Artifi cial Collapse of Blastocysts Before Vitrifi cation: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts

The purpose of this study was to systematically examine blastocoelic fl uid reduction prior to vitrifi cation and its potential benefi ts. In addition, we compared artifi cial collapse (AC) by laser pulse to a mechanical method. Mouse and dicarded human blastocysts were used in this study. Blastocysts were collapsed using either a 10 ms pulse with a laser (LAC) or else mechanical puncture with a microneedle (MAC). Blastocysts were vitrifi ed on cryoloops using a two-step ethylene glycol/dimethyl sulfoxide protocol. We examined the effects of AC on specifi c outcome parameters such as overall survival, reexpansion, cell proliferation, and DNA damage. Unlike others, we report overall high survival rates with expanded blastocysts even without fl uid reduction. We did detect a signifi cant increase in blastomeres showing signs of DNA damage in the control group (13%) in comparison to blastocysts AC prior to vitrifi cation (LAC 3%, MAC 5%; p < 0.001). Control blastocysts exhibited a lower rate of reexpansion. Within 3 h of warming, 73% and 81%, respectively of mechanically or laser collapsed blastocysts were fully reexpanded as compared to only 53% of control blastocysts (p < 0.001). Overall blastomere count was also signifi cantly lower in control blastocysts (CT 103+32, MAC 121 + 37, LAC 134 + 35; p < 0.0001). Early blastocysts with smaller blastocoelic volumes did not benefi t from any further reduction of fl uid volume. AC can reduce DNA damage and enhance postwarming reexpansion and cell proliferation in expanding blastocysts. The laser method also appeared to be effective and may offer some advantages over mechanical collapse.