All these steps were carried out in 20 μL microdrops at 39 °C under mineral oil. Afterwards the embryos were cultured individually in CR2aa medium under 5% CO2 and 39 °C for 120 min (T120). Pictures of embryos from each culture media were captured at 0, 5, 10 and 120 min (T0, T5, T10 and T120, respectively), with a CCD camera connected to an inverted microscope and saved in a computer using the Pinnacle Studio software, v. 7.11 (Pinnacle, Mountain View, CA, USA). The images were analyzed by the ImageJ software v. 1.40 (National Institute of Health, USA). For embryo area measurement,

the zona pellucida MAPK Inhibitor Library and periviteline space were excluded. For area measurement, images were previously calibrated using a graduated glass slide. Measures of T0 area (T0 = 1) were used as a reference for further T5, T10 and T120 relative area determination. Dehydration was considered the T5 data and indicates the reduction in area immediately after embryo exposure to hypertonic medium (T5). T10 and T120 show the area recovery after 10 (T10) and 120 (T120) min in isotonic medium. Vitrification was performed by OPS method as first described by Vajta et al. [35]. Expanded blastocysts at 168 hpi, morphologically classified as good or excellent, were vitrified

using DMSO and EG as CPAs. The embryos were equilibrated into 10% DMSO plus 10% EG in PBS medium supplemented with 5% FCS (HM2) for 1 min followed by 30 s into 20% DMSO plus 20% EG, loaded into OPS and Sitaxentan plunged into liquid nitrogen. Warming was performed by immersing OPS

into HM2 with 0.25 M sucrose at 39 °C for 1 min, this website followed by two-step rehydration in 0.25 M and 0.15 M of sucrose for 5 min each one. All steps were at 39 °C. Afterwards, the embryos were washed in HM2. Vitrified-warmed embryos were cultured in CR2aa medium with granulosa cells monolayer for 72 h. Control group embryos were cultured simultaneously. Survival rate was assessed by blastocyst re-expansion and hatching at 72 h. Samples obtained from experiments 2 and 3 were used for RNA extraction and PCR analysis. Total RNA was extracted from pools of five embryos using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and treated with DNase. Messengers RNA were amplified (one round) using the MessageAmp™II aRNA Amplification Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions, in order to get enough material for transcript analysis. This procedure generated a final volume of 20 μL with concentration of ∼70 ng/μL of anti-sense amplified RNA (aRNA). The aRNA samples were reverse transcribed (RT) using the SuperScript III First-Strand Synthesis Supermix (Invitrogen, Carlsbad, CA, USA) and a random hexamer primer, according to the manufacturer’s instructions.