Effect of embryo morphology and morphometrics on implantation of vitrified day 3 embryos after warming: a retrospective cohort study

Abstract

BackgroundCharacteristics routinely used to evaluate embryo quality after thawing include number of blastomeres survived and presence of mitosis resumption after overnight culture. It is unknown to which extent symmetry and fragmentation affect implantation after warming and whether application of stricter criteria either before vitrification or after warming would improve implantation rate (IR) of vitrified/warmed embryos. This study aimed to find new parameters to improve selection criteria for vitrification and for transfer after warming.MethodsFirstly, we evaluated standard morphological characteristics (intact survival, mitosis resumption, number of blastomeres, symmetry and fragmentation) of 986 warmed day 3 embryos and, from a subset of 654, we evaluated morphometric characteristics (fragmentation, symmetry and volume change). Secondly, we tested the hypothesis that IR of day 3 vitrified/warmed embryos is influenced by morphometric characteristics. IR per embryo transferred was calculated using embryos that were transferred in a single embryo transfer (SET) or a double embryo transfer (DET) with either 0 or 100 % implantation (830/986). We investigated the significant differences in IR between the different categories of a specific characteristic. These categories were based on our standard embryo evaluation system. The statistical tests Chi-square, Fisher’s exact or Cochrane-Armitage were used according to the type and/or categories of the variable.ResultsThe 986 embryos were transferred in 671 FET cycles with 16.9 % (167/986) IR. After exclusion of DET with 1 embryo implanted, IR per embryo transferred was 12.4 % (103/830). Embryo symmetry, fragmentation and volume change in vitrified/warmed day 3 embryos were not associated with IR. However, when mitosis resumption was present after overnight culture, intact embryos reached significantly higher IR than non-intact embryos and only when the embryo compacted after overnight culture the number of cells damaged after warming had no effect on IR. Concretely, embryos with 8 cells after warming or >9 cells after overnight culture–including compacted embryos–reached the highest IR (>15 %) while embryos with <6 cells after warming or with ≤6 cells after overnight culture had extremely low IR (<1 %).ConclusionsIR of vitrified embryos is determined by the number of cells lost, by the occurrence of mitosis resumption, and by the specific number of blastomeres present but not by fragmentation, blastomere symmetry or volume change. Unselecting embryos for cryopreservation because of fragmentation >10 % and/or symmetry < 75 % only leads to unwanted loss of embryos with acceptable implantation potential.Trial registrationRetrospectively registered NCT02639715.

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study

“…The presence of asymmetrical blastomeres and the % fragmentation in fresh cleavage stage embryos have been negatively correlated with implantation rate (IR) [ 9 ] and positively correlated with the incidence of aneuploidy [ 24 – 27 ]. However, using a computer based morphometric analysis –that calculates both embryo symmetry and fragmentation based on individual blastomere volume and total cell volume (TCV) [ 28 ] –we did not observe any effect of neither symmetry (>50%) nor fragmentation (<40%) on IR in FET [ 29 ]. Yet, due to the fact that cryopreservation media cause exchange of water and medium through the cell membrane [ 8 , 30 ] and because of the different concentration of cryoprotectants present in slow freezing and vitrification media [ 30 ], we hypothesize that TCV might influence differently the survival of slow frozen-thawed embryos than of vitrified-warmed embryos.…”

“…Yet, due to the fact that cryopreservation media cause exchange of water and medium through the cell membrane [ 8 , 30 ] and because of the different concentration of cryoprotectants present in slow freezing and vitrification media [ 30 ], we hypothesize that TCV might influence differently the survival of slow frozen-thawed embryos than of vitrified-warmed embryos. Using morphometric evaluation we have previously shown that, indeed, vitrification can induce both an increase or decrease of TCV after warming, [ 29 ] and in fresh cycles, TCV has been associated with IR [ 28 ]. Currently, the effect of morphometric parameters on survival and IR after cryopreservation is still unexplored.…”

“…Two characteristics are routinely used: 1) the number of blastomeres survived and 2) the presence of mitosis resumption after overnight culture [ 8 ]. The IR is positively correlated with the number of blastomeres survived [ 21 , 29 , 31 ], even though there may be no difference in IR once at least 75% of blastomeres have survived [ 22 , 23 ]. At the same time, the IR is positively correlated with the presence of mitosis resumption [ 21 , 32 , 33 ] even if the embryo has degenerated blastomeres after thawing or warming [ 6 , 22 ].…”

“…At the same time, the IR is positively correlated with the presence of mitosis resumption [ 21 , 32 , 33 ] even if the embryo has degenerated blastomeres after thawing or warming [ 6 , 22 ]. In addition, we have previously observed that if compaction occurs during 20-24 h of overnight culture, there is no detrimental effect of the number of blastomeres degenerated after vitrification-warming on IR [ 29 ]. Despite the fact that the correlation between the number of blastomeres survived and the presence of mitosis resumption with IR in FET cycles has been extensively studied, the specific timing of mitosis resumption and compaction in cleavage stage embryos after thawing or warming has never been described and, more importantly, has never been related to IR in FET cycles.…”

“…Thus, we hypothesize that the time to mitosis resumption and time to compaction depends on the freezing method, and that this time interval may be shorter for embryos whose implantation leads to a pregnancy. Besides, the reported embryo TCV change caused by vitrification media [ 29 ] raises the question of whether slow freezing media also causes TCV change and, if contrarily to vitrification [ 29 ], has an effect on IR.…”