC1A, PPARG, RPS6KB1) and oxidative stress (GPX1) was measured by quantitative PCR. The H2AFZ and ACTB genes were used as housekeeping genes. Statistical analysis was assessed by one-way ANOVA. Nobiletin supplementation during MNEGA showed no differences in cleavage rate, whereas the blastocyst yield at Day 8 was higher (P