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What is the effect of three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid), when used individually and in combination, on mouse embryo development in culture, and subsequent fetal development post-transfer?A combination of antioxidants resulted in significant increases in blastocyst cell number, maintained intracellular glutathione (GSH) levels, supported earlier cleavage times from 5-cell stage to expanded blastocyst, and improved fetal developmental irrespective of incubator oxygen concentration.Acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid have been shown to have beneficial effects individually in several tissues, and most recently on developing embryos, in the presence of oxidative stress.Morphokinetics of mouse embryos were quantitated using time-lapse imaging. GSH levels in pronucleate oocytes were measured. Blastocysts underwent differential nuclear staining for inner cell mass and trophectoderm cells or were transferred to recipient females to assess implantation and fetal development.Pronucleate oocytes from F1 mice were cultured in 5 or 20% oxygen either individually or in groups of 10, in media G1/G2, in the presence or absence of 10 µM acetyl-L-carnitine /10 µM N-acetyl-L-cysteine /5 µM α-lipoic acid, either individually or in combination. Controls were embryos cultured without antioxidants. Intracellular levels of reduced glutathione were quantitated in pronucleate oocytes. Embryo development and viability were analysed through time-lapse microscopy and embryo transfers.Antioxidants significantly increased mouse blastocyst cell numbers compared with control when used individually (P< 0.05) and to a greater effect when all three were used in combination (P< 0.01) in 20% oxygen. The combination of antioxidants resulted in faster development rates to 5-cell cleavage stage, which continued until the expanded blastocyst stage when cultured in 20% oxygen. The beneficial effects of combining the antioxidants were greater for embryos cultured individually as opposed to in groups of 10 and for those embryos cultured in 20% compared to 5% oxygen. Levels of GSH were significantly decreased in control embryos that were incubated in the absence of antioxidants in 20% oxygen (P< 0.01), compared with in vivo flushed embryos. However, when embryos were cultured with antioxidants the level of GSH was not different to that of in vivo developed embryos. Embryos cultured in the presence of antioxidants in 20% oxygen and transferred resulted in significantly longer crown-rump length (11.6 ± 0.1 mm versus 11.3 ± 0.1 mm; P< 0.01), heavier fetuses (209.8 ± 11.8 mg versus 183.9 ± 5.9 mg; P< 0.05) and heavier placentas (103.5 ± 3.1 mg versus 93.6 ± 2.7 mg; P< 0.01) compared with controls (all data are mean ± SEM). Further, a post-implantation benefit of the antioxidant combination was also evident after culture in 5% oxygen.Embryo development and implantation was only examined in the mouse.These findings show that a combination of antioxidants in the culture media has a highly beneficial effect on mouse preimplantation embryo development in vitro and on subsequent fetal development post-transfer. These data indicate a potential role for the inclusion of specific antioxidant combinations in human embryo culture media irrespective of oxygen concentration. However, before application to human embryos, a proper evaluation of this approach in prospective, preferably randomized, trials will be required.This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.