Rapid Thawing and Culture of Human Embryos

Human embryos were thawed by a two-step rapid thawing protocol using Quinn's Advantage Thaw Kit (CooperSurgical, Trumbull, CT, USA) as previously described (1 (link),5 (link)). In brief, either cryostraws or vials were removed from the liquid nitrogen and exposed to air before incubating in a 37°C water bath. Once thawed, embryos were transferred to a 20 µl microdrop of either Quinn's Advantage Cleavage Medium (CooperSurgical) supplemented with 10% SPS between Days 1–3 or Quinn's Advantage Blastocyst Medium (CooperSurgical) with 10% SPS after Day 3 under mineral oil (Sigma, St Louis, MO, USA). Approximately half of the embryos were also cultured in the presence of a growth factor cocktail containing 10 ng/ml BDNF (PeptroTech Inc., Rocky Hill, NJ, USA), 40 ng/ml IGF-I (Sigma-Aldrich, St Louis, MO, USA), 5 ng/ml EGF (R&D Systems, Inc., Minneapolis, MN, USA), 2 ng/ml GM-CSF (R&D Systems, Inc.), 0.5 ng/ml FGF2 (R&D Systems, Inc.) and 10 ng/ml of GDNF (R&D Systems, Inc.). All embryos were cultured at 37°C with 6% CO₂, 5% O₂ and 89% N₂ and embryo development was monitored daily by microscope for up to 7 days.