1. Cryopreservation Slow Freezing Patrick Quinn PhD, HCLD patrick.quinn@coopersurgical.com Sage In-vitro Fertilization, Inc Redmond, Oregon, USA

2. DISCLOSURE I am Vice President of research and Development at Sage In-Vitro Fertilization, Inc. We produce a range of commercial ART media products

3. Sage IVF Production @ Pasadena Redmond Cooper Surgical, Trumbull

4. Sage IVF Southern California Central Oregon

5. Slow-freezing method Vitrification Attention to the size Ice Crystal Formation Equilibration Seeding Slow freezing

6. Slow cooling requires a minimum of ~140 minutes. Greatest problems: extra & intracellular ice causing fracture osmotic swelling & shrinking solution effect chemical toxicity of CPAs expensive equipment

7. Cryoprotectants The mechanism of the protective action of cryoprotectants is the same, but their toxicities are different; Permeability is different with different cryoprotectants and temperatures; Therefore, the toxicity of cryoprotectants must be considered for freezing;

8. There are osmotic change before and after freezing in cryopreservation solution; These osmotic changes may cause the death of cells, normally it is referred to ‘osmotic injury’; Cryoprotectants

9. Thawing Solutions Hypertonic solution is required, i.e. non- permeable sucrose is added to prevent swelling and shrinkage of the cells;

10. Chilling injury; Cryoprotectant (toxicity and temperature); Osmotic injury; Speed of freezing and thawing; Factors affecting successful frozen-thawing

11. Step by Step Use of Sage Embryo Cryopreservation Media

12. Cryopreservation of 2PN/Cleaving Embryos/Blastocysts QA Embryo Freeze Kit (ART-8014) PPD, PPD+0.1M sucrose, Diluent – all with 12 mg/mL HSA. QA Blastocyst Freeze Kit (ART-8015) 5% Glycerol, 9% Glycerol + 0.2 M Sucrose, Diluent QA Thaw Kit (ART-8016) 0.5 M Sucrose, 0.2 M Sucrose, Diluent

13. Freezing of Zygotes, D2 & D3 Embryos Use QA Embryo Freeze Kit ART-8014. Prepare 0.5 M & 1.0 M propanediol (PPD) solutions. Move embryos through solutions in following sequence. 0.9 mL/well + oil, 37 o C. PPD ConcentrationmL 1.5 M PPDmL Diluent 0.5 M0.3 mL0.6 mL 1.0 M0.6 mL0.3 mL 1 2 3 4 1.0.5 M PPD, 5 minutes 2.1.0 M PPD, 5 min 3.1.5 M PPD, 10 min 4.1.5 M PPD + 0.1 M Sucrose 5minutes, load into straw and place in freezing machine at room temperature. Can use steps 3 & 4. Recommend 1, 2, 3 & 4.

14. Freezing of Blastocysts; Fully Expanded Blastocysts (FEB) of good quality Use QA Blastocyst Freeze Kit ART-8015. Move FEBs through solutions in following sequence. 1.0 mL/well + oil, 37 o C. 1 2 3 Diluent 5 minutes 5% Glycerol, 10 minutes 9% Glycerol/0.2 M Sucrose, 10 minutes. Load into straw and place in freezing machine at room temperature.

15. Cooling Ramps for Cryopreservation 1.Place cryocontainer (straw) in freezing mchine (Planar) at room temperature. 2.Cool from 22 o C to - 7 o C at 2 o C/minute. 3.Hold for 5 minutes at - 7 o C. 4.Seed. Check that seeding is progressing after 3-4 minutes. 5.Hold for 10 minutes at - 7 o C after seeding. 6.Cool at 0.3 o C/minute to -35 o C. 7.Transfer to liquid nitrogen.

16. Thawing of Embryos Applies to zygotes, cleaving embryos and blastocysts. Use QA Thaw Kit ART-8016. Thawing: Straw: Hold in air for 30 seconds, then agitate in a water bath at 35-37 o C until ice has melted. Vial: Agitate in a water bath at 35-37 o C until ice has melted. Use 35 mm dishes with 2.5 – 3.0 mL of solution/dish, covered with oil. Empty cryocontainer contents into a dry dish 0.5 M Sucrose 37 o C 10 minutes 0.2 M Sucrose 37 o C 10 minutes 60 mm dish. 100 uL drops of Diluent, covered with oil. 37 o C 1 minute in each drop, pipetting back and forth. ET or culture

17. COOLING PROTOCOL Sage DFU 1.Embryo to Freezing Medium, 20 min, RT o C 2. Load into straw or vial. Cool to -7 o C at 2 o C/min. 3. Hold at seeding temp for 5 min, seed, hold for another 10 min then cool at about 0.3ºC/min to around –35ºC and then transferred to LN2.

18. THAWING PROTOCOL I Sage DFU 1. Hold straw in air, 30-40 secs 2. Water bath, 30-35 o C, until ice melted 3. Transfer container contents to a dry dish and find embryo(s) 4. 0.5 M sucrose, 10 min, RT o C 5. 0.2 M sucrose, 10 min, RT o C All at room temperature

19. THAWING PROTOCOL II Sage DFU 6. W1 = Wash Medium, 37 o C, 5 min 7. W2 = Wash Medium, 37 o C, 5 min 8. 3 x 30 uL equilibrated Culture Medium All at 37 o C

20. Results from Conventional Human Blastocyst Freezing Menezo et al, 2001 # Thawing cycles 943 # Transfer cycles 857 (91%) # Thawed blastocysts 1,993 # Transferred blastocysts 1,596 (80%) # Clin preg/ET 204 (24%) # Ongoing preg/ET 171 (20%) Clinical IR 14.6% Ongoing IR 12.5% Live births/frozen blast. 10%

21. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 Routine Freezing of Blastocysts Original blastocyst cryopreservation protocol: modified by Menezo & Veiga (1997) and it became extremely convenient. Differing clinics have had inconsistent results with this protocol. Much of this has probably been due to inexperience on the part of many embryologists, both with selecting blastocysts of sufficient quality to freeze, and also understanding the subtleties of the cryopreservation technique. The most common practice to attempt improved consistency has been to reintroduce one or two glycerol concentration steps in the thaw, with one or two extra sucrose dilutions.

22. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 Selection Criteria for Human Blastocysts for Cryopreservation: Expanding blastocyst growth rate: day-5 > day-6 > day-7 Overall cell number > 60 cells (depending on day of development) Relative cell allocation to trophectoderm / inner cell mass Original quality of early stage embryo: pronuclear formation, blastomere regularity and mono-nucleation, fragmentation

23. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 Shady Grove Fertility, Maryland: Cryopreservation Outcomes for 2000 (to June) Zygote Cleavage Stage Blastocyst Total Thaw 10 10 58 78 Transfer 9 8* 54 71 Viable Pregnancy 2 5 18 25 %/Thaw 20% 50% 31% 32% %/Transfer 22% 62.5% 33% 35% * thaw at the cleavage stage with culture and transfer at the blastocyst stage.

24. COMPARISON OF BLASTOCYST CRYOPRESERVATION SLOW COOLING VERSUS VITRIFICATION (Cryotop)

25. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 Figure 1. Hatching Blastocyst Post-Thaw.

26. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 Routine Freezing of Blastocysts Original blastocyst cryopreservation protocol: modified by Menezo & Veiga (1997) and it became extremely convenient. Differing clinics have had inconsistent results with this protocol. Much of this has probably been due to inexperience on the part of many embryologists, both with selecting blastocysts of sufficient quality to freeze, and also understanding the subtleties of the cryopreservation technique. The most common practice to attempt improved consistency has been to reintroduce one or two glycerol concentration steps in the thaw, with one or two extra sucrose dilutions.

27. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 BLASTOCYST CRYOPRESERVATION FREEZE 1.Holding Medium: modified HTF + 20% HSA. 2.Freeze expanding, fully-expanded, and/or hatched or hatching blastocysts on Day-5/6 (unless fertilization delayed). 3.Embryos into modified HTF + 20%HSA @ 37 o C, then move onto cool bench (22 o C), and wash through several droplets for about 1 to 2 min. 4.Move into 5% glycerol for 8 min. 5.10% glycerol + 0.2M sucrose for 8 min (including loading time). Load straws / cryo-vials. (1.2ml Nunc cryo-vials containing 0.3ml medium after rinsing). 6.Cool @ –2 o C/min to –7.0 o C; hold for 15min; “seed” after 5min; - 0.3 o C/min to –38 o C, then plunge into liquid nitrogen for storage.

28. Tucker (2001) ART Media, AAB CRB 5 th Annual Symposium, pp 95-116 BLASTOCYST CRYOPRESERVATION THAW 1. Room temperature for 1min. Water-bath @ 30 o C till ice gone. 2. Locate blastocyst in 10% glycerol + 0.4M sucrose for 30 to 40sec. 3. 5% glycerol + 0.4M Sucrose for 3min. 4. 2.5% glycerol + 0.4M Sucrose for 3min. 5. 0.4M Sucrose alone for 3min; move dish to warm microscope/bench. 6. 0.2M Sucrose for 3min. 7. 0.1M Sucrose for 3min 8. Modified HTF + 20% HSA @ 37 o C for three washes, then into culture of HTF + 15% HSA. 9.Undertake assisted hatching while blastocyst still collapsed post-thaw. 10.Culture for a minimum of 4hrs to observe re-expansion.

29. Thawing of Blastocysts More gentle removal of CPAs. For example, Dean Morbeck: 1. Reagents: 1. Quinn’s Advantage Blastocyst Freeze Solution (9% Glycerol, 0.2M Sucrose; ART-8011; part of Blastocyst Freeze Kit ART-8015) 2. Quinn’s Advantage Embryo Thaw Solution (0.5M Sucrose: ART-8005) 3. Quinn’s Advantage Embryo Thaw Solution (0.2M Sucrose: ART-8007) 4. Quinn’s Advantage Embryo Thaw Solution (Diluent) Products 2,3 & 4 are in the Embryo thaw kit ART-8016. Solutions #Diluent0.2 M Sucrose0.5 M Sucrose9% Glycerol, 0.2M Sucrose 1001 mL 20 0 30 00 4 00 5 000

30. Thawing of Blastocysts Dean Morbeck 2. Make a separate dish for each solution. Make 2 x 200 ul drops each of solutions 1-4 and cover with 8- 10 ml oil. For solution 5, make 8-12 x 25-50 ul drops and cover with oil. Using the 300 um stripper, transfer blastocysts to a 200 ul drop of solution #1 under oil (4.5% glycerol, 0.35M sucrose) at room temperature. Leave blastocysts in solution #1 for 4 minutes and transfer to subsequent solutions according to the following protocol: Use a new, rinsed 300 um Stripper tip to rinse blastocysts through 6 drops of equilibrated blastocyst media under oil. Place blastocysts in the drops designated for culture. View blastocysts and return dish to incubator. Solutions #Time (minutes)SucroseGlycerol 140.35 M4.5% 230.35 M0 320.2 M0 410.1 M0 5Rinse through drops 00

31. Sage DFU: possible variations HEPES-HTF w/12 mg/mL HSA SAME Diluent 5 min 37 o C 5% Gly 10 min 37 o C 10% Gly/0.2M Suc 10 min 37 o C 30-35 o C to -6 o C @ 2 o C/min SAME 2 min seed + 8-13 min total=10-15 min -7 o C to -35 o C @ 0.3 o C/min -35 o C to -180 o C @ 50 o C/min or direct to LN from -35 o C straw to RT 20-30 secs SAME; then variation of Tucker dilution protocol water @ 30-35 o C 30 secs 0.5M Suc 5 min 37 o C9% Gly + 0.2M suc, RT 30-40 secs 0.2M Suc 5 min 37 o C4.5% gly + 0.2 suc, 3min (1:1 9% G+0.2 M suc: 0.2 M suc) Wash 7 x 100 uL HEPES2.3% gly + 0.2 suc, 3min (1:1 4.5% G+0.2 M suc: 0.2 M suc) 1 min/drop @ 37 o CT 0.2 M suc, 3 min, move dish to 37 o C: 0.1 M suc, 3 min, x3 wash in diluent transfer to culture medium Freeze Cool Thaw

32. Wade et al., ASRM (2005) ActionRodney Wade FREEZING base mediumSAGE products (& uses 0.5 mL vials) step 1Place culture dish @ RT 5 min step 25% Gly 10 min RT step 39% Gly/0.2M Suc 10 min RT COOLING ramp 1RT to -7 C @ 2 C/min Hold5 min, then seed; hold another 15 min ramp 3-7 C to -35 C @ 0.3 C/min ramp 4 ramp 5to LN THAWINGampule held at RT 60 secs use 2.5 mL of each solution in 35 mm dish overlaid with 1.5 mL of oil water @ 37 C 2 min 0.5M Suc 5 min RT 0.2M Suc 10 min RT HEPES 10 min RT to 37 C transfer to culture medium

33. Wade et al., ASRM (2005) ROOM TEMP AIR WATERBATH 37°0.5M SUCROSE0.2 SUCROSEM-HTF to 37°IN CULTURE GROUP 11 Minute2 – 3 Minutes10 Minutes 15 Minutes4 – 8 Hours GROUP 21 Minute2 – 3 Minutes5 Minutes10 Minutes15 Minutes4 – 8 Hours # CPET# TRANSFERS# EMBRYOS THAWED # EMBRYOS TRANSFERRED + PREGNANCYFHT GROUP 1123108 (87.8%)470269 (59.23%)43 (39.8%)37 (34.26%) GROUP 2141126 (89.4%)529277 (52.4%)83 (65.9%)62 (49.2%) P=.02P=.001P=0.025 THAW PROCEDURES

34. Wade et al., ASRM (2005)

35. The Two Biggest Factors in Slow Freezing v. Vitrification 1. Success rates 2. Time of procedure

36. COMPARISON OF BLASTOCYST CRYOPRESERVATION SLOW COOLING VERSUS VITRIFICATION (Cryotop)

37. Timing of Slow Cooling vs. Vitrification oCoC +20 0 -20 -40 -60 Time minutes 30 6090120 Slow cooling Vitrification

38. Timing of Slow Cooling vs. Vitrification EventSlow CoolingVitrification (10 oocytes) Strip cumuluss 5-10 min Equilibrate in CPA20 min5 + 1 = 6 min Load devicestraw/vial 2-3 mineg OPS 15 secs. Cool to seeding T o 15 min Seed2 min Plunge in LN2 & store5 min HANDS ON TIME20-30 min for all 20 oocytes 10 min per group 1-2 oocytes: X5= 50 min Several patientsCan be done together Each has to be done individually

39. Final Word From Lynette Scott Fertility Center of New England, Boston I am still not convinced about Vit at the cost…. Patrick, kits are way over conventional and straw systems prohibitive if you are doing single embryo cryo, which I am as I do single embryo ET and FET in 20-30% of cases. And if my conventional FET rates = fresh rates, even on IR, why would I increase my cost…… Devils advocate. Give me a really good reason because time is not one, I am freezing 1-5 patients, 2-10 embryos at a time and with conventional cryo my embryologists are multi- taskers, with vit Forget It!! They are it for a few hours. And you have to have a very “nice” system. Or a very “good” embryologist. I can teach even junior techs to freeze-thaw in conventional systems. As my FET system works so well, 90% survival, FHB as good as fresh, what are my incentives to change??

40. Final Word My personal opinion 1.For both systems, need to optimize culture system, choose the best embryos, and optimize cryopreservation system. 2.There is a place for both systems: i. Vitrification for oocytes, and in cases where there are only a few embryos and/or 1 or 2 patients. ii. Slow freezing where there are many embryos and/or patients.