Liquid Nitrogen Vapor Sealing of Straw Containers can be Unsafe and Detrimental to Embryo Survival

Background: Aseptic closed vitrification (VTF) systems have been proving their clinical effectiveness in recent years. Although the risk of pathogenic contamination between samples in liquid nitrogen storage has been a debatable issue among open VTF systems users, there is growing interest to hybridize systems. In short, some open system users aim to achieve the ultra-rapid cooling rates of direct LN2 exposure and then seal the device into a plastic straw container. Specialized commercial LN2 baths have been developed to assist in these hybrid-device systems. We strived to 1) determine whether LN2 vapor sealing of straws presents safety and reliability concerns that create potentially harmful laboratory practices; and 2) reveal a validation method which verifies the competency of seals as a quality control practice. Materials and Methods: Using a repeated VTF (rVTF) model on research consented, discard embryos, human blastocysts were randomly assigned to either Control (n=19) or ultra-rapid cooling treatment (UR-TRT; n=22). Standard micro Secure-VTF (μS-VTF) warming of flexipettes was first performed without extraction/elution, then dried with sterile gauze. Re-VTF was performed at 1 min post-warming, by either: 1) Control μS-VTF; or 2) UR-TRT where flexipettes were dipped into LN2 (5 sec), inserted in straws held in LN2 and sealed closed for storage. Subsequently, all straws were warmed using standard μS-VTF procedures and elution in sucrose solutions. Following isotonic equilibration and 24h in vitro culture, blastocyst survival and development, respectively, was assessed. Results: Following the fatal rupturing of the first 3 UR-TRT straws, warming procedures had to be modified for possible LN2 accumulation inside straws due to incomplete seals. By allowing for 15 sec of N2 out gassing, the remaining 19 straws warmed without incident, yet 6 did reveal evidence of LN2 seepage (41% incomplete seals). No difference in blastocyst survival at 0 h was evident between treatments; however development was reduced in the UR-TRT group at 24 h (86.7% vs. 62.5%). Conclusion: The hybridization of a UR-closed VTF system has proven to be a potentially unreliable, unsafe and less effective procedure in our rVTF model system. The inability to guarantee complete seals of super-cooled straws or the possible entry of N2 vapors inside a straw upon sealing creates significant risks which are unnecessary quality control variables absent in standard aseptic, closed VTF methods.