Safety of oocyte cryopreservation: Lessons from donor banking

Zsolt Peter NAGY

Reproductive Biology AssociatesAtlanta, USA

Alpha Meeting Budapest 2010

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

1986: Slow freeze, DMSO (Chen, Australia)1987: Slow freeze, DMSO (Van Uerm, West Germany)1989: Slow freeze, PROH and DMSO (Siebzegnrubi, West Germany)

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)

Oocyte Freezing History

Eight years

8 years

Oocyte Freezing History

Reported Live Births

0

20

40

60

80

100

120

140

160

180

200

1986 1987 1989 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006Year

2007

Vitrification n=253Slow Freezing n=233

6

• Government restrictions on the number of eggs that can be

Rationale for Oocyte Freezing

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

zona pellucidazona pellucidahardeninghardening

membrane membrane permeabilitypermeability

Cytoplasmic and Cytoplasmic and Cytoskeleton Cytoskeleton

damagedamage

Meiotic spindle Meiotic spindle depolymerizationdepolymerization

Impact on oocyte Impact on oocyte physiologyphysiology

Polar body Polar body degeneration/fusiondegeneration/fusion

Safety IssuesSafety Issues

Ghetler et al., 2006

Fresh

Frozen

No evidence of cortical granule discharge in cryopreserved oocytes

Failed FertilizedFailed Fertilized

Frozen Non-frozen

Gook et al., 1993

“The immunostaining examination for CG of the frozen–thawed oocytes did not reveal evidence of the premature release of CG.”

Li et al., 2005

Safety IssuesCortical granule

3h 373h 37°°CCTS2TS2TS1TS1 PBSPBSTS3TS3

THAWING

PBSPBS FS1FS1 FS2FS2

FREEZING

� � �

� � �

Rienzi et al., 2004

Safety IssuesSafety IssuesMeiotic spindle depolymerizationMeiotic spindle depolymerization

Coticchio et al., 2004

VITRIFICATION

SLOW FREEZING

Safety IssuesSafety IssuesOsmotic toxicity / Dehydration injury / Chilling injuryOsmotic toxicity / Dehydration injury / Chilling injury

Experimental Data

33/3323/23

Vitrification Slow freezing

33/3323/23

Vitrification Slow freezing

28/28 34/34

Thawing in the Fixative

Exposure to the Freezing

Sol.

Vitrification Slow freezing

21/21

9/29

Post Thaw 30 min

20/29

Post Thaw Vitrification Slow freezing

19/19 (100) 23/30 (76.7)

13/13 (100) 26/32 (81.3)

28/28 (100) 34/40 (85.0)

1 h

2 h

4 h

VITRIFICAT. / WARMING

Intact spindle

Diminished

spindle

Fresh control MII oocytes

100% (25/25)

0 (0/25)

Equilibration sol. 15 min, RT

100% (20/20)

0 (0/20)

Vitrification sol. 1 min, RT

0 (0/23) 100% (23/23)

Warming into fixative

0 (0/28) 100% (28/28)

0 min post warm. proced.

0 (0/28) 100% (28/28)

15 min (37 °C) post warming

100% (28/28)

0 (0/28)

30 min (37 °C) post warming

100% (39/39)

0 (0/39)

1 h (37 °C) post warming

100% (19/19)

0 (0/19)

2 h (37 °C) post warming

100% (13/13)

0 (0/13)

4 h (37 °C) post warming

100% (28/28)

0 (0/28)

SLOW-FREEZE /RAPID THAW

Bi-polar spindle

Disorganized spindle

Absent spindle

Anaph/ Teloph spindle

Fresh control MII oocytes

100% (33/33)

0 (0/33) 0 (0/33) 0 (0/33)

Freez. Sol.-Equilibr. 20 min, RT

100% (35/35)

0 (0/35) 0 (0/35) 0 (0/35)

Fixed immediately upon thawing*

100% (31/31)

0 (0/31) 0 (0/31) 0 (0/31)

Thawing-(0.5 M Sucr.)10 min, RT

100% (22/22)

0 (0/22) 0 (0/22) 0 (0/22)

Thawing-(0.2 M Sucr.)10 min, RT

0 (0/19) 100% (19/19)

0 (0/19) 0 (0/19)

Washing-(0 M Sucr.) 10 min, RT

0 (0/23) 100% (23/23)

0 (0/23) 0 (0/23)

30 min (37 °C) post thawing

0 (0/29) 69.0% (20/29)

31% (9/29)

0 (0/29)

1 h (37 °C) post thawing

76.7% (23/30)

16.6% (5/30)

6.7% (2/30)

0% (0/30)

2 h (37 °C) post thawing

81.2% (26/32)

6.3% (2/32)

0 (0/32) 12.5% (4/32)

4 h (37 °C) post thawing

85.0% (34/40)

0 (0/40) 10.0% (4/40)

5.0 % (2/40)

100 100 100 98.198.595.8

91

68.7

29.6

91

73.1

49.3

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

Control-No freezing/ThawingVitrificationSlow Freezing

PN BL/D4 BL/D5 CLEAV

% of examined oocytes

55

/55

67

/67

71

/71

54

/55

50

/55

50

/55

66

/67

46

/67

49

/67

68

/71

21

/71

35

/71

Developmental Potential After Oocyte Freezing/Thawing

Lane and Gardner., 2001Lane and Gardner., 2001

Pyru

vate

Upt

ake

(pm

ol/o

ocyt

e/h)

0

1

2

3

4

5

a

b

c

Control Vitrification Slow-freezing

Safety IssuesSafety IssuesImpact on oocyte physiologyImpact on oocyte physiology

Techniques

Slow freezingSlow freezing

VitrificationVitrification

2222--3737°°CC

2222--3737°°CC

--196196--210210°°CC --196196--210210°°CC

Physiologicalsolution

Cryoprotectantsolution

Vitrificationsolution

Beforecooling

Duringcooling

In LN2

Ice seeding

Slow cooling

Rapid cooling

Slow FreezingSlow Freezing VitrificationVitrification

Ultra rapid cooling0.3ºC/min

200,000ºC/min

Techniques

Vitrification is a process that produces a glasslike Vitrification is a process that produces a glasslike solidification of living cells not by crystallization but by solidification of living cells not by crystallization but by an extreme elevation of viscosity during the coolingan extreme elevation of viscosity during the cooling

WHAT IS VITRIFICATION?WHAT IS VITRIFICATION?

Base medium

Base medium + CryoprotectantBase medium + Cryoprotectant

Slow Freeze

• Lower cryoprotectant

concentration

• Longer exposure time

• Cryomachine

• Longer to perform

• Technically easier

Does one method cause more cryodamage than the other?

Techniques

Vitrification

• Higher cryoprotectant

concentration

• Shorter exposure time

• Shorter to perform

• More precise timing

• More clinical expertise

• Open containers

PermeatingPermeating

Affect / pass through cell membranesAffect / pass through cell membranes

Interact with and replace HInteract with and replace H22OO

Lower freezing pointLower freezing point

Toxicity with TToxicity with Too and Concentrationand Concentration

PROHPROH

DMSODMSO IncreasedIncreased

GlycerolGlycerol PermeabilityPermeability

Ethylene GlycolEthylene Glycol

TechniquesCryoprotective Agents

Non-PermeatingDo not pass through cell membranesCreate osmotic gradient / Dehydration(High MW: >1000)

GlucoseSucroseFicoll

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

IRVINE SCIENTIFICIRVINE SCIENTIFIC

Perform at Room Temperature (10 min)

Oocyte vitrification freeze protocolOocyte vitrification freeze protocol

KeyH = HEPES buffered Culture Medium

with protein (eg. mHTF + SSS)ES = Equilibration Solution (3 drops)VS = Vitrification Solution (4 drops)

B=Bottom, C=Center, T=Top

KeyTS = Thawing Solution (1 drop)DS = Dilution Solution (2 drops)WS = Washing Solution (3 drops)

B=Bottom, T=Top

20 μl drops

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.

3. Transfer specimen(s) from merged drop to 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-equilibrated culture medium for recovery (2-3 hours) prior to subsequent manipulations.

IRVINE SCIENTIFICIRVINE SCIENTIFIC

Oocyte vitrification warm protocolOocyte vitrification warm protocolPerform at Room Temperature

GOLD GRIDGOLD GRID

3 mm

European Hospital European Hospital -- Rome, ItalyRome, Italy

VITRIFICATION VITRIFICATION -- ToolsTools

Nylon loop (20µm wide; 0.5-0.7 mm in diameter)

Thin film of cryoprotectant solution by surface tension

Oocytes are placed by pipette

VITRIFICATION VITRIFICATION -- ToolsTools

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.

VITVIT--MASTERMASTER

European Hospital European Hospital -- Rome, ItalyRome, Italy

Results

Summary of clinical outcomes from oocyte cryopreservation using various protocols

115.33.46.12.49.12.3

Implantations per 100 thawed oocytes

1416112151710Implantation

(%)

76333129495323Embryos per 100 thawed

oocytes

928386100909385Cleavage (%)

91685856738054ICSI fert (%)

91 (628)59 (190)62 (329)52 (127)74 (4902)72 (926)50 (3537)

Vitrification 2.7 M EG + 2.1 M DMSO + 0.5 M

sucrose

1.5 M PROH + 0.3 M sucrose

(Na depleted)

1.5 M PROH +

0.2 M sucrose

(Na depleted)

1.5 M PROH +

0.1 M sucrose

(Na depleted)

1.5 M PROH +

0.3 M sucrose

1.5 M PROH +

0.2 M sucrose

1.5 M PROH +

0.1 M sucrose

Survival (%) (#)

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 decumulationVitrification media: Ethylene glycol & DMSO

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.

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

RBA experience on oocyte freezingCryo Egg Bank (donor)

• ET 247 Transfers211-D5 / 36-D3

• +FCA 147 (60%)• No of FCAs 219 (44%)

Singleton x 93Twin x 54Triplet x 6Miscarriage x 12

Nagy et al.,RBA 2009

RBA experience on oocyte freezingCryo Egg Bank (donor)

Current comparisonVitrified egg vs fresh (same donor) May 2006- March 2009

Cryo oocyte Fresh oocyte PNumber of donors 81 81 NANumber of recipients 100 91 NAMean age (±SD) of recipients 40.9 (±4.9) 41.2 (±4.7) NSMean number of oocytes per recipients 7.1 25.28 <.001

Mean number of oocytes for ICSI 6.0 15.0 <.001Average 2PN ICSI fertilization rate 77% 57% <.001

Implantation Rate 52% 56% NSMean number of embryos cryopreserved 1.5 (±1.5) 12.5 (±8.8) <.001

Clinical pregnancy rate 67% 69% NS

Nagy et al.,RBA 2009

Controln=16

Vitrificationn=10

P

Female AgeMean (+/-SD) 29.4 (+/-5.4) 28.0 (+/-1.0) NS# Cells per BlastMean (+/-SD) 43.0 (+/-13.4) 38.0 (+/-32.8) NSNormal cells per blastMean (+/-SD) 23.0 (+/-14.7) 20.0(+/-28.5) NSTotal # of normal cells 368 200 NS

% Normal cell 56.5 52.6 NS

Total # of cells 688 380 NS

COMPARISON OF ANEUPLOIDY RATES OF BLASTOCYST STAGE EMBRYOS DERIVED FROM FRESH AND VITRIFIED OOCYTES

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 9* Four of these embryos were biopsied in the first cycle, then vitrified

Frozen Embryos From Frozen Eggs

RBA experience on oocyte freezingCryo Egg Bank (donor)

RBA experience: IVF patients 32–38 years

15 patients (34 y mean age) Cryo Egg Fresh EggM2 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 48Clinical Pregnancies 11 out of 15Implantation rate / FCA 15 (42%)

Antagonist Antagonist + HCG trigger + Lupron trigger

P value

# 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

(mean±SD)1325 (6.40±1.99) 108 (5.68±0.94) P=0.1205

# 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

HCG versus Lupron trigger

D

R1R2R3R4R5

D

R1R2R3R4

DR1R2R3

DR1R2

Efficiency of donor egg distribution among multiple recipients

Donors n=25; Age: 27.0±2.6

Recipients n=59; Age: 40.7±5.1

20 sets

2 sets

2 sets

1 set

Total warmed eggs= 418 (7.1 ± 2.1 eggs per recipient)

Donor #

Survival# Fert # (%) BL # (%)

Sperm count X106 Motile (%)

6 6 (100) 1 (16)* 20 701

5 5 (100) 5 (100)* 12 45

6 5 (83) 3 (60) 160 202

3 3 (100) 2 (66) 60 20

5 5 (100) 3 (60) 67 903

5 5 (100) 3 (60) 95 85

5 4 (80) 4 (100) 75 154

6 5 (83) 3 (60) 40 70

5 4 (80) 3 (75) 17 655

7 6 (85) 5 (83) 40 70

4 4 (100) 4 (100) 35 406

5 5 (100) 5 (100) 35 30

8 7 (87) 5 (71) 6 407

7 5 (71) 3 (60) 81 80

6 5 (83) 2 (40) 3 338

8 7 (87.5) 4 (57) 50 80

5 4 (80) 3 (75) 70 459

6 4 (66) 4 (100) 4 50

6 5 (83) 4 (80) 87 7010

6 5 (83) 5 (100) 60 70

Donor # Survival

# Fert # (%) BL # (%)

Sperm count X106 Motile (%)

6 6 (100) 4 (66) 2 511

4 4 (100) 2 (50) 120 70

4 4 (100) 4 (100) 50 4012

6 6 (100) 5 (83) 35 45

12 11 (91) 11 (100)* 49 5513

6 6 (100) 2 (33)* 140 70

5 4 (80) 2 (50) 5 4014

6 5 (83) 2 (40) 60 90

6 6 (100) 5 (83) 40 5015

4 4 (100) 2 (50) 30 50

6 6 (100) 3 (50) 160 5016

6 5 (83) 4 (80) 31 1

6 5 (83) 4 (80) 9 0.517

6 4 (66) 3 (75) 3 33

4 3 (75) 3 (100) 57 5518

6 5 (83) 3 (60) 70 75

7 7 (100) 2 (28) 38 7019

7 6 (85) 4 (66) 65 25

9 9 (100) 8 (88) 139 7020

8 8 (100) 7 (87) 61 80

Results DR1

20 setsR2

1. Sperm count<20 million/ml: with a difference by > 20 million/ml2. Sperm motility (%) differed by > 30%

Results

Donor #

Survival

#Fert # (%) BL # (%)

Sperm count X106

Motile (%)

8 8 (100)a 6 (75)‡ 20 50

8 8 (100)a 6 (75)‡ 50 7021

9 9 (100)a 9 (100)‡ 70 75

6 6 (100)a 6 (100)‡ 38 35

4 3 (75)a 3 (100)‡ 110 4522

4 4 (100)a 3 (75)‡ <1 20

Donor #

Survival# Fert # (%) BL # (%)

Sperm count X106

Motile (%)

23

5 5 (100)a 3 (60)‡ 70 55

9 7 (77)a 5 (71)‡ 55 67

6 5 (83)a 4 (80)‡ 13 30

7 6 (85)a 4 (66)‡ 56 20

24

5 5 (100)a 2 (40)‡ 100 60

4 4 (100)a 4 (100)‡ 34 40

5 4 (80)a 2 (50)‡ 90 75

5 3 (60)a 2 (66)‡ 110 60

DR1R2R3

2 sets D

R1R2R3R4

2 sets

1. Sperm count<20 million/ml: with a difference by > 20 million/ml2. Sperm motility (%) differed by > 30%

Results

D

R1R2R3R4R5

1 set

Donor # Survival# Fert # (%) BL # (%)Sperm count X106

Motile (%)

10 9 (90)a 7 (77)‡ 18 65

6 5 (83)a 3 (60)‡ 67 30

6 4 (66)a 4 (100)‡ 115 55

5 3 (60)a 3 (100)‡ 82 37

25

6 4 (66)a 3 (75)‡ 8 50

1. Sperm count<20 million/ml: with a difference by > 20 million/ml2. Sperm motility (%) differed by > 30%

Practical approachesThe use of sibling oocytes to compare oocyte vitrification outcomes with different cryoprotectant components: a formula without DMSOC.-C. Chang, and Nagy - Fertility and Sterility September 2009

In-vivo vs. rescued in-vitro maturation metaphase II oocyte vitrification outcomes in humanC.-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 hoursC.-C. Chang Nagy - Fertility and Sterility September 2009

The oocyte spindle is preserved by 1,2-propanediol during slow freezingChing-Chien Chang, and Nagy Fertility and Sterility 15 March 2010 (Vol. 93, Issue 5, Pages 1430-1439)

Oocyte cryopreservation birth 'case reports‘ 1986–2008

Cryopreservation methodParameter

Slow-freeze Vitrification Both

No. of embryo transfers 1974 834 19

No. of liveborn babies 282 285 12

Baby gender (gender information available for 168 slow-freeze, 189 vitrification and 12 both methods)

99 female, 69 male 86 female, 103 male

8 female, 4 male

Birth defects 1 ventricular septal defect, 1 choanal atresia, 1 Rubenstein-Taybi syndrome

2 ventricular septal defect, 1 biliary atresia, 1 clubfoot, 1 skin haemangioma

None

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].

FRESHEGG

FROZENEGG

PATIENT AGE 39.86+5.59

39.72+5.48

DELIVERIES 58 96LIVEBIRTHS 91 146TERM DELIVERY 37 WK 28 66

PRETERM < 37 WK 29 2934-36 WK 23 1832-33WK 1 728-31 WK 5 3

ABNORMALITY 3Down sy. 2xHemangioma

4brain hemm.; club foot; VSD; pelvic kidney

Live Birth Data from Egg Cryo from RBA

Live Birth Data from Egg Cryo from RBA

FRESHEGG

FROZENEGG

NO. OF PATIENTS 58 96LIVEBIRTHS 91 146XX NA 62XY NA 80WEIGHT (gr)AVG

2659.36+690.97

2708.61+801.11

SINGLETON/Tw/Tr 26/31/1 47/47/2WEIGHT (gr) AVGSINGLETONS

3161.55+808.17

3320.06+773.00

WEIGHT (gr) AVGTWINS

2483.16+465.17

2397.73+624.28

WEIGHT (gr) AVGTRIPLETS

1606.5+56.67

1809.68+564.56

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

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!!!.

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

AcknowledgmentAcknowledgment