L26/P-160 Perinatal outcome after warmed transfer of blastocysts exhibiting different collapse patterns during pre-vitrification equilibration: a prospective cohort study

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Abstract Study question Does spontaneous blastocyst collapse during pre-vitrification equilibration affect perinatal outcomes after vitrified–warmed embryo transfer when embryos are hierarchically classified by morphology? Summary answer Live birth rates (LBRs) decreased as the degree of collapse progressed from partial to complete, whereas perinatal outcomes were comparable, except for the sex ratio. What is known already Previous time-lapse studies indicate that blastocysts exhibiting strong contraction during culture are associated with reduced hatching in animal models and lower implantation in humans, suggesting that blastocyst contraction negatively affects reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival or pregnancy rates by facilitating cryoprotectant permeation into the blastocoel. However, shrinkage is not induced in widely used cryoprotectant combinations, including our protocol, because viability is already high, demonstrating the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha Consensus Meeting on Cryopreservation has not issued any recommendations regarding artificial shrinkage. Study design, size, duration A single-centre 2 × 3 factorial trial enrolled 1,860 patients undergoing autologous IVF/ICSI with a freeze-all strategy followed by 2,844 single vitrified–warmed blastocyst transfers between April 2018 and May 2022. Warmed embryos were selected using a hierarchical approach based solely on morphological assessment during culture; the collapse patterns observed during pre-vitrification equilibration were not considered. Transferred blastocysts were classified as high- or low-quality based on the ICM and TE cell number and integrity. Participants/materials, setting, methods Blastocysts were vitrified and warmed using in-house solutions and Rapid-i carriers. For vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (EG; 15 min at 37 °C). Spontaneous collapse was assessed under an inverted microscope before vitrification in 15% EG, 15% dimethyl sulfoxide (DMSO), and 0.5 M sucrose. Collapse was defined as the separation of the TE from the zona pellucida. Blastocysts with and without a blastocoel cavity were classified as partially and completely collapsed, respectively. Main results and the role of chance Overall, 240 high-quality non-collapsed (HN), 441 high-quality partially collapsed (HP), 122 high-quality completely collapsed (HC), 303 low-quality non-collapsed (LN), 645 low-quality partially collapsed (LP), and 315 low-quality completely collapsed (LC) day-5 blastocysts were transferred (embryo survival rate, 99.7%; no transfer cancellations). Maternal age (years: HN, 34.5 ± 3.8; HP, 35.1 ± 3.7; HC, 35.2 ± 3.9; LN, 35.6 ± 3.8; LP, 36.1 ± 3.8; LC, 36.6 ± 4.1; P < 0.001) and LBRs (HN, 52.7%; HP, 48.7%; HC, 48.4%; LN, 38.5%; LP, 35.3%; LC, 29.5%; P < 0.001) differed significantly among groups. These trends were observed in day-6 blastocyst transfers (n, 778), although the LBRs were lower. Day-5 blastocyst transfers yielded 847 live births. The proportion of female infants differed significantly among groups (HN, 37.3%; HP, 44.3%; HC, 50%; LN, 48.3%; LP, 48.7%; LC, 61.7%; P = 0.01), whereas other perinatal outcomes, including gestational age (weeks: HN, 38.7 ± 0.2; HP, 38.7 ± 0.1; HC, 38.8 ± 0.2; LN, 38.8 ± 0.1; LP, 38.7 ± 0.1; LC, 38.8 ± 0.2), and birth weight (g: HN, 3097 ± 50; HP, 3070 ± 33; HC, 3054 ± 52; LN, 3080 ± 41; LP, 3045 ± 29; LC, 3038 ± 51), were comparable. Limitations, reasons for caution The cryoprotectant concentration in the pre-vitrification equilibration solution was relatively low. Therefore, caution is warranted when interpreting collapse patterns, as osmolarity may influence blastocyst shrinkage. The higher proportion of female infants in collapsed embryos may partly reflect slower preimplantation development compared to males, although the underlying mechanism remains unclear. Wider implications of the findings Spontaneous blastocyst collapse during pre-vitrification equilibration influenced LBRs but not perinatal outcomes, except for the sex ratio. Among morphologically comparable blastocysts vitrified using EG and DMSO, less-collapsed embryos should be preferentially warmed for transfer to increase IVF success rates. Follow-up studies are crucial for confirming infant health. Trial registration number No

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