Antioxidants in IVF culture media leads to more blastocysts, higher live birth rates

A 2024 study found that supplementing IVF culture media with antioxidants improved fertilization, blastocyst formation rates and live birth rates for patients aged 35-40.

Reactive oxygen species (ROS) are highly reactive molecules that can impact egg, sperm and embryo quality. ROS are generated by cells as part of their metabolism, but they can also be found in culture media (the nourishing liquid that gametes or embryos are stored in in the lab).

ROS can be neutralized using antioxidants, and some research has shown that supplementing antioxidants in culture media can improve embryo quality.

This post reviews a study by Mizumoto et al. (2024), where researchers added three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine, and α-lipoic acid) to culture media used for preparing eggs and sperm and for growing embryos. They measured how this changed IVF outcomes, like fertilization rates, as well as pregnancy outcomes after a single blastocyst was transferred.

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Study details

This section covers key details of how the study was performed, including patient characteristics, how they were treated, and other methods used. For those who aren’t interested in these details, and just want to see the results, you can go ahead and skip this part.

• This was a prospective randomized study that took place at a single IVF center in Japan between 2018 and 2019.
• Exclusions: No patients with frozen eggs, donor eggs, frozen sperm, donor sperm, surgically retrieved sperm, <8 retrieved eggs, >40 years old, PGT and multiple embryo transfer cycles (only single embryo transfers were permitted).
• This was a sibling egg study, where patients’ retrieved eggs were randomly split and cultured with or without antioxidants.
• The antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid) were included in the embryo culture media, and sperm/egg preparation medias before insemination.
• Embryos were grown up in a time-lapse incubator, and evaluated on day 3 and day 5/6. Blastocysts were frozen, and a single blastocyst was thawed and transferred. About 90% of cycles were ICSI.

In terms of sample size, there were a total of 127 patients that had a single egg retrieval: 66 were aged <35 and 61 were 35-40. A total of 1734 mature eggs resulted from these retrievals, with 872 being cultured without antioxidants (the control) and 862 being cultured with antioxidants. These women had 188 transfer attempts from embryos made from these eggs, with up to four transfers per patient.

In terms of baseline characteristics, everything seemed to be roughly equal between the two groups (no stats were done). The no antioxidant control seemed to have a higher proportion of patients with male factor (53 patients vs 40). What’s unusual is that both groups (<35 and 35-40) got an average of about 17.4 eggs after their retrieval. This seems pretty high for the 35-40 group, but I guess these were good prognosis (high AMH) patients.

IVF outcomes improve with antioxidant-supplemented culture media

Of the 127 patients that had an egg retrieval, there were 1734 mature eggs: 862 were cultured in media that had antioxidants and 872 were cultured in standard media without the antioxidants.

For all ages, with the antioxidant-supplemented media, there was:

• Higher fertilization (71.8% vs 65.3%, p= 0.0278)
• Higher number of day 3 embryos (6-10 cells, 50.8% vs 44.8%, p= 0.0341).
• Higher number of day 5-6 blastocysts (expansion 3 or higher, 39.7% vs 32.7%, p= 0.0007).

They then took this data and looked at subgroups based on age (<35 and 35-40).

For women <35, there was only an increase in the number of day 3 embryos (51.3% vs 47.3%, p= 0.0352).

For women 35-40, per mature egg there was:

• Higher number of day 3 embryos (50.2% vs 42.3%, p= 0.0384).
• Higher number of day 5 -6 blastocysts (36.3% vs 27.7%, p= 0.0112).
• Higher number of day 5-6 good quality blastocysts (3BB or higher, 23.8% vs 14.8%, p= 0.0021).

So the overall results were mainly attributed to the benefit in women aged 35-40.

All the results above were calculated per mature egg (ie. fertilization rate = number of fertilized eggs/number of mature eggs x 100%). When they did the analysis per fertilized egg, there weren’t many statistical differences (except in the number of day 5-6 good quality blastocysts for patients aged 35-40). This shows that including the antioxidants in the media used for egg and sperm preparation is important.

Most cycles were ICSI, and when comparing conventional IVF vs ICSI, the results were similar.

Improved pregnancy rates with antioxidant-supplemented culture media, for women aged 35-40

The blastocysts were then frozen and the single best embryo was thawed and transferred and pregnancy outcomes were reported. There were up to four transfer attempts per patient, with a total of 94 transfers for each of the two groups (188 in total — 90 for women <35 and 98 for women 35-40). The results below are cumulative, and include results after up to four transfers per patient.

The only statistically significant result was in the 35-40 age group. With antioxidant-supplemented culture media, there was:

• Higher beta-hCG pregnancy rate (73.2% vs 45.2%, p= 0.0049).
• Higher pregnancy rate by fetal heart beat (48.2% vs 28.6%, p= 0.0493).
• No change in pregnancy loss rates.
• Higher live birth rate (42.9% vs 23.8%, p= 0.0499).
• No differences in birth weights.

They also looked at results for the first transfer and second transfer onwards, with no differences between the groups for any of the pregnancy outcomes. This analysis didn’t compare ages, which is unfortunate (I would have liked to see just the first transfer results). There were mostly similar proportions of patients doing a second, third and fourth transfer.

Conclusions

This study found that patients that used antioxidant-supplemented media had higher fertilization rates and formed more blastocysts, which was especially true for patients aged 35-40.

An analysis showed that including the antioxidant supplements in the egg/sperm preparation media was important for these outcomes.

Patients had a single blastocyst transfer and the antioxidant-supplemented group showed higher pregnancy and live birth rates, but only for those aged 35-40.

So why the effect on age?

The authors mention that aging reduces the ability of gametes to protect themselves from ROS damage. By including antioxidant supplements, this helped to neutralize ROS and protect gametes and embryos.

While these results are promising, the researchers point out that the safety and stability of the antioxidants in the culture media needs to be studied before clinical application, along with more studies on the long-term effects in newborns.

Related studies

To learn more about this topic, you can check out a number of studies referenced in this study below (3 links):

Reference

Mizumoto, Shigetoshi, Yozo Nagao, Hitomi Watanabe, Keiko Tanaka, and Takeshi Kuramoto. 2024. “Culture Media with Antioxidants Improved Fertilization, Preimplantation Embryo Development and Clinical Outcomes of Advanced Age Patients.” Reproductive Biomedicine Online 0 (104415): 104415.

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About Embryoman

Embryoman (Sean Lauber) is a former embryologist and the founder of Remembryo, an IVF research and fertility education website. After working in an IVF lab in the US, he returned to Canada and now focuses on making fertility research more accessible. He holds a Master’s in Immunology and launched Remembryo in 2018 to help patients and professionals make sense of IVF research. Sean shares weekly study updates on Facebook, Instagram, and Reddit regularly. He also answers questions on Reddit or in his private Facebook group.

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