1. Oocyte cryopreservation: slow freezing vs. vitrification
Ana Cobo
IVI-Valencia, Spain

2. Contents Cryopreservation Techniques Clinical outcome 3. Conclusions
Slow freezing
Vitrification
Cryo-injury
Clinical outcome
Slow Freezing
Slow Freezing vs. Vitrification
Vitrification vs. Fresh oocytes
3. Conclusions
Conclusions

3. Cryopreservation Techniques
Slow freezing
Vitrification
Cryo-injury

4. Cryopreservation techniques
Equilibrium Cryopreservation Procedures:
When the osmotic equilibrium between the intra and extra cellular media is reached by applying very slow
cooling rates.
Slow Freezing:
The cooling rate must be slow enough to allow a huge dehydration of the cell, thus avoiding the chance of intracellular ice formation.

5. Cryopreservation techniques
Non Equilibrium Cryopreservation Procedures:
It is not necessary to establish the osmotic balance between the intra and extracellular media along the period of cooling of the cells.
Vitrificacation:
The super-cooled intracellular water does not crystallize but turns into a very viscous solid of similar consistency to the glass.

6. Cryopreservation options
Ice
(Freezing)
No Ice
(Vitrification)
Freeze
Sol.
Vitri

7. Benefits and disadvantages
Slow freezing Vitrification
Ice crystal / ++ -
Chilling / +
Toxic
Osmotic
Mechanical
Equipment / ++
Skills
Speed
Contamination / (???)
Vajta
Oocyte vitrification in ART

8. Injuries during cryopreservation
Biological Factors (size, meiotic spindle)
Technical Factors (cooling/warming rates, CPAs)
Species, stage of development and origin of the samples
Oocyte Vitrification in ART

9. Chilling: lipids of the cell membrane, meiotic spindle.
Injuries during cryopreservation
2. Mechanisms of injury
Chilling: lipids of the cell membrane, meiotic spindle.
Ice Formation: from-5 and-15ºC to-80ºC.
Intracellular ice formation (IIF)
Osmotic damage
Osmotic stress

10. Strategies to minimize injuries
Slow-rate freezing:
An attempt to maintain a balance between various sources of injury by using low CPA concentration and controlled ice formation
Vitrification:
Radical elimination of ice formation,
and to reduce toxic and osmotic damage caused by high CPA concetrations
Oocyte Vitrification in ART

11. Practical ways to achieve vitrification in embryology
Cryoprotectant.
concentration
Practical ways to achieve
vitrification in embryology
Cooling-warming rates
Oocyte vitrification in ART

12. Increasing the cooling rates
How to perform?
Use minimum volume in the loading solution allows to achieve extremely high cooling rates
(Arav et al 1992)
Establish direct contact
Open Systems
Oocyte vitrification in ART

13. Loading of Oocyte in minimum volume VS on Cryotop
Cryotop Sheet
X not minimum VS
〇　Minimum VS
Oocyte in VS
Cryotop
Oocyte in VS
Cryotop

14. Method of choice Cooling rate °C/min 20,000 2,000
Straw Cryotip Grid&loop OPS SOPS Cryotop
Oocyte vitrification in ART

15. Clinical outcome Slow Freezing Slow Freezing vs. Vitrification
Vitrification vs. Fresh oocytes
Vitrification

16. Slow Freezing 1st generation protocols 2nd generation protocols
EQ: 1.5M PROH / 0.3 M suc
Dilution:
PROH (1.0; 0.5; 0.2 M) /0.3 Msuc.
Fabbri et al, 2001
Preg
Impl
EQ: 1.5M PROH / 0.1 M suc
Dilution:
PROH (1.0; 0.5; 0.2 M) /0.2Msuc
Lasalle et al, 1985
Ref.
Al-Hasani et al., 1987
Gook et al., 1994
Tucker et al., 1998
Porcu et. al, 2000
Borini et.al, 2004
Surv. (%)
33.9
41.0
24.0
46.8
37*
Nº ovo
159
134
311
1640
737
1st generation protocols
Pregnancy 25.4% Implantation 16.4%

17. Slow Freezing 2nd Generation Protocols 3rd Generation Protocols
Replacement of Na+ for Choline
EQ: 1.5M PROH HTF+ o PBS+/0.3M suc
(26,3)
(37,5)
2nd Generation Protocols
3rd Generation Protocols
EQ: 1.5M PROH / 0.3 M suc
Dilution:
PROH (1,0; 0.5; 0,2 M) /0.2 M suc.
Fabbri et al, 2001 %
Bianchi, et.al., 2007

19. Eight centers
Cycles involving MII cryopreservation
between January 2006 and April 2008
“Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is
reduced compared with fresh cycles”.

20. Slow Freezing vs. Vitrification
Human oocyte cryopreservation:comparison between slow and ultrarapid methods.
Paffoni et. Al. RBM online 2008
Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and Vitrification.
Cao et. al .Fertil Steril
Slow freezing or vitrification of oocytes: Their effects on survival and meiotic spindles, and the time schedule for clinical practice.
Chen et.al. Taiwan J Obstet Gynecol
Human oocyte cryopreservation: comparison between slow and ultrarapid methods.
Fadini et. al. RBM on line 2009.
Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification.
Smith et.al. Fertil. Steril
Oocyte vitrification is associated with a better outcome than the slow-freezing method.

21. Vitrified vs. Fresh oocytes
Delivery rate using cryopreserved oocytes is comparable to conventional in vitro fertilization using fresh oocytes: potential fertility preservation for female cancer patients
Griffo et al. 2009
Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.
Cobo et.al. Fertil. Steril
Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking
Nagy et.al. Fertil. Steril
Embryo development of fresh ‘versus’ vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study.
Rienzi et.al. Human Reprod
Embryo development and gestation using fresh and vitrified oocytes
Almodin et.al. Human Reprod
Use of cryo-banked oocytes in an ovum donation programme: a prospective,
randomized, controlled, clinical.
Cobo et.al. Human Reprod
Oocyte vitrification does not impair the outcome as compared with fresh oocytes

22. To identify randomized controlled trials that assessed the efficiency of oocyte vitrification compared to SF cryopreservation methods and fresh cycles (PRISMA statement principles).
Oocyte vitrification in ART

23. Clinical application of oocyte vitrification:
a systematic review and meta-analysis
of randomized controlled trials
2,799 records
16 assessed for eligibility
5 included
Uni-centric studies (4282 vitrified oocytes, 3524 fresh oocytes and 361 slow-frozen oocytes) and Only MII oocytes.
Three vitrification protocols using different cryo-protectant combinations and devices were analyzed: Cryotip® (349 oocytes), Cryotop® (2645 oocytes) and Cryoleaf®
Cobo, Díaz, Fertil. Steril. 2011

24. Clinical application of oocyte vitrification:
a systematic review and meta-analysis
of randomized controlled trials
Oocyte vitrification in ART

25. Clinical application of oocyte vitrification:
a systematic review and meta-analysis
of randomized controlled trials
There were no differences regarding the fertilization rate when vitrified oocytes were compared to fresh ones (OR 1.02, 95% CI [0.91, 1.13]; heterogeneity: p=0.17, I2=44%, fixed effects model) but higher fertilization rates were observed when vitrification was compared to SF (OR 1.50, 95% CI [1.07, 2.11]; heterogeneity: p=0.56, I2=0%, fixed effects model) (Figure 3).
Coclusions
This review supports the hypothesis that the potential of fertilization, embryogenesis and pregnancy from oocytes derived from vitrification/warming cycles is not significantly different to that of fresh oocytes. It also suggests that oocytes coming from vitrification/warming cycles could result in better survival and fertilization rates than those coming from SF/thawing cycles.
Vitrification also resulted in a higher rate top-quality embryo (22.4% vs. 8.0%, OR 3.32, 95% CI [ ], p<0.01) and embryo cleavage rate (day 2: 64.6% vs. 47.7%, OR 2.00, 95% CI [ ], p<0.01; day 3: 53.0% vs. 33.3%, OR 2.25, 95% CI [ ], p<0.05) as compared to SF.
In conclusion, based on the evidence provided by the randomized studies available, vitrification appears to be an efficient method to cryopreserve oocytes. However, more large-scale controlled trials, aimed at evaluating clinical outcomes after oocyte vitrification, will be required to further strengthen this conclusion.
Oocyte vitrification in ART

26. To compare the outcome of vitrified-banked oocytes to the gold standard procedure employing fresh oocytes.
Primary end point. Ongoing pregnancy rate (OPR) per intention to treat (ITT) population.
Secondary outcomes.
clinical pregnancy rate
implantation rate
fertilization rate
embryo quality

27. Results
Cobo et al Hum Reprod, 2010

28. Results
0.921
( )
Superiority of the OPR of the fresh group was not established
Non-inferiority of vitrified group was shown with a margin of 0.667
(above the non-inferiority limit of 0.660)
Cobo et al Hum Reprod, 2010

29. Donors base line characteristics Recipients base line characteristics
4 years-experience of an ovum donation program using cryo-banked oocytes
Donors base line characteristics
Recipients base line characteristics
95% CI
Number of donors or recipients
1187 NA
1602
Nº donation cycles
1856
Mean donations/donor or recipient
1.6
1.2
Age (years)
27.1
41.1
BMI (Kg/m2)
22.5
23.7
Days of stimulation/endometrial preparation
12.2
15.4
rFSH dose (IU)
1761
E2 on day of hCG (pg/ml)
2726.6
329.5
P4 (ng/ml)
0.9
0.4
MII oocytes retrieved or received/donation cycle (mean)
22741 (12.2)
Survival rate (%)
20669 (90.1)
Mean oocyte survival /donation cycle
20669 (11.1)
Cobo et al ESHRE 2011.
4 years-experience of an ovum donation program using cryo-banked oocytes.

30. Results IVF outcome 95%CI Mean MII oocytes injected/donation cycle
20669 (11.1 )
Fertilization rate
15163(73.4)
Cleavage day-2
14346 (94.6)
Cleavage day-3
12028 (83.8)
Top quality day- 2 /cleaved embryo
5998 (41.8)
Top quality day- 2 /injected oocyte
5998 (29.0)
Top quality day- 3/cleaved embryo
6323 (52.6)
Top quality day- 3/injected oocyte
6323 (30.5)
Blastocyst rate/embryo subjected to extended culture
4779 (58.9)
Good quality blastocyst
1773 (37.8)
Top quality blastocyst
1747 (36.5)
Cobo et al ESHRE 2011.
4 years-experience of an ovum donation program using cryo-banked oocytes.

31. Results Clinical outcome 95% CI Number of embryo transfers/donation
1607 (86.6)
Number of day-3 transfers
766 (47.7)
Number of blastocyst transfers
841 (52.3)
Number of embryos replaced (Mean ± SD)
3037 (1.6 ± 0.7)
Number of embryo cryopreservation cycles/donation cycle
1181 (63.6)
Mean number of re-vitrified embryos (Mean ± SD)
3491 (1.9 ± 2.0)
Implantation rate
1274 (41.9)
Implantation rate/day-3 embryo transfer
545/1447 (37.7)
Implantation rate/blastocyst embryo transfer
729/1590 (45.8)
Ongoing pregnancy /cycle
767 (41.3)
Ongoing pregnancy /embryo transfer
767 (47.7)
Ongoing pregnancy / day-3 embryo transfer
348 (45.4)
Ongoing pregnancy /blastocyst embryo transfer
419 (49.8)
Number of Cryo-transfers
423/1181 (35.8)
Mean number of embryos replaced
2.2 ± 0.7
Ongoing pregnancy rate
178/423 (42.1)
Number of babies born (deliveries) “fresh ET”
604 (465)
Number of babies born (deliveries) “Cryo-transfers”
97(75)
1274 (41.9)
P<0.05
767 (41.3)
767 (47.7) NS
178/423 (42.1)
Cobo et al ESHRE 2011.
4 years-experience of an ovum donation program using cryo-banked oocytes.

32. Results
COPR according to the number of oocytes consumed. Kaplan-Meier plotting
Oocytes consumed
b. Day-3 and Blastocyst transfer
Day-3
Blastocyst
Day-3 cesnsored
Blastocyst censored
Oocytes consumed
a. Overall data
Survival fuction
Censored
35 oocytes=
COPR 97.6% *
*P<0.05 (Long rank Breslow and Tarone-Ware tests)
20 oocytes=
COPR 76.1%
13 oocytes=
COPR 69.9%
16 oocytes=
COPR 70.7%
15 oocytes=
COPR 58.8%
13 oocytes=
COPR 50%
Cobo et al ESHRE 2011.
4 years-experience of an ovum donation program using cryo-banked oocytes.

33. “The excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg-cryobank and makes it likely that this approach becomes the future standard of care”.

34. Oocyte vitrification in infertile patients
N (mean SD)
Rank
Nº patients
308
Age
35.1 ± 4.3
26-44
Nº aspirated oocytes
3534 (14.8 ± 7.9)
1-46
Nº vitrified oocytes
3104 (10.7 ± 5.0)
1-25
Nº warmed oocytes
2986
1-18
Survival N(%)
85.8
Per transfer
Per cycle
+BHGC
CPR
OPR IR
Oocyte vitrification in ART

35. Oocyte vitrification in LR patients. Accumulation of oocytes
COPR depending on the number of oocytes used, including fresh and warmed (accumulated)
Kaplan Meier survival Curves
Overall outcome
<39 years old
≥39 years old
Cobo et al., submitted
Oocyte vitrification in ART

36. P=0,006
Oocyte vitrification in ART

37. Absolute number Mean (95%CI) % (95%CI)*
High, consistent and predictable pregnancy rates after oocyte vitrification: a prospective longitudinal multicentric cohort study
Rienzi L. 1, Cobo A. 2, Paffoni A. 3 et al., 1GENERA Centre for Reproductive Medicine, Rome, Italy; 2IVI Valencia, Spain; 3Fondazione Ca' Granda Ospedale Maggiore Policlinico, Infertility Unit, Milan, Italy
 Characteristics
Absolute number
Mean (95%CI)
% (95%CI)*
Cycles
486
Warmed oocytes
2721
5.62 ( )
Survived oocytes
2304
4.78 ( )
84.7 ( )
Inseminated oocytes
2182
4.53 ( )
94.7 ( )
Fertilized oocytes
1642
3.41 ( )
75.2 ( )
Top quality embryos
796
1.89 ( )
48.1 ( )
Embryos transferred
929
1.91 ( )
Embryos cryopreserved
184
0.38 ( )
Delivery rate per cycle
per transfer
128/488
128/450
26.3( )
29.4 ( )
Newborn
147
15.8 ( )
Rienzi et al; ESHRE 2011

38. Decisional making model based on recursive partitioning analysis (per patient basis)% N
0.000
71.6
322
1.000
28.4
128
Total
100.0
450
0 = delivery not obtained
1 = delivery obtained
Number of vitrified MII
≤ 8 MII
> 8 MII
By recursive partitioning analysis it can be estimated that more than 8 oocytes vitrified are required to improve the outcome (22.6 vs 46.4% DR, respectively). % n
0.000
77.4
263
1.000
22.6 77
Total
75.6
340 % n
0.000
53.6 59
1.000
46.4 51
Total
24.4
110
Rienzi et al; ESHRE 2011
Reproductive clock
≤ 38 years
> 38 years
≤ 3 days
> 3 days % n
0.000
72.5
168
1.000
27.5 63
Total
50.9
229 % n
0.000
87.4 97
1.000
12.6 14
Total
24.7
111 % n
0.000
59.3 48
1.000
40.7 33
Total
18.0 81 % n
0.000
37.9 11
1.000
62.1 18
Total
6.4 29
Fig.1 Decisional making model based on recursive partitioning analysis (per patient basis)

39. Decisional making model based on recursive partitioning analysis (per patient basis)
≤ 8 MII
> 8 MII
Female age
Day of transfer
≤ 38 years
> 38 years
≤ 3 days
Blastocyst % n
0.000
72.5
168
1.000
27.5 63
Total
50.9
229 % n
0.000
87.4 97
1.000
12.6 14
Total
24.7
111 % n
0.000
59.3 48
1.000
40.7 33
Total
18.0 81 % n
0.000
37.9 11
1.000
62.1 18
Total
6.4 29
Rienzi et al; ESHRE 2011

40. Perinatal outcome of child born after oocyte vitrification
Perinatal outcome of child born after oocyte vitrification. Paired cohorts study.
Perinatal outcome
Vitrified
Fresh
Nº Deliveries
160
262
Nº New born
212
315
Perinatal mortality
3 (1.4)
Mean maternal age at delivering
39.2 ± 4.9
39.6 ± 5.3
Mean gestational age (weeks)
37.4 ± 2.5
38.0 ± 2.3
No. of deliveries at <37 weeks (%)
37 (23.1)
47 (17.9)
No. of deliveries at <34 weeks (%)
9 (5.6)
12 (4.6)
Birth weight (mean ± SD)
2,718 ± 0.668
2,896 ± 0.658
No. of LBW (%) <2500gr.
65 (17.4)
75 (23.8)
No. of VLBW (%) <1500 gr.
14 (1.9)
13 (4.1)
Median Apgar score at 1 min.
8.9 ± 0.6
8.9 ± 0.8
Median Apgar score at 5 min.
9.3 ± 0.6
9.6 ± 0.3
Sex of new born. Female.
113 (53.7)
172 (54.6)
Sex of new born. Male.
99 (46.3.)
143 (45.4)
Incidence of major congenital anomalies
4(1.8)
2 (0.6)
This slide shows the perinatal outcome of 147 new born after OV NS
Cobo, Serna. Hum Reprod 2011

41. Conclusions
Vitrified oocytes are currently offering excellent clinical outcomes either for ovum donation programs or for infertile patients.
The safety of the strategy is being confirmed by the obstetric and perinatal outcome assessment.

42. Making possible the establishment and well functioning of this program