A randomized controlled trial comparing embryo vitrification with slush nitrogen to liquid nitrogen in women undergoing frozen embryo transfer: embryology and clinical outcomes

Abstract STUDY QUESTION Does the use of slush nitrogen (SN) for embryo vitrification improve embryo transfer outcomes compared to liquid nitrogen (LN)? SUMMARY ANSWER SN is a safe method for embryo preservation and significantly improves post-warming survival rates during repeated vitrification–warming cycles; however, after a single freeze–thaw cycle, pregnancy outcomes are not improved when embryos are vitrified with SN compared to LN. WHAT IS KNOWN ALREADY SN is a combination of solid and LN, with a temperature lower than regular LN, and it is an alternative to conventional LN in achieving a faster cooling speed. Studies have shown that SN improves survival in non-human embryos and human oocytes. However, it is unknown whether the use of SN reduces blastocyst damage in humans during vitrification—as indicated by increased survival across multiple vitrification–warming cycles—or whether it enhances pregnancy outcomes in a single vitrification–warming cycle. STUDY DESIGN, SIZE, DURATION Following the pre-clinical trial assessing embryo survival after repeated freeze–thaw cycles using SN and LN on 50 donated embryos per group, a randomized controlled trial was performed, where 253 patients were enrolled between September 2020 and January 2022, and 245 underwent an IVF stimulation, which resulted in at least one blastocyst for cryopreservation. Of those, 121 were allocated to the SN (study), and 124 were allocated to the LN (control) group. Randomization occurred on the day of blastocyst biopsy using a computer-generated block schema. Groups were assigned via opaque envelopes, opened by the embryologist on vitrification day. The patient, physician, and clinical team were blinded to the intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS All couples with female aged between 18 and 42 years old undergoing IVF stimulation at one university-affiliated infertility center, with plan for preimplantation genetic testing for aneuploidy and subsequent single, frozen embryo transfer (FET) were eligible for inclusion in this study. MAIN RESULTS AND THE ROLE OF CHANCE The pre-clinical trial demonstrated significant improvements in blastocyst survival, with the SN group achieving a mean of 7.5 survived vitrification–warming cycles (range: 3–22), significantly surpassing the mean of 3.0 cycles (range: 0–10) in the LN group (P < 0.0001). Following the pre-clinical trial, 223 patients randomized to SN or LN underwent single FET. Baseline characteristics were similar between groups, as were embryology outcomes, including the number of oocytes retrieved, mature oocytes, fertilization rate, and total blastocysts biopsied. No significant differences were observed between the two groups in pregnancy rate, clinical pregnancy rate, sustained implantation rate, or miscarriage rate (P = 0.16, 0.80, 0.49, and 0.74, respectively, using Student’s t-test). A futility analysis indicated no value in continuing recruitment and therefore the study was closed. LIMITATIONS, REASONS FOR CAUTION Neonatal or birth outcomes were not assessed. Termination of the study based on futility analysis precludes a conclusion of equivalence between SN and LN. WIDER IMPLICATIONS OF THE FINDINGS This study demonstrates that SN is a safe alternative to traditional LN for vitrification; however, it did not demonstrate improvements in the reproductive potential of vitrified embryos. STUDY FUNDING/COMPETING INTEREST(S) The project was funded by the Foundation for Embryonic Competence TRIAL REGISTRATION NUMBER NCT04496284. TRIAL REGISTRATION DATE 3 August 2020. DATE OF FIRST PATIENT’S ENROLLMENT 5 September 2020.