Safety of oocyte cryopreservation- Lessons from donor banking_ALPHA_Nagy_Peter_01.05.2010

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