Authors: Schiewe MC, S Zozula , R VanTol, A Jones, RE Anderson

Study question: Is the efficacy of vitrified human blastocyst post-warming dilution using a universal sucrose dilution approach comparable to standard manufacturer procedures?

Summary answer: Human blastocysts vitrification is equally effective using a simple sucrose step down dilution approach independent of the type of cryoprotective agent used.

What is known already: Blastocyst vitrification is a global standard in the IVF industry. Numerous vitrification systems are being marketed and successfully used. Each commercial company has its own recommended thawing solution. As more patients move their cryopreserved embryos between laboratories, it has become increasingly difficult and cost inefficient to maintain a stock of different thaw solutions. Although proprietary in formulation, it is safe to assume that all manufactured thaw solutions have one thing in common, declining sucrose concentrations. Sucrose is an effective non-permeating solute that safely removes intracellular cryoprotective agents (EG, DMSO, Glycerol) and allows for isotonic equilibration by gradual rehydrating dilution steps.

Study design, size, duration: Research consented, discard slow frozen (SF) embryos (N=363; n=328 cleavage stage, n=35 blastocysts) were thawed, and fair-good quality blastocysts were randomly assigned within batch groups to 1 of 2 vitrification treatments: 1)control Glycerol/EG solution,\, I.C.E. >7.9M;or 2)15% DMSO/15%EG solution. All vitrified blastocysts were warmed rapidly according to standard operating procedures (SOP) then randomly assigned to either stepwise SOP dilution or universal sucrose step down dilution (n=31 embryos/group; 2 x 2 factorial arrangement of treatments).

Participants/materials, setting, methods: All vitrified blastocysts were rapidly cooled (>1500C/min) and warmed (>6000C/min) in an aseptic closed system. All warmed embryos underwent stepwise dilutions by manufacturer SOP or a universal 4-step sucrose dilution (21 ºC) of 1.0M for 5 min and 0.5M, 0.25M and 0.125 M for 3 min each before isotonic equilibration for 5 min at 37ºC. Embryo survival and development was determined and differences in %survival and continued development were statistically compared by Chi-square analysis (p<0.05).

Main results and the role of chance: Sucrose diluted, SF embryos yielded 124 (34.2%) fair to good quality blastocysts for study revitrification, whether derived from cleaved embryos (n=113, 34.5%) or blastocysts (n=14; 40%). Day 5 SF blastocysts had superior sustained development (67%) in contrast to Day 6 (20%). There was no differences in survival or sustained development of vitrified blastocysts between the combined dilution treatments, whether SOP thawing (93.5%) or standard sucrose dilutions (90.3%) was applied. Interestingly, cryoprotective agent treatments were significant, with 98.4% (n=61) of the Glycerol/EG treated blastocysts being viable post-dilution and culture compared to 86.9% for DMSO/EG exposure (n=53). Only a single I.C.E.-sucrose treated embryo degenerated, whereas degeneration occurred in 4 DMSO-SOP and 5 DMSO-sucrose exposed embryos. Although some DMSO/EG treated blastocyst appeared to be more sensitive to osmotic injury, the overall viability of the intact blastocysts was excellent.

Limitations, reasons for caution: Possible differences in blastocyst quality may have contributed to VTF survivability of blastocyst exposed to the EG/DMSO vitirification solutions, as DMSO exposure to the lipid bilayer of vulnerable tropectoderm cells may be more sensitive to osmotic stress. Alternatively, 30%EG/DMSO solutions may be unstable under closed vitrification/warming conditions, being less reliable.

Wider implications of the findings: This study has demonstrated that a cost-effective “universal” approach involving stepwise sucrose dilutions is an acceptable thawing method for vitrified blastocysts independent of the vitrification solution used. This standardized procedure allows for continuity of patient care within and between laboratories vitrifying embryos by different methods.