STUDY QUESTION Does one-step warming (OW), a simplified embryo warming protocol, adversely affect survival and developmental potential in vitrified cleavage or blastocyst stage embryos compared to standard multi-step warming (SW)? SUMMARY ANSWER OW showed no detrimental effects on survival and developmental potential compared to SW in cleavage and blastocyst stage embryos. WHAT IS KNOWN ALREADY While standard embryo warming protocols involve a multi-step procedure using a stepwise osmotic solution to avoid a rapid influx of water into the embryo, recent studies suggest that eliminating the stepwise warming process does not reduce embryo survival and embryo transfer outcomes. However, previous reports have focused primarily on pregnancy rates, and a more detailed analysis of the effects of rapid osmotic pressure changes on embryos is necessary to standardize the protocol. STUDY DESIGN, SIZE, DURATION This preliminary study includes donated 377 vitrified human embryos (177 cleavage and 200 blastocyst stage) from 210 patients approved for discard at the patient’s consent. The embryos were randomly allocated and warmed using either SW or OW protocols. In the SW protocol, embryos were rinsed with a stepwise osmotic solution (thawing, dilution, and washing solutions), and the process was completed with a 13-min warming period. In the OW protocol, embryos were only rinsed in a single solution (thawing solution) for 1 min. PARTICIPANTS/MATERIALS, SETTING, METHODS Post-warming embryos were cultured using a time-lapse incubator. Survival rate and developmental potential, including the occurrence of abnormal morphokinetics and the time required for blastocyst formation after warming of cleavage stage embryos, were compared between SW and OW. Embryos that developed into the blastocyst stage were morphologically evaluated. In the warming of blastocyst stage embryos, the survival rate was determined by the presence of blastocoel expansion, and the proportion of full re-expanded blastocysts was observed at 3- and 24-h post-warming. An in vitro adhesion assay was also performed on blastocysts after culture, and adhesion rate and outgrowth area were measured 24, 48, and 72 h after culture with fibronectin-precoated dishes. MAIN RESULTS AND THE ROLE OF CHANCE OW did not negatively impact survival rates in either cleavage (100% in both OW and SW groups) or blastocyst stage embryos (99% in both groups). Cleavage stage embryos warmed by OW had superior or comparable rates of morulation (96 vs 85%, P = 0.0387), blastulation (78 vs 73%, P = 0.4044), full-blastocyst formation (60 vs 53%, P = 0.3196), and expanded-blastocyst formation (56 vs 49%, P = 0.4056) compared to those warmed by SW. Time-lapse monitoring analysis revealed that the frequency of collapses was reduced in OW (30 vs 50%, P = 0.0410). Additionally, all other abnormal morphokinetics were equivalent between OW and SW (P > 0.05); moreover, the time required for blastocyst formation (P > 0.05) and the morphological quality after development into the blastocyst stage (P > 0.05) were not significantly different between OW and SW. In warming of blastocyst stage embryos, the time required for full re-expansion was longer with OW (3.20 ± 3.03 h vs 2.14 ± 2.17 h, P = 0.0008), but there was no significant difference in the proportion of full re-expanded blastocysts at 3- (67 vs 75%, P = 0.2417) and 24-h (98 vs 97%, P = 1.0000) post-warming. The in vitro adhesion assay showed no significant differences in adhesion rate and outgrowth area at all observation points (P > 0.05). LIMITATIONS, REASONS FOR CAUTION This study was carried out as a preliminary trial using discarded embryos, which limited the number of embryos analyzed. Additionally, the impact on embryo transfer outcomes, such as clinical pregnancy and livebirth rates, remains unclear. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that OW is a safe and efficient alternative to SW protocols and may improve the efficiency of IVF laboratory workflow without impairing embryo potentials. STUDY FUNDING/COMPETING INTEREST(S) No funding was obtained for this study. The authors have no conflicts of interest to declare related to this study. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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玻璃化人卵裂和囊胚期胚胎的一步加热不会对胚胎存活率和随后的发育潜力产生不利影响


研究问题 与标准的多步加温 （SW） 相比，一步加温 （OW） 是一种简化的胚胎加温方案，是否会对玻璃化卵裂或囊胚期胚胎的存活和发育潜力产生不利影响？总结答案 与卵裂和囊胚期胚胎的 SW 相比，OW 对存活和发育潜力没有不利影响。已经知道的虽然标准的胚胎加热方案涉及使用逐步渗透溶液的多步骤程序，以避免水快速流入胚胎，但最近的研究表明，消除逐步加热过程不会降低胚胎存活和胚胎移植结果。然而，以前的报告主要集中在妊娠率上，有必要更详细地分析快速渗透压变化对胚胎的影响，以标准化该方案。研究设计、规模、持续时间 这项初步研究包括捐赠的 377 个玻璃化人类胚胎（177 个卵裂期和 200 个囊胚期），这些胚胎来自 210 名经患者同意批准丢弃的患者。胚胎被随机分配并使用 SW 或 OW 方案加热。在 SW 方案中，用逐步渗透溶液（解冻、稀释和洗涤溶液）冲洗胚胎，并在 13 分钟的升温期内完成该过程。在 OW 方案中，胚胎仅在单一溶液（解冻溶液）中冲洗 1 分钟。参与者/材料、设置、方法 使用延时培养箱培养加热后的胚胎。比较 SW 和 OW 的存活率和发育潜力，包括异常形态动力学的发生和卵裂期胚胎升温后囊胚形成所需的时间。 对发育到囊胚阶段的胚胎进行形态学评估。在囊胚期胚胎的加热中，存活率由囊胚腔扩增的存在决定，并且在加热后 3 小时和 24 小时观察到完全再膨胀的囊胚的比例。培养后还对囊胚进行了体外粘附试验，并在用纤连蛋白预包被培养皿培养后 24 、 48 和 72 小时测量粘附率和生长面积。主要结果和机会 OW 的作用没有对卵裂 （OW 和 SW 组均为 100%） 或囊胚期胚胎 （两组均为 99%） 的存活率产生负面影响。与 SW 加热的胚胎相比，经 OW 加热的卵裂期胚胎的死亡率 （96 vs 85%，P = 0.0387）、爆破率 （78 vs 73%，P = 0.4044）、全囊胚形成率 （60 vs 53%，P = 0.3196） 和扩大囊胚形成率 （56 vs 49%，P = 0.4056） 具有更高或相当的水平。延时监测分析显示，OW 中塌陷的频率降低 （30 vs 50%， P = 0.0410）。此外，OW 和 SW 之间的所有其他异常形态动力学是等效的 （P > 0.05）;此外，OW 和 SW 之间囊胚形成所需的时间 （P > 0.05） 和发育到囊胚期后的形态质量 （P > 0.05） 差异不显著。在囊胚期胚胎的升温中，OW 完全再扩增所需的时间更长 （3.20 ± 3.03 h vs 2.14 ± 2.17 h，P = 0.0008），但在升温后 3 小时（67 vs 75%，P = 0.2417）和 24 小时 （98 vs 97%，P = 1.0000） 完全再扩增的囊胚比例没有显着差异。 体外粘附试验显示所有观察点的粘附率和生长面积无显著差异 （P > 0.05）。局限性，谨慎的原因 这项研究是使用丢弃的胚胎进行的初步试验，这限制了分析的胚胎数量。此外，对胚胎移植结果的影响，例如临床妊娠率和活产率，仍不清楚。研究结果的更广泛意义我们的研究结果表明，OW 是 SW 方案的一种安全有效的替代方案，并且可以在不影响胚胎潜力的情况下提高 IVF 实验室工作流程的效率。研究资金/竞争利益 本研究没有获得资金。作者没有与本研究相关的利益冲突需要声明。试验注册号 N/A。