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Safety of oocyte cryopreservation lessons from donor banking alpha-nagy_peter_01.05.2010

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Safety of oocyte cryopreservation- Lessons from donor banking_ALPHA_Nagy_Peter_01.05.2010

Safety of oocyte cryopreservation- Lessons from donor banking_ALPHA_Nagy_Peter_01.05.2010

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  • 1. Safety of oocyte cryopreservation: Lessons from donor banking Alpha Meeting Budapest 2010 Zsolt Peter NAGY Reproductive Biology Associates Atlanta, USA
  • 2. Learning Objectives - To review briefly the history - To review the need/indications - To review safety issues - To review existing techniques - Current results of egg freezing - Future Perspectives / Conclusions
  • 3. Oocyte Freezing History 1986: Slow freeze, DMSO (Chen, Australia) 1987: Slow freeze, DMSO (Van Uerm, West Germany) 1989: Slow freeze, PROH and DMSO (Siebzegnrubi, West Germany) Eight years 1997: Slow freeze, PROH and Sucrose - ICSI (Porcu, Italy) 1998: Slow freeze, PROH and Sucrose - Immature/Donor oocytes ( Tucker, USA) 1999: Vitrification, EG and Sucrose - open pulled straws (Kuleshova, Australia) 2000: Vitrification: EG and Sucrose - electron microscope grid (Toon, Cha, Korea) 2003: Vitrification, EG, DMSO and Sucrose - CryotopTM (Katayama, USA) 2003: Slow freeze, Choline-based medium (Quintans, Argentina)
  • 4. Oocyte Freezing History 200 Reported Live Births 180 160 140 120 100 80 60 8 years 40 20 0 1986 1987 1989 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Slow Freezing n=233 Year Vitrification n=253
  • 5. Rationale for Oocyte Freezing • Government restrictions on the number of eggs that can be fertilized • Fertility Preservation • Women with malignant / premalignant conditions offered treatment that may negatively impact their future ability to have children (50,000 per year under 40 years) • Women delaying childbearing – Career – Partnership status – Psychological / emotional • Donor oocyte banking • Male unable to produce semen sample day of retrieval • IVF patients egg freeze instead of embryo freeze 6
  • 6. Safety Issues membrane Cytoplasmic and permeability Cytoskeleton damage zona pellucida Polar body hardening degeneration/fusion Meiotic spindle Impact on oocyte depolymerization physiology
  • 7. Safety Issues Cortical granule No evidence of cortical granule discharge in Fresh cryopreserved oocytes Gook et al., 1993 Failed Fertilized Frozen Non-frozen Frozen “The immunostaining examination for CG of the frozen–thawed oocytes did not reveal evidence of the premature release of CG.” Li et al., 2005 Ghetler et al., 2006
  • 8. Safety Issues Meiotic spindle depolymerization Rienzi et al., 2004 PBS FS1 FS2 FREEZING THAWING TS1 TS2 TS3 PBS 3h 37°C
  • 9. Safety Issues Osmotic toxicity / Dehydration injury / Chilling injury Coticchio et al., 2004 VITRIFICATION SLOW FREEZING
  • 10. Experimental Data
  • 11. Vitrification Slow freezing Exposure to the Freezing Sol. 23/23 33/33 Vitrification Slow freezing Thawing in the Fixative 28/28 23/23 34/34 33/33
  • 12. Vitrification Slow freezing Post Thaw 30 min 9/29 20/29 21/21
  • 13. Post Thaw Vitrification Slow freezing 19/19 (100) 23/30 (76.7) 1h 13/13 (100) 26/32 (81.3) 2h 28/28 (100) 34/40 (85.0) 4h
  • 14. VITRIFICAT. / Intact Diminis SLOW-FREEZE / Bi-polar Disorg Absent Anaph/ WARMING spindle hed RAPID THAW spindle anized spindle Teloph spindle spindle spindle Fresh control 100% 0 (0/25) Fresh control MII 100% 0 (0/33) 0 (0/33) 0 (0/33) MII oocytes (25/25) oocytes (33/33) Equilibration 100% 0 (0/20) Freez. Sol.-Equilibr. 100% 0 (0/35) 0 (0/35) 0 (0/35) sol. 15 min, RT (20/20) 20 min, RT (35/35) Vitrification sol. 0 (0/23) 100% Fixed immediately 100% 0 (0/31) 0 (0/31) 0 (0/31) 1 min, RT (23/23) upon thawing* (31/31) Warming into 0 (0/28) 100% Thawing-(0.5 M 100% 0 (0/22) 0 (0/22) 0 (0/22) fixative (28/28) Sucr.)10 min, RT (22/22) 0 min post 0 (0/28) 100% Thawing-(0.2 M 0 (0/19) 100% 0 (0/19) 0 (0/19) warm. proced. (28/28) Sucr.)10 min, RT (19/19) 15 min (37 °C) 100% 0 (0/28) Washing-(0 M Sucr.) 0 (0/23) 100% 0 (0/23) 0 (0/23) post warming (28/28) 10 min, RT (23/23) 30 min (37 °C) 100% 0 (0/39) 30 min (37 °C) post 0 (0/29) 69.0% 31% 0 (0/29) post warming (39/39) thawing (20/29) (9/29) 1 h (37 °C) 100% 0 (0/19) 1 h (37 °C) post 76.7% 16.6% 6.7% 0% post warming (19/19) thawing (23/30) (5/30) (2/30) (0/30) 2 h (37 °C) 100% 0 (0/13) 2 h (37 °C) post 81.2% 6.3% 0 (0/32) 12.5% post warming (13/13) thawing (26/32) (2/32) (4/32) 4 h (37 °C) 100% 0 (0/28) 4 h (37 °C) post 85.0% 0 (0/40) 10.0% 5.0 % post warming (28/28) thawing (34/40) (4/40) (2/40)
  • 15. Developmental Potential After Oocyte Freezing/Thawing % of examined oocytes 100 100 100 100 98.198.5 95.8 91 91 90 55 54 50 50 80 / / / / 73.1 70 55 55 55 68.7 55 60 67 66 46 49 49.3 Control-No freezing/Thawing 50 / / / / Vitrification 67 67 67 67 40 Slow Freezing 29.6 30 71 68 21 35 20 / / / / 71 71 71 71 10 0 1 2 3 4 PN CLEAV BL/D4 BL/D5
  • 16. Safety Issues Impact on oocyte physiology 5 4 a Pyruvate Uptake (pmol/oocyte/h) 3 b 2 c 1 0 Control Vitrification Slow-freezing Lane and Gardner., 2001
  • 17. Techniques Vitrification 22-37°C Slow freezing 22-37°C -196-210°C -196-210°C
  • 18. Techniques Slow Freezing Vitrification Physiological solution Before cooling Cryoprotectant solution Vitrification solution Ice seeding Ultra rapid cooling During cooling 0.3ºC/min Slow cooling 200,000ºC/min Rapid cooling In LN2
  • 19. WHAT IS VITRIFICATION? Vitrification is a process that produces a glasslike solidification of living cells not by crystallization but by an extreme elevation of viscosity during the cooling Base medium + Cryoprotectant Base medium
  • 20. Techniques Vitrification Slow Freeze • Higher cryoprotectant • Lower cryoprotectant concentration concentration • Shorter exposure time • Longer exposure time • Shorter to perform • Cryomachine • More precise timing • Longer to perform • Technically easier • More clinical expertise • Open containers Does one method cause more cryodamage than the other?
  • 21. Techniques Cryoprotective Agents Permeating Non-Permeating Affect / pass through cell membranes Do not pass through cell Interact with and replace H2O membranes Lower freezing point Create osmotic gradient / Dehydration Toxicity with To and Concentration (High MW: >1000) PROH DMSO Increased Glucose Glycerol Permeability Sucrose Ethylene Glycol Ficoll
  • 22. Oocyte vitrification freeze protocol Perform at Room Temperature (10 min) 1. Incubate specimen in H for 1 min 2. Merge ES1 with H for 2 min 3. Merge ES2 with H + ES1 for 2 min 4. Transfer from merged drops to BOTTOM (B) of ES3 for 3 min 5. Transfer from ES3 to CENTER (C) of VS1 and proceed as shown in diagram Key H = HEPES buffered Culture Medium with protein (eg. mHTF + SSS) ES = Equilibration Solution (3 drops) IRVINE SCIENTIFIC VS = Vitrification Solution (4 drops) B=Bottom, C=Center, T=Top
  • 23. Oocyte vitrification warm protocol Perform at Room Temperature 1. Rinse CryoTip™ by aspirating an equal volume (~1µl) of TS and dispensing next to CryoTip contents. 2. Merge content and rinse drop and wait 1 minute. 20 μl 3. Transfer specimen(s) from merged drop to drops BOTTOM (B) of TS for 1 minute. 4. Transfer specimen(s) to BOTTOM of DS1 and DS2 drops for 2 minutes each. 5. Transfer Oocyte(s) (2 min) or through each WS1 (B),WS2 (T) and WS3 (T) as indicated 6. Then transfer specimen(s) to pre- Key equilibrated culture medium for recovery TS = Thawing Solution (1 drop) (2-3 hours) prior to subsequent DS = Dilution Solution (2 drops) manipulations. WS = Washing Solution (3 drops) B=Bottom, T=Top IRVINE SCIENTIFIC
  • 24. VITRIFICATION - Tools GOLD GRID 3 mm European Hospital - Rome, Italy
  • 25. VITRIFICATION - Tools Nylon loop (20µm wide; Thin film of cryoprotectant 0.5-0.7 mm in diameter) solution by surface tension Oocytes are placed by pipette
  • 26. Open or Closed system? CLOSED SYSTEMS (heat sealing): - More “easily accepted” in daily IVF use – Prevention of “contamination. -It works for slow-freezing for embryo/oocyte, reasonably for vitrifiation of embryos – but questionable for vitrification of oocytes. - It may be questionable if closed system truly prevents (biological) particles passing through (material feature at -196C?) OPEN SYSTEMS: -It’s use is questioned because of “contamination” “risk”. – However, no proven evidence that contamination occurred with oocyte/embryo storage. - It works well both for embryos and oocytes.
  • 27. VIT-MASTER European Hospital - Rome, Italy
  • 28. Results
  • 29. Summary of clinical outcomes from oocyte cryopreservation using various protocols 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M PROH + PROH + Vitrification 2.7 PROH + 0.3 PROH + PROH + PROH + 0.1 M 0.2 M M EG + 2.1 M M sucrose 0.1 M 0.2 M 0.3 M sucrose sucrose DMSO + 0.5 M (Na sucrose sucrose sucrose (Na (Na sucrose depleted) depleted) depleted) Survival (%) (#) 50 (3537) 72 (926) 74 (4902) 52 (127) 62 (329) 59 (190) 91 (628) ICSI fert (%) 54 80 73 56 58 68 91 Cleavage (%) 85 93 90 100 86 83 92 Embryos per 100 thawed 23 53 49 29 31 33 76 oocytes Implantation (%) 10 17 5 21 11 16 14 Implantations per 100 2.3 9.1 2.4 6.1 3.4 5.3 11 thawed oocytes
  • 30. RBA experience on oocyte freezing: cryo egg bank (donor) Donor selection: young (<35; mean 28y.) & healthy Stimulation: rFSH with antagonist or agonist Egg collection: 36 h post hCG and decumulation Vitrification Ethylene glycol & DMSO media: Warming: Three steps; 1.0 M, 0.5 M, 0 M sucrose ICSI: 3 h post thaw / ET on Day 5 Nagy ZP. Personal Communication. September 2009.
  • 31. RBA experience on oocyte freezing Cryo Egg Bank (donor) 119 Don. 139 cl. (25.8y.) 2779 M2 Vit (20/don) 247 Recipients 1592 Warmed M2 (6.4/R.) •Survived 1386 (87%) •Fertilized 1206 (87%) •Blastocysts-d5 (211) 723 / 1069 (67%) •No of Es for ET 494 (2.0 / Recip.) •No of Es for Cryo 322 (1.3 / Recip.) Nagy et al.,RBA 2009
  • 32. RBA experience on oocyte freezing Cryo Egg Bank (donor) • ET 247 Transfers 211-D5 / 36-D3 • +FCA 147 (60%) • No of FCAs 219 (44%) Singleton x 93 Twin x 54 Triplet x 6 Miscarriage x 12 Nagy et al.,RBA 2009
  • 33. Current comparison Vitrified egg vs fresh (same donor) May 2006- March 2009 Cryo oocyte Fresh oocyte P Number of donors 81 81 NA Number of recipients 100 91 NA Mean age (±SD) of recipients 40.9 (±4.9) 41.2 (±4.7) NS Mean number of oocytes per 7.1 25.28 <.001 recipients Mean number of oocytes for 6.0 15.0 <.001 ICSI Average 2PN ICSI fertilization 77% 57% <.001 rate Implantation Rate 52% 56% NS Mean number of embryos 1.5 (±1.5) 12.5 (±8.8) <.001 cryopreserved Clinical pregnancy rate 67% 69% NS Nagy et al.,RBA 2009
  • 34. COMPARISON OF ANEUPLOIDY RATES OF BLASTOCYST STAGE EMBRYOS DERIVED FROM FRESH AND VITRIFIED OOCYTES Control Vitrification P n=16 n=10 Female Age Mean (+/-SD) 29.4 (+/-5.4) 28.0 (+/-1.0) NS # Cells per Blast Mean (+/-SD) 43.0 (+/-13.4) 38.0 (+/-32.8) NS Normal cells per blast Mean (+/-SD) 23.0 (+/-14.7) 20.0(+/-28.5) NS Total # of normal cells 368 200 NS % Normal cell 56.5 52.6 NS Total # of cells 688 380 NS
  • 35. RBA experience on oocyte freezing Cryo Egg Bank (donor) Frozen Embryos From Frozen Eggs 30 patients (from Cryo Egg Bank) Cryo Embryo Number of warmed embryos 67 Survived 65 (97%) No of Es for ET (x) 60 (2.0)* Pregnancies (Clinical) 21 (70%) Implantation / FCA 25 (42%) Miscarriages 5 Live births 17 Girls * Four of these embryos were biopsied in the first cycle, then vitrified 9
  • 36. RBA experience: IVF patients 32–38 years 15 patients (34 y mean age) Cryo Egg Fresh Egg M2 Eggs (Sibling Eggs) 116 (7.7) 112 (7.5) Survived 99 (85%) - Fertilization Rate 87% 84% Blastocyst Rate 64% 62% Number of Embryos Transferred 36 (2.4) 0 Number of Embryos Frozen 27 48 Clinical Pregnancies 11 out of 15 Implantation rate / FCA 15 (42%)
  • 37. HCG versus Lupron trigger Antagonist Antagonist P value + HCG trigger + Lupron trigger # of donor (mean age±SD) 93 (26.35±2.9) 9 (26.57±2.54) P=0.8265 # of recipient (mean age±SD) 207 (41.05±4.75) 19 (39.47±4.04) P=0.1619 # of egg warmed 1325 (6.40±1.99) 108 (5.68±0.94) P=0.1205 (mean±SD) # of egg survived (%)* 1150 (86.8%) 103 (95.3%) P=0.0064 # of egg fertilized (%) 999 (86.8%) 93 (90.3%) P=0.3604 # of embryo cleaved (%) 976 (97.7%) 92 (98.9%) P=0.7144 # of ET (mean±SD) 419 (2.02±0.43) 35 (1.84±0.37) P=0.0790 # of (+) hCG (%) 149 (71.9%) 13 (68.4%) P=0.7916 # of Clinical PR(%) 126 (60.8%) 11 (57.9%) P=0.8102 # of Implantation (%) 193 (46.0%) 12 (34.3%) P=0.2168
  • 38. Efficiency of donor egg distribution among multiple recipients R1 Donors n=25; Age: D R2 20 sets Recipients n=59; Age: 27.0±2.6 40.7±5.1 R1 D R2 2 sets R3 R1 R2 D R3 2 sets R4 R1 R2 D R3 1 set R4 R5 Total warmed eggs= 418 (7.1 ± 2.1 eggs per recipient)
  • 39. R1 Results D R2 20 sets Sperm Sperm Donor Donor Survival count Survival count # # # Fert # (%) BL # (%) X106 Motile (%) # Fert # (%) BL # (%) X106 Motile (%) 6 6 (100) 1 (16)* 20 70 6 6 (100) 4 (66) 2 5 1 11 5 5 (100) 5 (100)* 12 45 4 4 (100) 2 (50) 120 70 6 5 (83) 3 (60) 160 20 4 4 (100) 4 (100) 50 40 2 12 3 3 (100) 2 (66) 60 20 6 6 (100) 5 (83) 35 45 5 5 (100) 3 (60) 67 90 12 11 (91) 11 (100)* 49 55 3 13 5 5 (100) 3 (60) 95 85 6 6 (100) 2 (33)* 140 70 5 4 (80) 4 (100) 75 15 5 4 (80) 2 (50) 5 40 4 14 6 5 (83) 3 (60) 40 70 6 5 (83) 2 (40) 60 90 5 4 (80) 3 (75) 17 65 6 6 (100) 5 (83) 40 50 5 15 7 6 (85) 5 (83) 40 70 4 4 (100) 2 (50) 30 50 4 4 (100) 4 (100) 35 40 6 6 (100) 3 (50) 160 50 6 16 5 5 (100) 5 (100) 35 30 6 5 (83) 4 (80) 31 1 8 7 (87) 5 (71) 6 40 6 5 (83) 4 (80) 9 0.5 7 17 7 5 (71) 3 (60) 81 80 6 4 (66) 3 (75) 3 33 6 5 (83) 2 (40) 3 33 4 3 (75) 3 (100) 57 55 8 18 8 7 (87.5) 4 (57) 50 80 6 5 (83) 3 (60) 70 75 5 4 (80) 3 (75) 70 45 7 7 (100) 2 (28) 38 70 9 19 6 4 (66) 4 (100) 4 50 7 6 (85) 4 (66) 65 25 6 5 (83) 4 (80) 87 70 9 9 (100) 8 (88) 139 70 10 20 6 5 (83) 5 (100) 60 70 8 8 (100) 7 (87) 61 80 1. Sperm count<20 million/ml: with a difference by > 20 million/ml 2. Sperm motility (%) differed by > 30%
  • 40. Results R1 R1 R2 D R2 2 sets D R3 2 sets R3 R4 Sur Sperm Sperm Donor Motile Donor Survi Motile vival Fert # (%) BL # (%) count # Fert # (%) BL # (%) count # (%) val# X106 (%) # X106 8 8 (100)a 6 (75)‡ 20 50 5 5 (100)a 3 (60)‡ 70 55 21 8 8 (100)a 6 (75)‡ 50 70 9 7 (77)a 5 (71)‡ 55 67 23 9 9 (100)a 9 (100)‡ 70 75 6 5 (83)a 4 (80)‡ 13 30 6 6 (100)a 6 (100)‡ 38 35 7 6 (85)a 4 (66)‡ 56 20 22 4 3 (75)a 3 (100)‡ 110 45 5 5 (100)a 2 (40)‡ 100 60 4 4 (100)a 3 (75)‡ <1 20 4 4 (100)a 4 (100)‡ 34 40 24 5 4 (80)a 2 (50)‡ 90 75 5 3 (60)a 2 (66)‡ 110 60 1. Sperm count<20 million/ml: with a difference by > 20 million/ml 2. Sperm motility (%) differed by > 30%
  • 41. Results R1 R2 D R3 1 set R4 R5 Sperm Donor # Survival# Fert # (%) BL # (%) count Motile (%) X106 10 9 (90)a 7 (77)‡ 18 65 6 5 (83)a 3 (60)‡ 67 30 25 6 4 (66)a 4 (100)‡ 115 55 5 3 (60)a 3 (100)‡ 82 37 6 4 (66)a 3 (75)‡ 8 50 1. Sperm count<20 million/ml: with a difference by > 20 million/ml 2. Sperm motility (%) differed by > 30%
  • 42. Practical approaches The use of sibling oocytes to compare oocyte vitrification outcomes with different cryoprotectant components: a formula without DMSO C.-C. Chang, and Nagy - Fertility and Sterility September 2009 In-vivo vs. rescued in-vitro maturation metaphase II oocyte vitrification outcomes in human C.-C. Chang, Z.P. Nagy - Fertility and Sterility September 2007 High survival rates of vitrified human oocytes are maintained after exposure to transport conditions in the vapor phase of liquid nitrogen in dry shipper for 60 hours C.-C. Chang Nagy - Fertility and Sterility September 2009 The oocyte spindle is preserved by 1,2-propanediol during slow freezing Ching-Chien Chang, and Nagy Fertility and Sterility 15 March 2010 (Vol. 93, Issue 5, Pages 1430-1439)
  • 43. Oocyte cryopreservation birth 'case reports‘ 1986–2008 Parameter Cryopreservation method Slow-freeze Vitrification Both No. of embryo transfers 1974 834 19 No. of liveborn babies 282 285 12 Baby gender (gender 99 female, 69 male 86 female, 103 8 female, information available for 168 male 4 male slow-freeze, 189 vitrification and 12 both methods) Birth defects 1 ventricular septal 2 ventricular None defect, 1 choanal septal defect, 1 atresia, biliary atresia, 1 1 Rubenstein-Taybi clubfoot, 1 skin syndrome haemangioma Adapted Noyes N, Porcu E Borini A. Reprod BioMed Online 2009. http://www.rbmonline.com/Article/3971 [e-pub ahead of print on 8 April 2009].
  • 44. Live Birth Data from Egg Cryo from RBA FRESH FROZEN EGG EGG 39.86 39.72 PATIENT AGE +5.59 +5.48 DELIVERIES 58 96 LIVEBIRTHS 91 146 TERM DELIVERY 37 28 66 WK PRETERM < 37 WK 29 29 34-36 WK 23 18 32-33WK 1 7 28-31 WK 5 3 ABNORMALITY 3 4 Down sy. 2xHemangioma brain hemm.; club foot; VSD; pelvic kidney
  • 45. Live Birth Data from Egg Cryo from RBA FRESH FROZEN EGG EGG NO. OF PATIENTS 58 96 LIVEBIRTHS 91 146 XX NA 62 XY NA 80 WEIGHT (gr) 2659.36 2708.61 AVG +690.97 +801.11 SINGLETON/Tw/Tr 26/31/1 47/47/2 WEIGHT (gr) AVG 3161.55 3320.06 SINGLETONS +808.17 +773.00 WEIGHT (gr) AVG 2483.16 2397.73 TWINS +465.17 +624.28 WEIGHT (gr) AVG 1606.5 1809.68 TRIPLETS +56.67 +564.56
  • 46. Conclusions on Egg Banking - Similar outcomes with fresh and frozen eggs - Eliminating difficulty of synchronization - Decreasing risks of disease contamination - May prevent most of the moral/ethical questions of extra embryos - More cost effective – 5 frozen eggs / implantation Current results validate the use of oocyte cryo- banking for egg donation purposes
  • 47. Conclusions - Oocyte cryopreservation had historically low efficiency. - Recent reports indicate improved survival and implantation outcomes. - Vitrification may emerges as a more efficient technique vs slow freeze – not only for eggs but also for embryos. - Safety of oocyte cryopreservation has to be demonstrated – REGISTRY!!!.
  • 48. Acknowledgment EMBRYOLOGISTS PHYSICIANS Jeremy Chang PhD Hilton Kort, MD Graham Wright, BSc Carlene Elsner, MD Stacey Jones, BSc Dorothy Mitchell-Leef, MD Diana Patricia Bernal, DVM Andrew Toledo, MD Ann Fisher, BSc, MPH Scott Slayden, MD Wendy Brockman, BSc Robert Straub, MD Thomas Elliott, BSc Micheal Witt, MD