1. Embryo Cryopreservation Slow freezing vs. Vitrification
BASAK BALABAN BSc.
American Hospital of Istanbul
Assisted Reproduction Unit
AMERICAN
HOSPITAL

2. Cryopreservation Slow freezing of human oocytes Vitrification
of bovine
oocytes
İn small
drops
Slow
freezing
of human
embryos
Slow
freezing
of domestic
animal
embryos
Vitrification of
mouse oocytes
OPS
Vitrification
of bovive
oocytes and
embryos
Vitrification of
bovine
blastocysts
Slow
freezing
of mouse
embryos
Vitrification
of mouse
embryos
1972
1973/74
1983
1985
1986
1989
1993
1996
1997/98

3. Cryopreservation Techniques
Slow conventional freezing
Traditional Vitrification
Ultrarapid vitrification

4. From Kasai et al. RBM Online 2004
Vitrification solutions
DMSO+Acetamide+ propylene glycol
Ethylene glycol+ Ficoll+Sucrose
Ethylene glycol+ DMSO
Ethylene glycol+ glycerol
Slow Freezing solutions
DMSO /1-2 PROH + Sucrose
Glycerol+
Sucrose
Base medium +
Cryoprotectant
From Kasai et al. RBM Online 2004

5. Differences of slow freezing and vitrification
high levels of cryoprotectants
very fast cooling rates
(~20,000oC/min)
fast cooling rates result in solidification of solution into glass-like structure (no crystallization)
takes seconds
Slow-freezing
low levels of cryoprotectants
slow controlled rates of cooling (0.3oC/min)
slow dehydration to minimize ice-crystal formation
takes hours

6. Vitrification & Slow-cooling
Control of solute penetration
Yes No
Control of dehydration rate
Duration out of the incubator
10min.
3 hrs.
Prolonged temperature shock
Fracture of ZP
Possible
Capture by growing ice crystals
Equipment and running costs
Inexpensive
Expensive
Kuleshova et al. F&S 2002

7. Vitrification of Human Embryos
Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos.
Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M.
Hum Reprod :2874-9
Implantation rate of 17% for best embryos
Implantation rate of 9.8% for all embryos

8. Problems Associated with Traditional Vitrification Procedures
High levels of cryoprotectants are toxic to embryos
(4-10 M compared to M)
Procedure must be performed at 4oC
Technically demanding
Advantages of Ultra-Rapid Vitrification
Increases in cooling rates alleviates toxicity of high levels of cryoprotectants
Can be performed at room temperature or 37oC

9. Variables in Vitrification
Cooling &warming rates:Ideal vitrification protocol must pass rapidly through the critical temperature zone of 15 to – 5ºC to decrease chilling injuries. High warming rates by directly plunging cells into the warming solution is suggested (-196 to 37ºC)

10. Variables in Vitrification
Concentration of the cryoprotectant:
To achieve high cooling rates requires the use of high concentrations of the cryoprotectant solution which depresses ice crystal formation, so a critical concentration is required but in some cryoprotectants, this minimal concentration (Cv) can lead to either osmotic or chemical toxicity

11. Variables in Vitrification
Sample size and carrier systems
Sample size should be minimized to reduce the duration of vapour coat and to increase the cooling rate, minimizing the volume of the vitrification solution as much as possible is necessary to facilitate vitrification by higher cooling rates
To minimize the volume of the vitrification solution special carriers are used for vitrification process
** Open pulled straws
** Flexipet- denuding pipette
** Microdrops
** Electron-microscopic copper grids
** Hemistraw system
** small nylon coils or nylon mash
** Cryotop,cryotip
** Cryoloop

12. Carriers for vitrification
Cryotop
Cryotip
Cryotip
Kuwayama et al.,RBM Online 2005

13. Cryoloop Hampton Research, Laguna Niguel, CA, USA Nylon loop
(20µm wide; mm in diameter)
Thin film of cryoprotectant solution by surface tension
Embryos are placed by pipette
Hampton Research, Laguna Niguel, CA, USA

14. Advantages of Cryoloop Vitrification
Lack of thermoinsulating layer maximizes heat transfer (>20,000oC/min)
Easy manipulations
Constant visualization of embryo
Cryoloop stored within cryovial
Procedure is performed at 37oC

15. Concerns with Regards to Sterility of Liquid N2 storage
Tedder et al., 1995
Hepatitis B transmission
Bielanski et al., 2000
Viral contamination
Bielanski et al., 2003
Microbial contamination, no viral cross contamination
Kyuma et al., 2003
No microbial or viral cross contamination

16. Clinical Results on Ultra-Rapid Vitrification of
Human Cleavage Stage Embryos
Author
Carrier
Cryosurvival(%)
CPR%
IR%
Mukaida 1998
Conventional
straws
71.8
15.5
4.9
Saito 2000
16.0
Danasouri 2001
OPS
49.3
30.5
10.4
Kuwayama 2005
CRYOTIP
98.0 27
Raju 2005
Locally prepared cryovial
95.3
35.0
14.9
Desai 2007
CRYOLOOP
85.0
44.0
20.0
Balaban 2008
92.2
29.7

17. Cryopreservation of human embryos by vitrification or
slow freezing: A systematic review and meta-analysis
Pubmed search: 873, only 4 included!!,
Primary outcome: Postthaw survival rate,
Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR
Loutradi et al., F&S 2008

18. Heterogenity of the studies included
Loutradi et al., F&S 2008

19. Pooled data on cleavage, blastocyst
development &hatching, CPR, IR, and LBR
were NOT feasible
**Valojerdi et al.,JARG 2009 retrospective study: higher CPR/IR,
Kolibianakis et al.,Curr.Op.Obst.Gyn review no dif. İn CPR

20. Cryopreservation Results with Slow Freezing
Urman&Balaban et al. F&S 2007

21. CBS High security straw
Heat
sealed
Heat
sealed
Filling
noozle
part
Connection
part with
aspiration
system
Identification plug
Hydrophobic
Safety plug e
Seeding point
Conventional straw e
Seeding point
Cotton plug
connected to
the aspiration
system
Plastic plug
Air bubble
Medium

22. More Successful Results in Slow Freezing Cycles with HSS
Balaban et al., F&S 2007

23. First Trials of ultra-rapid vitrification on human day 3 cleavage stage embryos
GIII/G5 series (Vitrolife, Sweden) sequential culture system was used for fresh embryo culture
Day 3 embryos of good quality that were donated for research with concent were used
Ethylene glycol and 1,2-propanediol was used as the cryoprotectant. All procedures were performed at 37ºC (Lane M and Gardner DK (2001) Vitrification of mouse oocytes using a nylon loop. Mol. Reprod. Devel. 58: ).
Cryoloop was used as the carrier system
American Hospital

24. Cryosurvival rates of vitrified day 3 cleavage stage embryos
No.of day 3 embs. Vitrified (Total)
165
No.of 8c vitrified (%)
92(55.7)
No.of warmed embs.
No.of lost embs.during warmed (%)
3(1.8) (3-8c)
Total Cryosurvival (%)
152 (93.8) (152/162)
Cryosurvival of 8c top quality embs. (%)
89 (100) (89/89)
Embs. with 100% intact blastomeres after warming
104 (68.4)
8c with all blast.intact (%)
68 (76.4) (68/89)
American Hospital Data 2006

25. Blastocyst formation rates of vitrified day 3 cleavage stage embryos
Parameter
Total
Day 5
Day 6
Blastocysts from 100% survived embs.(%)
66(63.4)
Blastocysts (%)(Total)
84(55.2)
76(50.0)
8(5.2)
Good quality blasts.(≤3AA)(%)
39(46.4)
38(50.0)
1(12.5)
Blast from 8c (%)
55(59.7) -
Hatching blastocysts(%)(Total)
26(30.9)
20(26.3)
2(3.5)
H.Blasts. from 8C embs.(%)
18(32.7)
17(30.9)
1(1.8)
American Hospital Data 2006

26. A randomized controlled study on human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation
To compare the blastocyst development between embryos that were cryopreserved by either slow freezing or vitrification on day 3
To determine whether slow freezing and vitrification have different effects on human cleavage stage embryo metabolism
Human Reproduction 2008
Balaban B¹, Urman B¹, Ata B, Isiklar A¹, Larman MG², Hamilton B² and Gardner DK²

27. Table-1- Embryo development
Slow Freezing
Vitrification
95%CI of difference
P value
Cryosurvival (%)
206/232
(88.7)
222/234
(94.8)
+1 – 11%
0.02
Embs. with 100% blastomere survival (%)
106/206
(51.4)
173/222
(77.9)
+18 - %35
0.00
Blastocyst formation (%)
102/206
(49.5)
134/222
(60.3)
+1 – 20%
Blasts.≥ 3AA (%)
43/102
(42.1)
70/134
(52.2)
- 2.7 – 22.8%
0.12
Hatching blastocysts (%)
22/102
(21.5)
42/134
(31.3)
- 1.4 – 20.9%
0.09
Balaban & Gardner et al., HR 2008

28. Analysis of Embryo Metabolism
Non-invasive fluorometric assays based on the reduced pyridine nucleotides NADH or NADPH
Fluorescence
Pyruvate (mM)
Pyruvate + NADH + H Lactate + NAD+
Balaban & Gardner et al., HR 2008

29. Table 2- Metabolic Analysis5 10 15 20 25
Slow Freezing
Vitrification *
Pyruvate uptake
(pmol/embryo/h)
Balaban & Gardner et al., HR 2008

30. Table-3- Clinical Outcome with Vitrification
No.of patient’s warmed 73
No.of embryos vitrified(Mean)
314 (4.2)
No.of embryos warmed
241(3.3)
No.of warmed embs. with 8C
85(35.2)
No.of warmed embs. with >8C
49(20.3)
No.of warmed embs with <8C
107(44.3)
Cryosurvival (%)
222 (92.1)
No.of embs. with 100%survival
160(72.1)
No.of morula on day 4 (day of ET)
138(62.1)
Balaban & Gardner et al., HR 2008

31. Table-3- Clinical Outcome with Vitrification
No.of ET (Mean)
168(2.3)
No.of morula ET (%)
146(86.9)
CPR(%)
36(49.3)
IR(%)
50(29.7)
OPR(%)
33(45.2)
MPR(%)
13(36.1)(1-triplet, 12 twins)
Abortion(%)
3(8.3)
Deliveries(%)
8(24.2:8/33)(2-twins, 6 singleton)
Balaban & Gardner et al., HR 2008

32. Conclusion
Human embryo metabolism is significantly depressed following cryopreservation by slow freezing
In contrast, embryos that were vitrified had a metabolism activity almost twice that of the frozen embryos
Subsequent embryo development appeared to be elevated following vitrification
These data therefore support the hypothesis that vitrification is associated with less cellular trauma when slow freezing and should be considered as the primary method of human embryo cryopreservation
Balaban & Gardner et al., HR 2008

33. Clinical Outcome with Embryo Vitrification
No.of patient’s warmed
339
No.of embryos vitrified (Mean)
1458 (4.3)
No.of embryos warmed (Mean)
1051 (3.1)
No.of warmed embs. with 8C(%)
354 (33.6)
No.of thawed embs. with >8C(%)
210 (19.9)
No.of thawed embs with <8C (%)
487 (46.3)
Cryosurvival (%)
955 (90.8)
No.of embs. with 100%survival(%)
640 (67.0)
No.of morula on day 4 (day of ET)(%)
546 (57.1)
American Hospital of Istanbul Data:

34. Clinical Outcome with Embryo Vitrification
No.of ET (Mean)
780 (2.3)
No.of morula ET (%)
553 (70.8)
CPR(%)
147 (43.3)
IR(%)
203 (26.0)
OPR(%)
133 (39.2)
MPR(%)
48 (32.6) (4-triplet, 44-twins)
Abortion(%)
14 (9.5)
Deliveries(%)
52 (39.0) (19-twins, 33-singleton)
American Hospital of Istanbul Data:

35. First trials with the Closed System-RAPID I
CARRIER USED
RAPID-I
CRYOLOOP
No.of embryos vitrified 64 66
No.of embryos warmed
No.of warmed embs. with 8C
24 (37.5)
24 (34.8)
No.of warmed embs. with >8C
11 (17.1)
12 (18.1)
No.of warmed embs with <8C
29 (45.3)
30 (45.4)
Cryosurvival (%)*
52 (81.2)
60 (90.9)
No.of embs. with 100%survival
28 (53.8)
39 (65.0)
No. of blastocysts (day 5)*
23 (44.2)
31 (51.6)
Good quality blasts. (≤3AA)*
10 (43.4)
13 (42.0)
Hatching blasts (day 5)*
7 (30.4)
9 (29.0)
*NS, Fisher’s exact test
Larman & Balaban Oral presentation ASRM 2009

36. Neonatal Outcome of Vitrified Cleavage Stage Embryos
Rama Raju et al. F&S 2009
No.sig. dif. for neonatal parameters:
Mean gestational age, birth weights for singleton & MPR,
PR induced complications,
Incidence of birth defects ( major & minor malformations)

37. Necessity of blastocyst vitrification ?
Increasing application of BT especially for some selected cases results with supernumerary blastocysts for freezing to increase cumulative pregnancy rates per oocyte retrieval
A reliable procedure for the cryopreservation of blastocysts is needed, because after fresh ET, only small number of supernumerary blastocysts are likely to be available for cryopreservation
Based on the published cochrane data (2008), vitrification appears to result in significantly higher survival and pregnancy rates

38. Blastocyst vitrification
First pregnancy after human blastocyst vitrification was achieved by Yokota et al., HR 2000
EG- based vitrification solutions are widely used as it has a low toxicity with rapid diffusion into the cell through ZP and cellular membrane
1st. Vit.sol. EG+DMSO
2nd. EG+DMSO+Ficoll+ Sucrose,
Warming: Decreasing concentrations of Sucrose sol. are preferred
Concentration of cryoprotectants are decreased to 7.5% from 25% over the years of experience

39. > blasts. vitrified
Youssry et al.,RBM Online 2008

40. Blastocyst vitrification
Is it the most effective and successful method to cryopreserve embryos at blastocyst stage???

41. Faster re-expansion after thawing with vitrification method
Stehlik et al.,RBM Online 2005

42. Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBR
were NOT feasible

43. Artifical shrinkage by microneedle Artifical shrinkage by laser
Large blatocoele of more developed blastocysts may disturb
the efficacy of vitrification due to
inappropriate Dehydration and permeation of cryoprotectant,
which may cause ice crystal formation in the rapid cooling and
warming steps of vitrification. Ice crystal formation can be a
voided by reducing fluid content of the blastocoele of more
developed blastocysts
Mukaida et al., HR 2006
1st.clin.appl- Vanderzwalmen et al.,
HR 2002,3,4

44. Takahashi et al.,F&S 2005
Liebermann et al., F&S 2006 also reported no adverse effect

45. Vitrification results with higher cryosurvival rates
for biopsied human embryos
Poor cryosurvival rates (approx. 30%) and clinical outcome reported after conventional slow freezing
of biopsied cleavage stage embryos ( Joris et al., HR 1999, Magli
et al., HR 1999)
Escriba et al., F&S 2008 had shown similar cumulative OPR/OPU for PDG
and non-PGD group with blastocyst vitrification
Zheng et al.,HR 2005

46. RESULTS
Vitrification as a cryopreservation method has many primary advantages and benefits based on the published data
Vitrification protocols are now starting to enter the mainstream of human ART
The reports of successfully completed pregnancies following vitrification are encouraging for further research
More studies on vitrification and warming procedures are needed to develop more efficient and optimal vitrification methods

47. Concerns regarding Vitrification
LN2 still remains to be a potential source of contamination since the technique is based on direct contact between the vitrification solution containing cryoprotectant agents and LN2. So from a clinical point of view:
Is there a need to sterilize LN2? How is it possible to maintain its sterility
Cross contamination with viruses?? ( No publication since 1985, about 450 publications)
Closed systems should be used in clinical human IVF in the future to avoid this concern.(Like CBS HS vitrification straws, Cryotip……) New clinical trials with safer closed systems should be applied
Low toxicity vitrification solutions must be designed in the future
Genetical structure of the vitrified cell?? Chromosal abnormalities, gene expressions More studies are needed to prove the safety of the technique

50. MAIN TOPICS
I. Basic Cryobiology and Cryopreservation Overview 1. Principles of cryobiology 2. The evolution of cryopreservation techniques: Slow-freeze, vitrification, freeze drying and beyond…. 3. Cryopreservation for wildlife conservation: Lessons for human IVF 4. What can we learn from the livestock industry? 5. The future of cryopreservation in assisted reproduction II. Oocyte Cryopreservation 1. What, when, and how to cryopreserve oocytes? Lessons from oocyte physiology 2. Cryopreservation of oocytes for social or medical reasons: Is there a difference? 3. The rise of the new banking system? Cryopreservation of donor oocytes 4. Recovery of high quality oocytes for cryopreservation: Optimising clinical protocols 5. To vitrify or not to vitrify? Molecular markers to assess cryopreservation techniques 6. Safety of oocyte cryopreservation: Outcomes and long-term follow-up III. Sperm Cryopreservation 1. Principles of sperm cryopreservation: Cryopreservation methods and effectiveness 2. Frozen versus fresh sperm: Are the outcomes really any different? 3. Cryopreservation of testicular and epididymal sperm: Outcomes, safety and long term follow-up 4. Cryopreservation of sperm for oncology patients: Pre- and post-treatment quality, utilization, success and safety 5. Molecular assessment of frozen-thawed sperm: Cytogenetics, DNA fragmentation, methylation and more… 6. Safety of sperm cryopreservation: Outcomes and long-term follow-up? IV. Embryo Cryopreservation 1. Assessing frozen embryo survival after thawing: Revisiting the 50% rule 2. Embryological criteria for successful cryopreservation: What stage and grade of embryos should be cryopreserved? 3. Slow-freezing versus vitrification: Are molecular markers or success rates driving cryopreservation techniques? 4. Optimizing frozen-thaw embryo cycles: Clinical aspects 5. A critical assessment of assisted hatching and lysed cell removal on frozen-thawed embryos 6. Safety of embryo cryopreservation: Outcomes and long-term follow-up V. Cryopreservation of Ovarian Tissue 1. Cryopreservation of whole ovaries or ovarian tissue: Clinical indications and outcomes 2. Collection, processing and storage of ovarian tissue: A review of best practice 3. Ovarian tissue transplantation: What constitutes success? 4. In Vitro Maturation from cryopreserved ovarian tissue: As good as fresh? VI. Regulation, Law and Ethics of Cryopreservation 1. Regulatory aspects of cryopreservation around the world: Global village or dystopia? 2. Posthumous collection and storage of gametes: Do the living have any rights? 3. Whose embryo is it anyway? Storage terms, future dispositions, effective consent, joint ownership and other dilemmas 4. An embryologist’s dream: The perfect consent form VII. Training and Quality Management Aspects of Cryopreservation 1. Optimizing cryopreservation techniques – What’s critical, what’s not? 2. Keeping a lid on cryopreservation: Key performance indicators in the IVF lab 3. Open forum for delegates to share their experiences (with panel discussion)

51. Material & Methods Clinical protocols
Hipothalamopituitary axis downregulation with GnRH analogues started on midluteal phase and discontinued after 10 days of estradiol treatment
Endometrium is prepared using incremental doses of Estradiol valerate (2- 8 mg)
Progesteron vaginal gel (%8) is started for luteal supplementation
Balaban HR 2006, Urman 2007, Balaban 2007

52. No difference in Neonatal parameters for fresh&vitrified embryos
Rama Raju et al.,
F&S 2009

53. No difference in Neonatal parameters for fresh&vitrified embryos
Rama Raju et al.,
F&S 2009

54. Slow Freezing & Vitrification of Cleavage stage embryos
No sig. differences between 2 carriers:
Cryotop, Cryotip,
V.Sol: EG+DMSO+ Sucrose-Vit.kit(Ir.S)
Kuwayama et al.,RBM Online 2005

55. Similar OPR after ET of biopsied vitrified blastocysts
Escriba et al., F&S 2008
Retrospective study,
Carrier: 0.25ml. French straws

56. Vitrification of blastocysts after PGD yields similar cumulative OPR
Escriba et al., F&S 2008