A prospective randomized controlled trial of antioxidants in human IVF and embryo culture media

Abstract

Study question: Does the addition of three antioxidants to culture media during gamete collection, insemination, and embryo culture increase the clinical pregnancy rate from fresh blastocyst transfers?

Summary answer: The clinical pregnancy rate from fresh blastocyst transfers was not increased by the addition of antioxidants to IVF and embryo culture media.

What is known already: Addition of antioxidants to media is beneficial in mouse IVF, embryo culture, and cryopreservation. Prospective clinical trials of sibling human oocytes found an improvement in embryo quality and increased pregnancy rates from frozen blastocyst transfers in older patients.

Study design, size, duration: Single-centre, prospective randomized controlled trial, superiority study comparing media with or without the addition of antioxidants from January 2019 to November 2021. A total of 1482 patients were randomized before egg collection. Patients and their doctors were blinded to the treatment group.

Participants/materials, setting, methods: Patients undergoing IVF/ICSI cycles and intending to undergo a fresh transfer of a single blastocyst were recruited. Exclusion criteria were previous participation in the study, use of cryopreserved oocytes/embryos, artificial oocyte activation, freeze-all cycle, or extraction of sperm from testicular biopsy. Seven hundred thirty-nine patients were randomized to control media and 743 patients to media containing the 'A3' antioxidant combination of acetyl-L-carnitine, α-lipoic acid, and N-acetyl-L-cysteine (treatment group).

Main results and the role of chance: The clinical pregnancy rate per randomized patient per cycle from fresh embryo transfer was not different between the control and antioxidant media (26.1% vs 22.9%; P > 0.05; RR 0.88 (95% CI 0.73-1.05)). In the Per Protocol population, which excludes patients with protocol violations or without a fresh transfer due to freeze-all or no embryo available, there was also no difference in between the control and antioxidant media in clinical pregnancy rate (36.7% vs 33.2%; P > 0.05; RR 0.90 (95% CI 0.76-1.07)) and live birth rate (32.4% vs 29.5%, P > 0.05). In the Intention-to-Treat population, antioxidant media produced a significant increase in the fertilization rate from 59.2 ± 26.3% to 64.5 ± 25.4% (P < 0.001) compared to control media. Blastocyst development rate per fertilized oocyte was not affected by antioxidant media, but the higher fertilization rate resulted in more fertilized oocytes per patient and therefore more blastocysts utilized per patient in the antioxidant group compared to the control (2.70 ± 2.59 vs 3.09 ± 2.96, P < 0.01). The increase in fertilization rate was observed in a subgroup analysis of ICSI cycles (57.9 ± 27.2% vs 68.3 ± 24.7%, P < 0.0001), and a decrease in the number of cycles with failed fertilization from 8.0 to 3.7% with antioxidant media (P < 0.01). In contrast, there was no effect of antioxidant media on fertilization rate in cycles with IVF insemination.

Limitations, reasons for caution: This was a single-centre study, so the effects of antioxidant media in clinics with different protocols are unknown. Patient oxidative stress, which may be influenced by inflammation, diet, smoking status, antioxidant supplement consumption, and other lifestyle factors, was not accounted for. Any potential effect of renewing the antioxidants in the media during culture was not examined.

Wider implications of the findings: Addition of antioxidants to culture media did not affect pregnancy rates from fresh single embryo transfers. An increase in fertilization rate was observed, which resulted in more blastocysts available for transfer and cryopreservation. There was no effect of antioxidants on blastocyst development rate or grade. Further studies are needed to validate the observed effect of antioxidants on fertilization rate following ICSI.

Study funding/competing interest(s): Culture media and an independent statistician were funded by Vitrolife AB. R.L.K has received travel funding and a speaker's honorarium from Vitrolife. D.K.G. has received research grants from Vitrolife at the University of Melbourne. N.-G.P. has received consulting fees from Vitrolife for work related to the study. All other authors have nothing to declare.

Trial registration number: ACTRN12618001479291.

Trial registration date: 4 September 2018.

Date of first patient’s enrolment: 28 January 2019.

Keywords: N-acetyl-L-cysteine; ICSI; acetyl-carnitine; culture media; fertilization; oxidative stress; reactive oxygen species; α-lipoic acid.