Introduction

Embryos can be vitrified at any point of development from pronuclear (PN) to the blastocyst stage1. The vast majority of published papers report results for one particular stage of development. Differences in vitrification and warming methodologies between laboratories hamper the accurate comparison of outcomes for embryos vitrified at different stages.

Vitrification has been used for all embryo cryopreservation at this clinic since October 2007, using the same cryoprotectant and closed storage system (Irvine Scientific Vit Kit and CryoTips). When vitrification was first introduced, blastocysts were vitrified with no additional intervention. Since September 2009 all blastocysts have been collapsed just prior to vitrification with the use of an ICSI needle.

The purpose of this study was to compare the survival rates and implantation potential of vitrified-warmed embryos according to the day of vitrification, and to evaluate whether collapsing blastocysts prior to vitrification is beneficial.

Materials and Methods

This was a retrospective data analysis of all frozen embryo transfer (FET) cycles involving the use of vitrified-warmed embryos from January 2008 to October 2012 (n=519). For embryos vitrifed on Days 1-4, if there were more embryos stored than required for transfer several were warmed and cultured on until the best could be selected for transfer with re-vitrification of surplus good quality blasts. Data were analysed using Fisher's exact test.

Results

Results are summarised in Table 1 and presented graphically in Figures 1-3. There were no significant differences between individual groups for any of the parameters measured. When results were grouped together to provide larger numbers for analysis, collapsed blastocysts showed a greater implantation rate than embryos vitrified on Days 1-4 (p<0.05; data grouped for Days 1-4 and Days 5-6). Again combining Day 5 and Day 6 results, a significant improvement in embryo survival is demonstrated following artificial collapse of the blastocoel prior to vitrification of blastocysts compared with no collapse (p<0.01).

None of the embryos vitrified at PN stage and cultured on after warming were transferred as blastocysts, however several patients with embryos vitrified on Days 2-4 had blastocyst transfer following culture (data not shown).

Discussion

This study shows good survival rates for all stages of development, especially if blastocysts are artificially collapsed before vitrification. However good embryo survival does not necessarily translate to good pregnancy or implantation rates, for example despite 68 PN embryos surviving and being cultured to select the best for transfer, only 2 implanted (of 16 transferred). It is possible that the large cellular volume of PN embryos may benefit from the stepwise addition of equilibration solution used during oocyte vitrification.

Inducing collapse of the blastocoel before vitrification has resulted in similar outcomes for Day 5 and Day 6 vitrified embryos. Blastocysts with large blastocoel volumes are thought to be more susceptible to ice crystal formation during vitrification or warming2; since Day 6 blastocysts are often at a later stage of expansion the benefit of artificial shrinkage may be greater than for Day 5.

This clinic now vitrifies all surplus embryos at the blastocyst stage, with artificial collapse beforehand. If patients are planned for elective vitrification of all embryos we now vitrify some PNs using the protocol for oocyte vitrification and vitrify the remainder on Day 2.

Conclusions

 Good survival rates can be achieved when vitrifying embryos at all stages of development from PN to blastocyst, although this does not necessarily result in good implantation potential.

 Collapsing blastocysts prior to vitrification improves their survival and implantation potential.

 Day 6 blastocysts, if artificially collapsed beforehand, show similar implantation potential to blastocysts vitrified on Day 5.

1 Edgar DH &Gook DA. A critical appraisal of cryopreservation (slow cooling vs vitrification) of human oocytes and embryos. Hum Reprod Update 2012; 18, 536-554

2 Kader AA, Choi A, Orief Y & Agarwal A. Factors affecting the outcome of human blastocyst vitrification. Reprod Biol Endocrinol 2009; 7, 99-109