Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems.

Abstract

This study evaluated and compared survival, re-expansion, and percentage of live cells of individual Days 5 and 6 human blastocysts that were vitrified and warmed with the Vit Kit Freeze/Thaw (Irvine Scientific, CA), or with two protocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada). Frozen/thawed Day 2-3 or discarded embryos were cultured to blastocyst (culture day 5-6). Group 1 blastocysts were vitrified with the Vit Kit (n = 29) and High Security Vitrification (HSV) devices. Group 2 (n = 47) and Group 3 (n = 48) blastocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws, using a direct plunge or a -100 °C holding step, respectively. Group 4 (Controls, n = 30) were not vitrified. Blastocysts were subsequently cultured for 24 h, assessed for survival and expansion, and then stained individually with propidium iodide and Hoechst. Live and total cell number was assessed with ImageJ (NIH), and the percentage of live cells calculated for each blastocyst. The percentage of live cells was not different between vitrified and control (non-vitrified) blastocysts, thus vitrification did not affect cell survival. Survival (following thawing and after 24 h culture), re-expansion, and percentage of live cells were not different for blastocysts vitrified and warmed between the two vitrification/warming kits, or between the two protocols for the Global Fast Freeze/Thaw Kits. Blastocyst vitrification can be achieved with equal success using simplified protocols and cheaper and easy to load freezing straws, providing simultaneously increased safety, and efficiency with lower cost, when compared with vitrification using specialized embryo vitrification devices.