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

Elia Fernandez Gallardo, Carl Spiessens, Thomas D'Hooghe, Sophie Debrock, Elia Fernandez Gallardo, Carl Spiessens, Thomas D'Hooghe, Sophie Debrock

Abstract

Background: Characteristics 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.

Methods: Firstly, 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.

Results: The 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 %).

Conclusions: IR 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 registration: Retrospectively registered NCT02639715 .

Keywords: Embryo morphology; Frozen embryo transfer; Implantation rate; Morphometrics; Vitrified/warmed embryo.

Figures

Fig. 1
Fig. 1
Proportion of intact and non-intact that resumed mitosis or compacted after overnight culture. Legend: There is a lower proportion of cleaving embryos and higher proportion of compacted embryos in intact versus non-intact embryos (Cochran-Armitage test p-value < 0.0001)
Fig. 2
Fig. 2
IR per embryo transferred in relation to the number of blastomeres degenerated after warming. Legend: Implantation rate (IR) (n embryos implanted/n embryos transferred) was calculated for embryos transferred in SET or DET with 0 % or 100 % implantation. Cochrane Armitage p-value = 0.02
Fig. 3
Fig. 3
IR per embryo transferred in relation to intact survival and mitosis resumption. Legend: Implantation rate (IR) (n embryos implanted/n embryos transferred) was calculated for embryos transferred in SET or DET with 0 % or 100 % implantation. There is significantly higher implantation rate in intact embryos than in non-intact embryos with mitosis resumption (fisher’s exact p-value = 0.03), but no difference between both groups for embryos without mitosis resumption or compacted embryos
Fig. 4
Fig. 4
IR per embryo transferred in relation to number of blastomeres, fragmentation and symmetry, evaluated manually. Legend: Implantation rate (IR) (n embryos implanted/n embryos transferred) was calculated for embryos transferred in SET or DET with 0 or 100 % implantation. P values were calculated using Cochrane-Armitage test. Significant p-values (<0.05) are marked with *. Higher number of blastomeres after overnight culture and higher blastomere symmetry after warming were significantly associated with higher IR. Embryos in morula stage after warming (n = 4) are included in the group of >9 blastomeres. Fragmentation and symmetry was not evaluated for embryos in M or EB stage (n = 4 after warming; n = 253 after overnight culture). Embryos with >25 % fragmentation after warming are not included in the graphs because of the low number (n = 5 after warming, n = 7 after overnight culture). M = morula; EB = early blastocyst

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