The effect of mechanically collapsing human blastocysts prior to cryopreservation

Abstract

Cryoprotectants and sucrose are used during embryo freezing to decrease the amount of water in cells and prevent ice crystal formation and subsequent damage. However, the blastocyst stage embryo contains a large fluid-filled central cavity called the blastocoele. If the fluid in the blastocoele is not lost during cryopreservation, damage may occur to the embryos. The objective of this study was to determine if artificially collapsing blastocysts prior to the addition of the cryoprotectant may decrease the amount of damage and result in a higher survival and developmental rate following cryopreservation. Embryos used in this study were donated by patients who had consented to the use of their surplus embryos for research. On Days 5 or 6, blastocysts demonstrating a blastocoele cavity comprising at least 50% of its diameter, were randomized to be mechanically-collapsed (MC) or served as controls. After cryopreservation and thaw, embryos were evaluated for survival and reexpansion of the blastocoele cavity at 6 hours and hatching after 24 hours. Blastocysts in the MC group (n=12) were held in place with a holding pipette (Humagen, Charlottesville, VA) and an ICSI pipette inserted at 3:00, opposite the inner cell mass. The blastocyst collapsed within seconds and was immediately cryopreserved. Control blastocysts (n=12) were not manipulated prior to freezing. Cryopreservation was performed using a two-step addition of glycerol (5 and 10%) in modified HTF-Hepes (Irvine Scientific, Irvine, CA) containing 2.0M sucrose. Cooling began at room temperature and proceeded at a rate of 2oC/min to -7oC where seeding occurred. Cooling continued at a rate of 0.3oC/min to -38oC, at which time the vials were plunged in liquid nitrogen. Thawing was performed by removing the vials from liquid nitrogen and letting them incubate in a 30oC water bath until all ice crystals had dissolved. Blastocysts were put through a 7-step dilution series to remove the glycerol and sucrose (10%Gly + 0.4M sucrose; 5% + 0.4M; 2.5% + 0.4M; 0.4M sucrose; 0.2M sucrose; 0.1M sucrose), rinsed in fresh medium and then cultured for an additional 24 hours. Upon thaw, 8/12 (67%) of the control embryos survived the procedure with 6/8 (75%) re-expanding after 6 hours in culture. None of the control embryos underwent hatching in vitro. Of the manipulated embryos, 9/12 (75%) survived the procedure with 8/9 (89%) re-expanding and 2/9 (22%) hatching. The rate of survival and reexpansion were not significantly different between the two groups. However, the performance of a likelihood ratio showed that the rate of hatching in the MC group was increased (p<0.05). These preliminary results suggest that artificially reducing the blastocoele cavity may improve the post-thaw development of blastocysts, possibly by reducing cryo-damage. We find these results promising and will continue the evaluations with additional embryos as they become available for research.