Fernandez Gallardo Elia, Spiessens Carl, D'Hooghe Thomas, Debrock Sophie
KU Leuven - University of Leuven, University Hospitals Leuven, Leuven University Fertility Center, Herestraat 49, B-3000, Leuven, Belgium.
Reprod Biol Endocrinol. 2017 Oct 3;15(1):79. doi: 10.1186/s12958-017-0299-5.
Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming.
During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing.
Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones.
Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies.
Retrospectively registered on December 15, 2015 NCT02639715 .
对体外培养的人类胚胎进行形态测量和形态动力学评估，可不受时间限制地进行评估，并减少观察者内部和观察者之间的变异性。尽管据报道这些技术可提高新鲜培养过程中卵裂期胚胎评估的质量，但在冷冻保存胚胎的评估中可能存在的优势却鲜有探索。本研究旨在比较慢速冷冻和玻璃化冷冻胚胎的形态测量和形态动力学参数，并确定它们与解冻/复温后胚胎存活及着床率（IR）的关系。
在新鲜培养期间，对286个解冻/复温后的胚胎测量其形态特征（总细胞体积（TCV）、对称性、碎片率和卵裂球数量）。同样，在解冻/复温后，对135个存活的胚胎测量类似的形态特征。此外，在90个胚胎解冻/复温后测量其形态动力学参数（有丝分裂恢复时间和致密化时间）。然后，使用线性回归，我们研究了玻璃化冷冻和慢速冷冻胚胎之间的差异以及所测量特征与胚胎存活和着床率的关系。应用统计校正以考虑数据聚类和多重检验。
玻璃化冷冻胚胎恢复有丝分裂并开始致密化的时间明显早于慢速冷冻胚胎。玻璃化冷冻胚胎的有丝分裂恢复率为82%，慢速冷冻胚胎为63%，有丝分裂恢复的中位时间分别为7.6小时和13.1小时（p = 0.02）。玻璃化冷冻胚胎的致密化率为62%，慢速冷冻胚胎仅为23%。玻璃化冷冻胚胎致密化的中位时间为18.1小时，但对于慢速冷冻胚胎，由于不到一半的慢速冷冻胚胎达到致密化，因此无法计算（p = 0.0001）。此外，无论冷冻方法如何，完整胚胎恢复有丝分裂的时间都明显早于不完整胚胎（恢复率分别为79%和66%，中位时间分别为7.6小时和14.6小时，p = 0.03）。关于形态测量，尽管卵裂球数量相似，但解冻后慢速冷冻胚胎的TCV较低，卵裂球对称性高于玻璃化冷冻胚胎。在慢速冷冻胚胎中，着床率与冷冻保存时的卵裂球数量有关，但在玻璃化冷冻胚胎中则不然。
有趣的是，玻璃化冷冻/复温后的胚胎恢复有丝分裂和致密化的时间明显早于慢速冷冻/解冻后的胚胎。然而，这种形态动力学参数的临床应用仍有待在更大规模的研究中进行调查。
于2015年12月15日进行回顾性注册，NCT02639715 。
