6-Step Sequential Dilution of Cryoprotectant Solution After Thawing Improves the Survival Rate of Vitrified Human Blastocysts

Abstract

Objective: Vitrification of blastocysts was not required an expensive equipments and time-wastage. The aim of this study was to investigate whether 6-step sequential dilution of cryoprotectant solution after thawing has influence on the survival rate of vitrified human blastocysts as compared to conventional method.Design: The surplus blastocysts produced by our culture method were vitrified using electron microscopic (EM) grid. Because the survival rate of vitrified-blastocysts diluted by conventional method after thawing was significantly lower than that of slow-frozen blastocysts, the dilution method of cryoprotectant solution was changed to 6-step sequential dilution method.Material methods: The blastocysts vitrified in this study were obtained by co-culturing zygotes with cumulus cells in a 10 μl YS medium containing 20% hFF for 4- to 5-days. The blastocyst embryos were also divided to ErB (early blastocyst), EEB (early expanding blastocyst), MEB (middle expanding blastocyst) and EdB (expanded blastocyst) according to their developmental morphology. Equilibration solution was supplemented with 20% ethylene glycol (EG) in m-DPBS containing 20% hFF, and vitrification solution was supplemented with 40% EG, 18% Ficoll and 0.3M sucrose in m-DPBS containing 20% hFF. The cryopreserved blastocysts were thawed, diluted and cultured on day 3 or 4 postovulation. The conventional dilution was performed by transferring thawed blastocysts from 0.3 M sucrose solution (1.5 min.) to culture medium, while 6-step sequential dilution was carried out by passing thawed blastocysts through 0.5M (3.0 min.), 0.4M (1.5 min.), 0.3M (1.5 min.), 0.2M (1.5 min), 0.1M (1.5 min.) to the culture medium. The survival rates of frozen-thawed blastocysts were assessed by observation of reexpanding blastocysts at 18h after co-culture with cumulus cells in a 10μl YS medium containing 20% hFF.Results: The survival rate of vitrified-blastocysts diluted by 6-step sequential dilution method after thawing (82.6%: 100/121) was significantly improved as compared to that (50.6%: 87/172) generated by conventional method. However, there was no difference between the survival rates of the blastocysts vitrified in accordance with the developmental stage.Conclusion: The present result indicates that to 6-step sequential dilution method after thawing could significantly improve the survival rate of vitrified-blastocysts as compared with conventional dilution method, probably by minimizing the osmotic damage during rehydration. Objective: Vitrification of blastocysts was not required an expensive equipments and time-wastage. The aim of this study was to investigate whether 6-step sequential dilution of cryoprotectant solution after thawing has influence on the survival rate of vitrified human blastocysts as compared to conventional method. Design: The surplus blastocysts produced by our culture method were vitrified using electron microscopic (EM) grid. Because the survival rate of vitrified-blastocysts diluted by conventional method after thawing was significantly lower than that of slow-frozen blastocysts, the dilution method of cryoprotectant solution was changed to 6-step sequential dilution method. Material methods: The blastocysts vitrified in this study were obtained by co-culturing zygotes with cumulus cells in a 10 μl YS medium containing 20% hFF for 4- to 5-days. The blastocyst embryos were also divided to ErB (early blastocyst), EEB (early expanding blastocyst), MEB (middle expanding blastocyst) and EdB (expanded blastocyst) according to their developmental morphology. Equilibration solution was supplemented with 20% ethylene glycol (EG) in m-DPBS containing 20% hFF, and vitrification solution was supplemented with 40% EG, 18% Ficoll and 0.3M sucrose in m-DPBS containing 20% hFF. The cryopreserved blastocysts were thawed, diluted and cultured on day 3 or 4 postovulation. The conventional dilution was performed by transferring thawed blastocysts from 0.3 M sucrose solution (1.5 min.) to culture medium, while 6-step sequential dilution was carried out by passing thawed blastocysts through 0.5M (3.0 min.), 0.4M (1.5 min.), 0.3M (1.5 min.), 0.2M (1.5 min), 0.1M (1.5 min.) to the culture medium. The survival rates of frozen-thawed blastocysts were assessed by observation of reexpanding blastocysts at 18h after co-culture with cumulus cells in a 10μl YS medium containing 20% hFF. Results: The survival rate of vitrified-blastocysts diluted by 6-step sequential dilution method after thawing (82.6%: 100/121) was significantly improved as compared to that (50.6%: 87/172) generated by conventional method. However, there was no difference between the survival rates of the blastocysts vitrified in accordance with the developmental stage. Conclusion: The present result indicates that to 6-step sequential dilution method after thawing could significantly improve the survival rate of vitrified-blastocysts as compared with conventional dilution method, probably by minimizing the osmotic damage during rehydration.