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Objective The global shipment of embryos has become commonplace, yet universal warming protocols for various vitrification systems (devices and solutions) are not widely used. The aim of our study is to validate effective protocols that can ease concerns regarding the warming of vitrified blastocysts sent to outside laboratories when alternatives to their typical commercial products are needed. Design Prospective, randomized study of patient consented research discard blastocysts (>2BB quality). An apriori arrangement of 4 sugar solution treatments was applied post-warming: A) fresh I.C.E. solutions; B) frozen-thawed I.C.E. solutions (i.e., stored in LN2); C) sucrose; and D) honey (commercial grade, multi-floral). Materials and methods Research consented blastocysts vitrified by the microSecure method in I.C.E. non-DMSO solutions (>7.9m glycerol/EG) were rapidly warmed in 1 of 4 solution treatment groups. The initial pilot study included 10 blastocysts per group accounting evenly for quality and batch effects before randomly assigning the treatment. Solutions A and B were a commercial I.C.E. product (T1-T4), whereas C and D were lab-made, filtered stock H-HTF+additive solutions (1M sucrose w/v and 10% honey v/v, respectively). The latter solutions both required heating and agitation over a 30 min period to completely mix into suspension. All treatments involved a 4-step dilution (3 min/step, 50% reduction/step, 21°C) prior to isotonic equilibration (LG-H+additives; 5 min at 37°C), followed by 18-24 hr in vitro group culture/microdroplet (5 embryos/drop). Survival, based on osmotic responsiveness and cellular integrity/fullness was visually assessed at 0 and +2 hr, followed by overnight blastocyst expansion and continued development. Results There was no difference in blastocyst survival (100%) or in-vitro development (90-95%) between treatments. Additional validation replicates are planned, but we do not anticipate any treatment effect. Discussion This study proves that non-permeating sugar-based dilution solutions can be effectively used to elute concentrated cryoprotective agents from blastomeres independent of source. We, and others, have previously advocated the use of 1M sucrose as a universal diluent. Furthermore, we have now proven that a natural product, honey, composed predominantly of fructose and glucose, is an efficient low-cost, readily available option for any lab around the world to safely extract potentially toxic cryoprotectants from cells. Finally, we have validated that thaw solutions stored in cryovials can be frozen in LN2 and maintain functionality, thus facilitating the potential shipment of vitrified material with procedurally compliant thaw solutions transported in the same LN2 dry-shipper. Disclosures JJS is the owner of I.C.E., LLC and MCS is one of his scientific advisors. Funding None |
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