Quinn s advantage serum protein substitute

Human embryos were cultured as described previously 30, 31. Thawed embryos were placed in a polystyrene dish containing 0.5 M sucrose solution for 10 minutes, then 0.2 M sucrose solution for a subsequent 10 minutes. Next, embryos were washed with Quinn's Advantage Medium with HEPES (Cooper Surgical, Trumbull, CT, http://www.coopersurgical.com) with the addition of 5% Quinn's Advantage Serum Protein Substitute (Cooper Surgical). Embryos were cultured in either Quinn's Advantage Cleavage or Blastocyst Medium (depending on stage) plus 10% Serum Protein Substitute (Cooper Surgical) under mineral oil (Sigma, St Louis, MO, http://www.sigmaaldrich.com) at 37°C with 6% CO₂, 5% O₂, and 89% N₂ under standard human embryo culture conditions and in agreement with current clinical IVF practice. The zona pellucida was removed by treatment with Acidified Tyrode's Solution (Millipore, Billerica, MA, http://www.millipore.com), and single blastomeres were collected by incubating in Quinn's Advantage Ca2+ and Mg2+‐free medium with HEPES (Cooper Surgical) for 5–20 minutes at 37°C with pipetting to break up into single cells. Blastomeres were tubed and flash frozen at −80°C until qRT‐PCR analysis.

Human embryos frozen at the two pronuclear stage by slow-freezing were thawed by a two-step process using the Quinn's Advantage Thaw Kit (CooperSurgical, CT, USA) as recommended by the manufacturer. The embryos were washed in Quinn's Advantage Cleavage Medium (CooperSurgical) supplemented with 10% Quinn's Advantage Serum Protein Substitute (CooperSurgical) and transferred to 100 μl drops of shared medium under mineral oil (Sigma, MO, USA). Embryos that did not survive the thaw procedure were discarded and excluded from further analysis, since this could influence the integrity of RNA and DNA within the cells, thereby affecting the results. Embryos were cultured in custom polystyrene Petri dishes (Auxogyn, CA, USA) with 12 individual microwells in the centre. Small markers (letters and numbers) were located at the edges to help with embryo identification. The dishes were prepared at least 5 h in advance and placed in the incubator to pre-equilibrate. The embryos were cultured at 37 °C with 6% CO₂, 5% O₂ and 89% N₂, standard human embryo culture conditions in accordance with current clinical IVF practice.

Approval from the University of Iowa’s Institutional Review Board (#200804752) was obtained prior to the performance of all experiments. Embryos used in our study had been specifically donated for use in research (#200109085) by patients who had undergone in vitro fertilization treatment, using either conventional insemination or intracytoplasmic sperm injection, and had supernumerary zygotes cryopreserved at the pronuclear stage. These embryos were cryopreserved and thawed as described previously [12 (link)]. For all experiments, embryos (n = 136) were cultured using commercially available embryo culture media supplemented with 20 % Quinns Advantage Serum Protein Substitute (Cooper Surgical) in 5.5–6.0 % CO₂ in air at 37 °C. Embryos designated for study on the third day post fertilization (D3) were cultured for 48 h, whereas embryos designated for study on the fifth day post fertilization (D5) were cultured for 96 h. All D3 embryos included in the studies (n = 27) exhibited 6–10 blastomeres with ≤20 % fragmentation, while D5 blastocyst-stage embryos selected for study (n = 27) were blastocysts with at least an “A” or “B” rating for both the trophectoderm and inner cell mass scores, as graded using the classification system outlined by Gardner and Schoolcraft [13 ].