Quinn s advantage blastocyst medium

All assigned ECM was collected from regular ICSI-cycles in which routine ICSI procedures were followed and during which all embryos were handled according to local regulations and standard operating procedures. Embryos were cultured in Sage medium (Quinn’s advantage cleavage medium, Cooper Surgical, USA) supplemented with 5% human serum albumin (HSA; Vitrolife, Göteborg, Sweden) from Day 1 to 3 after fertilization. Thereafter, embryos were transferred to fresh Sage medium (Quinn’s advantage blastocyst medium, Cooper Surgical, USA) supplemented with 5% HSA, on Day 3 after fertilization. Embryo morphology was assessed daily by an independent laboratory technician. Droplets (20 µl) from individually cultured embryos, now referred to as ECM, were collected in sterile 1.5 ml collection tubes after all embryos had been removed from the culture dishes for cryopreservation on Day 4. All ECM samples were collected on Day 4. Empty medium droplets, in which no embryo was grown but that otherwise underwent the same procedures, from the same dishes were used as controls. Samples were snap-frozen in liquid nitrogen and stored at −80°C.

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.