Cryopreservation of incomplete compacted morulae and preliminary biopsy of excluded fragments

Abstract

The complexity of predicting the embryo development potential at the cleavage stages and emergence of epigenetic risks during prolonged in vitro cultivation of pre-implantation embryos are advantageous when transferring the embryo at the morula stage to the uterine cavity. However, the criteria for embryos estimating at this stage which allow predicting the cryopreservation outcomes have been poorly described. All day 4 embryos (n=224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD= 100%, 75%, 50%, 25%, 0%, respectively) and then the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between the survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure with cryoprotective media and after freeze thawing they have been destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and excluded fragments biopsy of incomplete compacted morula prior to the embryo cryopreservation. This led to significant increase of their post-thawing survival rate up to (93.1 ± 4.1), (75 ± 8.8) % and blastocyst formation rate up to (85.2 ± 10.4), (59.4 ± 5.2)% in grades 2 and 3 respectively, and there was no significant difference in grade 4. Thereby using the blastomeres and excluded fragments biopsy in incomplete compacted morulae can improve the cryopreservation outcomes of embryos with BCD grade 2 and 3.