title: Effect of embryo morphology and morphometrics on implantation of vitrified day 3 embryos after warming: a retrospective cohort study.
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.
  
  
  BACKGROUND
  Retrospectively registered NCT02639715 .
authors.affiliation: University Hospitals Leuven, Leuven University Fertility Center, KU Leuven-University of Leuven, Herestraat 49, Leuven, B-3000, Belgium. elia.fernandezgallardo@kuleuven.be.
authors.forename: Elia
authors.identifier: http://orcid.org/0000-0002-7791-2644
authors.initials: E
authors.lastname: Fernandez Gallardo
authors.affiliation: University Hospitals Leuven, Leuven University Fertility Center, KU Leuven-University of Leuven, Herestraat 49, Leuven, B-3000, Belgium.
authors.forename: Carl
authors.initials: C
authors.lastname: Spiessens
authors.affiliation: University Hospitals Leuven, Leuven University Fertility Center, KU Leuven-University of Leuven, Herestraat 49, Leuven, B-3000, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: D'Hooghe
authors.affiliation: University Hospitals Leuven, Leuven University Fertility Center, KU Leuven-University of Leuven, Herestraat 49, Leuven, B-3000, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Debrock
mesh_terms: D000328:Adult; D015331:Cohort Studies; D015925:Cryopreservation / Q000379:methods* / Q000639:trends; D046149:Embryo Culture Techniques / Q000379:methods; D010064:Embryo Implantation / Q000502:physiology*; D005260:Female; D006358:Hot Temperature; D006801:Humans; D011247:Pregnancy; D012189:Retrospective Studies; D056826:Single Embryo Transfer / Q000379:methods* / Q000639:trends; D058989:Vitrification
keywords: Embryo morphology; Frozen embryo transfer; Implantation rate; Morphometrics; Vitrified/warmed embryo