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By Mai Truong, Oct 18, 2022

Age of Antioxidants - finding their way into the IVF laboratory

Antioxidants, long touted in cosmeceuticals for their anti-aging miracles, and in food industries for their health benefits, are now a new dynamic component in IVF media. While it has been demonstrated that the use of individual antioxidants has beneficial effects, their real power is manifest when used in combination, as is seen in vivo as part of an elegant antioxidant system. This blog post outlines the rationale for including antioxidants in IVF media and how the three antioxidants in the Gx Media system were selected and tested in the mouse model.

Gametes and embryos are exposed to oxidative stress in vitro

Exposure to 20% (atmospheric) oxygen has been shown to be a source of oxidative stress detrimental to the development of gametes and embryos. Although there are many well documented pathologies associated with culture at atmospheric oxygen [1, 2], it is still not uncommon for IVF clinics to utilise 20% oxygen in all or part of their human embryo culture system. In fact, even a brief exposure to atmospheric oxygen during the routine handling of embryos can have detrimental effects whereby the three combined antioxidants in handling media were found to improve embryo development and ameliorate the negative effects [3]. Furthermore, exposure to atmospheric oxygen is also unavoidable during sperm and oocyte collection, preparation and ICSI as well as biopsy. Exposure to atmospheric oxygen combined with ART processes that form high levels of reactive oxygen species (ROS), such as sperm preparation (i.e., centrifugation), ultimately means that the oxidative stress endured by gametes and embryos is increasingly heightened. Furthermore, even at 5 % oxygen gametes and embryos cannot escape ROS as it is generated through their oxidative metabolism.

Cryopreservation adds to the cumulative stress

Cryopreservation is not only conducted at atmospheric oxygen but also induces oxidative stress and ROS in a multi-pronged manner. The cells are exposed to extreme temperature changes and high cryoprotectant levels result in increased osmolalities. This in turn triggers the cells adaptive rescue systems such as DNA repair, but in doing so also produces ROS. As each stressor increases the susceptibility of gametes and embryos to a secondary stress with cumulative harmful impacts on embryo health [4, 5], the multiple sources of oxidative stress inevitably compound, causing further oxidative damage.

Selecting a combination of antioxidants

Over the past seven years we have investigated, with the mouse model, a combination of three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid) as they filled distinct yet complementary niches in oxidative stress responses. α-lipoic acid, known as the antioxidant of all antioxidants, is a key intracellular antioxidant with the capacity to recycle and reduce other cellular antioxidants. N-acetyl-L-cysteine is a powerful antioxidant with the ability to increase glutathione levels, which is a major intracellular antioxidant in itself. L-Carnitine protects against oxidative damage through scavenging free radicals and is most abundant in the epididymal fluid. These three antioxidants are powerhouses in their own right, but together were found to be even more effective in combating the multiple sources of oxidative stresses encountered by gametes and embryos. When added to the ART media system as a combined group, the beneficial effects to mouse embryo and foetal development and implantation rates were greater than when they were used on their own [6].

Antioxidants support an in vivo-like gene expression

When you look at the gene expression profiles of in vitro cultured and cryopreserved embryos there are significant perturbations compared to in vivo derived blastocysts [7]. Antioxidants in culture media substantially reduced the changes in gene expression, most strikingly those associated with increased risk for preeclampsia and intrauterine growth restriction (IUGR), particularly in the placenta [7]. Similar processes are affected following cryopreservation, though more genes are dysregulated overall. Cryopreservation therefore exacerbates the increased risk for preeclampsia and IUGR resulting from in vitro culture alone, once again highlighting the detrimental cumulative effects of stresses such as in vitro culture and cryopreservation. Combined antioxidants therefore may assist in maintaining a more in vivo-like gene expression profile following ART which may help maintain the viability of human embryos.

Still early days for the antioxidants – hopefully more success to come

Our animal studies show that cumulative oxidative stresses incurred during ART detrimentally impact gamete and embryo quality and viability. Combined antioxidants in ART media have a similar synergy in that their benefits are also cumulative and multi-factorial and ameliorate oxidative stress to resemble the in vivo environment more closely. Therefore, the addition of antioxidants during gamete handling, IVF, embryo culture/handling and cryopreservation will help combat the negative effects of oxidative stress and improve embryo development [3, 6, 8]. In the future such unique antioxidant enriched media could be an integral factor for increasing IVF outcomes and could herald a new age of ART media whereby a combination of three antioxidants is just the beginning.


Webinar with Prof. David K Gardner

If you are interested in learning more about antioxidants in IVF, this webinar, brought to you by the Vitrolife Academy, highlights the latest media innovations in ART. Professor David Gardner discusses the importance of antioxidants as an element of ART media, and the potential for improved embryo viability. He shares his experience leading to the development of culture media complemented with a unique mix of antioxidants, additionally discussing the results from current clinical studies.

Watch webinar



  1. Harvey, A.J., et al., Oxygen-regulated gene expression in bovine blastocysts. Biol Reprod, 2004. 71(4): p. 1108-19.
  2. Wale, P.L. and D.K. Gardner, Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development. Biol Reprod, 2012. 87(1): p. 24, 1-8.
  3. Truong, T. and D.K. Gardner, Antioxidants improve IVF outcome and subsequent embryo development in the mouse. Hum Reprod, 2017. 32(12): p. 2404-2413.
  4. Kelley, R.L. and D.K. Gardner, Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro. Reprod Biomed Online, 2016. 33(5): p. 537-549.
  5. Wale, P.L. and D.K. Gardner, The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update, 2016. 22(1): p. 2-22.
  6. Truong, T.T., Y.M. Soh, and D.K. Gardner, Antioxidants improve mouse preimplantation embryo development and viability. Hum Reprod, 2016. 31(7): p. 1445-54.
  7. Truong, T., A.J. Harvey, and D.K. Gardner, Antioxidant supplementation of mouse embryo culture or vitrification media support more in-vivo-like gene expression post-transfer. Reprod Biomed Online, 2022. 44(3): p. 393-410.
  8. Truong, T.T. and D.K. Gardner, Antioxidants increase blastocyst cryosurvival and viability post-vitrification. Hum Reprod, 2020. 35(1): p. 12-23.

Topics: Embryo culture & transfer

Written by Mai Truong

Mai Truong has been the laboratory manager of Professor David Gardner’s ART lab at the University of Melbourne for the past 15 years. Along with managing the ART laboratory and overseeing the team of staff and students, she has contributed to multi-centre collaborations across different institutes and published several papers on embryology. Her current research remains focused on the effects of antioxidants and culture conditions on IVF and embryo development.

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