Centre for Reproductive Medicine, UZ Brussel, Laarbeeklaan 101, Jette 1090, Belgium.
Hum Reprod. 2013 Nov;28(11):2943-9. doi: 10.1093/humrep/det356. Epub 2013 Sep 5.
Is the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos?
Vitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight.
After slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage.
STUDY DESIGN, SIZE, DURATION: Survival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed.
Survival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification.
LIMITATIONS, REASONS FOR CAUTION: This analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos.
Although it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed.
STUDY FUNDING/COMPETING INTEREST(S): none.
玻璃化冷冻和慢速冷冻第 3 天胚胎相比，细胞损失对进一步卵裂和植入的影响是否不同？
玻璃化冷冻的胚胎无论损失多少细胞，过夜发育都比慢速冷冻的胚胎好，但如果过夜继续分裂，它们的植入潜力相似。
在慢速冻融后，完整的 4 细胞胚胎或 1 个细胞受损的 4 细胞胚胎可以获得相似的植入率。对于第 3 天的慢速冷冻胚胎，如果至少有 25%的细胞丢失，则观察到较低的植入率。其他研究报告了 0、1 或 2 个细胞受损的 7-8 细胞胚胎具有相似的植入潜力。关于玻璃化冷冻胚胎与细胞损伤相关的进一步发育，尚无数据。
研究设计、规模、持续时间：回顾性评估了 7664 个慢速冷冻第 3 天的胚胎（研究期间：2004 年 1 月至 2008 年 12 月）和 1827 个玻璃化冷冻胚胎（研究期间：2010 年 4 月至 2011 年 9 月）。根据两种方案的冷冻前细胞阶段和冻融后细胞丢失情况，评估了过夜卵裂情况。在 780 个慢速冷冻和 294 个玻璃化冷冻的胚胎的单个胚胎转移（SET）亚组中，分析了细胞丢失与植入率之间的关系。
参与者/材料、设置、方法：使用含有二甲基亚砜（DMSO）的慢速控制性冷冻（冷冻保护剂）或含有 DMSO-乙二醇（EG）-蔗糖（S）的封闭玻璃化（冷冻保护剂）对具有≥6 个卵裂球和≤20%碎片的胚胎进行冷冻保存。只有解冻后≥50%的细胞完整的胚胎才能在夜间培养，并在进一步分裂后才能转移。对于每种结果，均进行逻辑回归分析。
玻璃化冷冻和慢速冷冻的存活率分别为 94%和 64%。逻辑回归分析显示，玻璃化冷冻的胚胎过夜卵裂率高于慢速冷冻（P < 0.001），且根据细胞损伤程度而降低（P < 0.001）。如果解冻后的胚胎继续分裂，则丢失的细胞数量或冷冻保存方法对其植入潜力没有影响。0、1 或 2 个细胞受损的胚胎的植入率分别为慢速冷冻的 21%（n = 114）、21%（n = 28）和 20%（n = 12），以及玻璃化冷冻的 20%（n = 50）、21%（n = 5）和 27%（n = 4）。
局限性、谨慎的原因：该分析是回顾性的，玻璃化冷冻和慢速冷冻的研究期间不同。玻璃化冷冻的 SET 数量有限。然而，有大量的玻璃化冷冻胚胎可用于分析存活胚胎的进一步分裂情况。
尽管从妊娠结局的角度来看，玻璃化冷冻胚胎在生存能力方面不一定比慢速冷冻胚胎更有优势，但玻璃化冷冻可提高存活率、更好的过夜发育和每个解冻胚胎的更高转移率。这增加了单个治疗周期的冷冻转移周期数量，并可能导致更好的累积临床结局。基于目前的数据，在夜间培养前解冻额外胚胎的政策取决于冷冻前的细胞阶段和解冻后的细胞损伤。对于 8 细胞胚胎，与 6-7 细胞胚胎相比，最多可以损伤两个细胞，而不是一个细胞，然后再解冻一个额外的胚胎。
研究资金/利益冲突：无。
