Vitrification with shortened equilibration across human embryonic developmental stages: a proof-of-concept study

Can vitrification with a shortened equilibration solution (ES) be safely applied to human embryos without compromising key embryological clinical outcomes?

Vitrification with shortened equilibration was associated with comparable observed survival, developmental progression, and blastocyst re-expansion rates when compared to standard vitrification.

Vitrification with shortened-equilibration exposure has been primarily evaluated in mouse models and in human oocytes at the germinal vesicle and metaphase II stages, where short exposure times have been shown to maintain post-warming survival and preserve mitochondrial integrity comparable to conventional vitrification. The European Commission (CE) approval exists for the use of shortened-equilibration vitrification protocols in human oocytes, although clinical experience remains limited. Conversely, evidence regarding the application of shortened-equilibration vitrification to human embryos is currently lacking, and there is currently no clinical regulatory approval for embryos or blastocyst vitrification using shortened-equilibration protocols.

Prospective proof-of-concept study performed in a single IVF laboratory from April 2025 to March 2026. It involved 45 human blastocysts, 49 cleavage-stage embryos, and 102 tripronuclear (3PN) zygotes. Blastocysts were allocated to standard, shortened-equilibration vitrification using full-volume (300 µl) or microdrop loading (50 µl) vitrification. 3PN zygotes were used as a model for early-stage embryo response and were vitrified immediately after fertilization or at the cleavage stage according to group allocation, using standard and shortened-equilibration protocols.

All blastocysts were identified as affected by preimplantation genetic testing for monogenic disorders (PGT-M) and used for research only; they were cryopreserved, then warmed using standard protocols, then re-vitrified per group using shortened-equilibration vitrification and subsequently warmed using one-step warming. 3PN zygotes were included as clinically relevant research material and assigned to standard vitrification or shortened-equilibration vitrification (300 µl, 2 min; microdrops 50 µl, 2-3 min). Outcomes included survival rate, day-3 development, blastocyst formation, iDAScore (morphokinetic assessment), and post-warming re-expansion.

3PN zygotes were allocated to four groups: standard vitrification and shortened-equilibration vitrification using full-volume loading (300 µl, 2-min) or microdrop loading (50 µl, 2 or 3-min). Baseline characteristics were comparable between groups. Post-warming survival rates ranged from 89.2% to 100% (P = 0.408). Day-3 development ranged from 47% to 60% (P = 0.868). Blastocyst formation ranged from 32.0% to 41.2% (P = 0.920). The mean iDAScore ranged from 2.25 to 4.07 (P = 0.281). In cleavage-stage embryos, survival ranged from 78.6% to 95.2% (P = 0.325), and blastocyst formation from 25.0% to 55% (P = 0.262). In blastocysts, survival ranged from 93.3% to 100% (P = 0.592). Re-expansion at 2 h ranged from 82% to 90% after first warming (P = 0.156) and from 82% to 85% after second warming (P = 0.784). Time to complete re-expansion ranged from 3.6 to 4.0 h (P = 0.893).

The sample sizes were limited, and the study may be underpowered to detect differences between groups; patient distribution was unbalanced due to random allocation of samples between groups. In addition, the use of 3PN zygotes limits direct comparison with established blastocyst quality benchmarks due to their inherently reduced developmental potential.

Shortened-equilibration vitrification demonstrated comparable post-warming survival, developmental progression, and blastocyst re-expansion to standard vitrification. These findings support the feasibility of shortening equilibration time without compromising key embryological outcomes. If validated in larger studies, this approach may contribute to optimizing and simplifying vitrification protocols in clinical practice.

This research received no specific external grant from funding agencies in the public, commercial, or not-for-profit sectors. Departmental and institutional resources from the Infertility and IVF Institute, Sheba Medical Center, supported the study and manuscript preparation. The laboratory also receives general research scholarship support from the Alrov Fund.

R.O. reports speaker's fees from Merck Group. The other authors declare that they have no competing interests.

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