Mitchel C Schiewe, PhD, HCLD Effect of Vitrification (VTF) Device System and Solution on Post-warming Viability of Re-Vitrified Human Blastocysts: A Randomized Crossover Study ASRM PosterPlus #172 Narrated by Mitchel C Schiewe, PhD, HCLD Newport Beach, CA Nashville, TN

Disclosures MC Schiewe, PhD developed µS-VTF without any commercial interests, and serves on the Scientific Advisory Board of Innovative CryoEnterprises.

Background - AAB 2016 Scientists at Ovation Fertility have previously shown that human blastocysts can be re-vitrified up to 5 times without a decline in survivability and 24 h development using a mS-VTF. Our metastable glycerol based VTF solution has compared favorably to commercially available solutions whose cryoprotective agent concentrations have been reduced to avoid cryotoxicity but to do so makes them unstable under slow warming conditions. We showed no difference in sustained blastocyst development between treatments after up to 5 min continuous exposure. Not surprisingly, differences began being apparent after 10min exposure, with the more concentrated solution being more toxic at >15 min exposure. 1 min 3 min 5 min 10 min 15 min 20 min VTF solution toxicity not overtly evident over 10 min * Significant reduction in EG/GLYC development at 15 &20 min exposures (Schiewe et al., 2016)

THERMODYNAMICS OF VITRIFICATION __ _ 0°C _ -20 _ -40 _ -60 _ -80 _ -100 _ -120 _ -140 It is important to understand the VTF system you are using: Unstable solutions form ice nuclei that will experience increasing ice growth if cooled or warmed too slowly (opaqueness can appear) Metastable solutions may form an abundance of small heterogeneous ice nuclei, but fail to experience additional ice growth, thus transparency is maintained under varying cooling / warming conditions. Equilibrium Metastable Unstable Tm Th Td Temperature (°C) Td Tg Brian Wowk developed a slow warming rate model to help us understand the relationship between CPA concentration and the stability of a VTF solution. Heterogeneous ice nucleation of solution impurities can be visualized and serve as a template for progressive ice growth (i.e., recrystallization) relative to the total concentration of CP A and warming rate. Metastable solutions are not dependent on rapid cooling and/or warming to minimize recrystalization. Whereas, the unstable solutions may form fewer but larger ice nuclei susceptible to further growth unless rapidly warmed. Both the type of VTF device and volume of solution used influences cooling and warming rates, and therefore, is of particular concern with “unstable non-equilibrium” VTF . Thus, this crossover study was conducted as assess the relationship between two established VTF device systems using a metastable or unstable solution. 0 10 20 30 40 50 60 70 80 90 100 Concentration (w/w %) Thermodynamic lines for melting points (Tm), heterogeneous nucleation (Th), devitrification (Td) and glass transition (Tg) are approximated trends. (Diagram adapted from Dr. Brian Wowk’s Talk; 50th Society for Cryobiology, 2013)

References: Schiewe, MC, S Zozula, RE Anderson, GM Fahy. Validation of microSecure vitrification (μS-VTF) for the effective cryopreservation of human embryos and oocytes. Cryobiol 2015; 71:264-72.  Schiewe, MC, S Zozula, N Nugent, K Waggoner, J Borba, L Gamboa and JB Whitney. Modified microSecure vitrification: A safe, simple and highly effective cryopreservation procedure for human blastocysts. J Vis Exp (2017); 121: e54871, doi:10.3791/54871. Schiewe, MC and RE Anderson. Vitrification: the pionneering past to current trends and perspectives of cryopreserving human embryos, gametes and reproductive tissues. J Biorepository Sci Appl Med (2017); 5:1-12. Schiewe, MC. Quality control factors influencing the successful and reliable implementation of oocyte and embryo vitrification. In: Marco-Jiménez, F and Akdemir, H (eds.) Cryopreservation (1st Ed.). InTech, Rijeka, Croatia, 2016. doi 10.5772/65332