Automation in the IVF laboratory: Results with a new device able to do both Vitrification/Rewarming of mice and bovine Oocytes and Embryos P. Patrizio 1, Y. Natan 2, P. Levi Setti 3, M. Leong 4, A. Arav 2 1 Yale Fertility Center, New Haven, USA 2 FertileSafe Ltd., Nes Ziona, Israel 3 Humanitas Reproductive Medicine, Rozzano, Italy 4 Women s Clinic, Hong Kong
Disclosure Co-Founder and Medical advisor FertileSafe Study sponsor: www.fertilesafe.com
Oocytes Vitrification Vs. Slow Freezing Cil et al., Fertil Steril 2013; 100: 492 99.
Senior Vs. Junior Embryologist JARG May 2018
Main Problems With Vitrification Time consuming Osmotic shock = short time exposure to ES Chemical toxicity = long time exposure to VS Mechanical damage due to manipulation (pipetting) Requires highly skilled personnel for producing small volume and high cooling and warming rates Lack of standardization of vitrification protocols and devices Results not reproducible
Mechanisms of Cryodamage Lipid phase transition Membrane leakage Water phase transition Water phase transition >3% intracellular crystallization Extracellular crystallization Mechanical damage Osmotic damage Cell death
Different Carriers for open system vitrification
Vitrification: Carriers Electron microscope grid; Cryotop; Cryoloop; Hemi-straw; Cryoleaf; Vitrification spatula; Nylon mesh; Plastic blade
Volume and cooling rate of different carriers
Cooling Rate of Different Carriers as Measured by Sound Meter APP 15,000C/min Vitrifit 18,000C/min Cryolock 20,000C/min Evit 29,000C/min CryoTop
What is Required from an Automatic Vitrification Device 1. Cooling and warming rates should be 20,000 C/min. 2. Volume on cryo-carrier should be <0.1µl (MDS). 3. Easy to operate. 4. Automate all vitrification (include plunging into LN) and warming steps. 5. Avoid mechanical damage. 6. Short time to complete procedure. 7. Usable for open or closed systems. 8. Storage of samples in standard LN tanks.
A Look Inside Sarah Mini stra ws Maxi straws 1. Sarah can vitrify up to 30 oocytes/embryos at once (in 6 Mini stra ws with 5 oocytes in each). 2. Sarah can vitrify up to 18 ova ria n tissue slice s (in 6 Ma xi stra ws with 3 samples (10 X 5mm) in e a ch).
Loading Straw and The Capsules
Materials and Methods VITRIFICATION [ES= 7.5% DM SO+ 7.5%EG + 20%FCS in TCM 199] [VS= 18% DM SO+ 18%EG+ 0.5M trehalose+ 20%FCS] Mice Oocytes/Embryos: Exposure to 3 ES (30,60,100%), 3 min each, followed by VS (100%) 1 min Bovine Oocytes/Embryos: Exposure to 6 ES (10,20,40,60,80,100%),1.5 min each, followed by VS (75, 100%) REWARM ING [WS= 1 M sucrose in TCM 199+ 20% FCS] Straws from LN into WS 100% at 37 C (5 sec)-followed by WS (100,50,25,12.5%) at RT (2.5 min each station). Oocyte survival mice: live/dead CYBR-14 PI fluorescent stain. Bovine: fertilization and Embryo cleavage/hatching.
Materials and Methods Results: M ice Oocytes and Embryos
Results: Bovine Oocytes Bovine Results % Cleavage % Blastocysts Vitrified Zygotes 54% (19/35) 9% (3/35) Fresh Zygotes 65% (13/20) 20% (4/20) Vitrified Oocytes 73% (61/84) 7% (6/84) Fresh Oocytes 83% (125/150) 11% (17/150) Table 3: Bovine zygotes and MII oocytes development after Vitrification/Warming with the Sarah system compared to the fresh controls. (p, NS) Arav et al., JARG e-pub May 26, 2018
Sarah vs. Gavi vs. Manual How long does it take? Sarah GAVI Manual 5 min 12 min 37min Sarah process Place solutions in cups Label straws and load oocytes Sarah equilibrate 6 straws with up to 30 oocytes or 6 embryos Total: 17 min FOR UP TO 30 OOCYTES Total: 37 min FOR 4 OOCYTES Total: 57 min FOR 4 OOCYTES
ONGOING STUDIES
Lamb M II oocytes results 90% (16/18) MII oocyte s surviva l with 44% (8/18) cleavage. 78% (15/19) MII oocytes survived with 47% (9/19) cleaved and 2 reached Morula and 1 blastocyst. Control group: 80% (16/20) Cle a ve d with 2 reached Morula and 1 reached blastocyst stage. Arav et al., unpublished
Spinal Cord and Dorsal Root Ganglia Slices Results A B C Regeneration of nerve fibers and migration of neuronal and glial cells from SC and DRG slices of rat fetuses cultured for 3 days (A,B) and 15 days (C) after vitrification/rewarming. (A,B) Phase-contrast microscopy, (C) immune-fluorescent double staining of migrated cells. Glial cells are in red (anti S-100 antibodies) and neurons in green (anti-neurofilament antibodies). Arav et al., Stem Cells Research and Therapy 2015
Human Ovarian Slices: preliminary results After 1 vitrification (control) After 2 vitrification (Sarah) pathologist unaware of methods, did not find differences between the ovarian slices
Vitrification of Testicular Tissue (ram)
Vitrification of Testicular Tissue A B C D Fig. A shows a testicular tissue inside Sarah. Fig. B shows the seminal tubules isolated from the testicular tissue after vitrification. Fig. C shows Hoechst vital stain of the tubules. Fig. D shows Hoechst vital stain of the spermatogonia cells. Arav et al., Theriogenology in preparation 16
Successful Vitrification of IVP Ovine Embryos at the Blastocyst Stage with E.Vit (much simpler) E.Vit vitrification Cryotop Vitrification Following vitrification and after 48h of culture the hatching rates of blastocysts (n=168) vitrified in E-Vit straws was 87.2%, comparable to Cryotop method (77.5%) and Fresh control group (85.3%) Ledda et al., IETS 2018
Direct Transfer Vitrified/Warmed Cattle Embryo
Summary and Conclusions Sarah can vitrify oocytes, embryos & tissue slices. Operates as a safe open system or a semi-closed system. Cooling and warming rates of 20,000 C/min. Is efficient (up to 30 oocytes simultaneously in 17 min). Automatic insertion into LN. Reduces osmotic and mechanical damages. One device dual function (vitrification/rewarming). Automation of both vitrification/warming will drastically reduce differences in survival/success rates among centers.
Sassari Thank You Hong Kong Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia