1. Frozen Dreams Vitrification, as becoming the key procedure in IVF Lab: Is 100% survival a reality?
Laura Rienzi
Senior Clinical Embryologist
GENERA Centres for Reproductive Medicine
Rome, Marostica, Umbertide, Napoli, Italy

2. Agenda
1. Review of different approaches utilized in the IVF Lab. for oocyte and embryo cryopreservation
2. The Revolution of Vitrification Approach
- New possibilities in in the Lab
- New clinical indications for cryopreservation
3. What do we expect for the future?
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3. Cryopreservation is essential in ART
Cryopreservation of gametes and embryos is an essential aspect of Assisted Reproductive Technologies (ART). Its widespread application has opened new clinical perspectives, allowing to increase efficacy and safety of IVF treatments. The proportion of cryopreserved embryo transfers compared with fresh cycles is growing in Europe. It has been estimated that overall cryopreservation contributed to 32% of the transfers in 2011 (compared to 28% in 2010). (Kupka et al., 2016). The observed differences are mainly due to specific legal situations that in some nations impose restricted embryo transfer policies aimed at reducing the incidence of multiple pregnancies (Coetsier and Dhont, 1998).
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4. Review of different approaches for oocyte and embryo cryopreservation

5. History of cryopreservation in ART
Slow freezing of domestic animal embryos
Slow freezing of human oocytes
Slow freezing of human embryos
Ultrarapid Vitrification with EM grids
OPS Ultrarapid Vitrification
Vitrification of mouse oocytes
Slow freezing of mouse embryos
Vitrification of mouse embryos
CryoLoop CryoTip Ultrarapid Vitrification
Vitrification of bovine blastocysts
1935
1948
1972
1973/1974
1983
1985
1989
1993
1996
1997
1999
“It is more difficult to destroy a prejudice than an atom”
Albert Einstein

6. PRONUCLEAR, CLEAVAGE STAGE and BLASTOCYST SLOW FREEZING HISTORY
DMSO
Slow-freezing
9% Glycerol +
0.2M sucrose
Slow freezing
1.5M PrOH +
0.1M sucrose
Slow freezing
1.5M PrOH +
0.2M/0.3M sucrose
1983
Trounson and Mohr
Cleavage-stage
First delivery
1984
Zeilmaker et al.
1985
Cohen et al.
Blastocyst
1995
Kaufman et al.
Blastocyst 83%
2002
Behr et al.
Blastocyst 98%
2003
Gardner et al.
Blastocyst 69%
2009
El-Toukhy et al.
Biopsied blastocyst 88%
1985
Lasalle et al.
1986
Testart et al.
Cleavage stage 87.5%
1990
Hartshorne et al.
Cleavage stage 80%
1991
Demoulin et al.
Pronuclear stage 54%
Cleavage stage 47%
1993
Veek et al
2.000 pronuclear stage 68%,
1996
Al Hasani et al
Pronuclear stage 77%
1998
Mandelbaum et al
Cleavage stage 73%
1999
Damario et al.,
Pronuclear stage 90%
Joris et al.
Biopsied cleavage stage
2001
Tiitinen et al.
Cleavage stage 76.6% SET
2002
Rienzi et al.
Cleavage stage
Blastomere removal
2007
Edgar et al.
Cleavage stage SET
2003
Jericho et al
Biopsied cleavage-stage
2009
Edgar et al.
Cleavage stage 92.6%
2011
Wood et al.
Cleavage stage 90%

7. The Vitrification Approach
Vitrification is a pseudo second order phase transition (IUPAC Compendium of Chemical Terminology, 1997) converting a material into a glassy amorphous solid that is free from crystalline structure.
Cooling/warming rates ×
Viscosity
Probability of vitrification ̴
Volume of the sample
Types of cells

8. Biophysical Aspects: Binary Phase Diagram
10-4 poise
CPs
Liquid phase T°
Equilibrium
0°C
freezing
Thermal hysteresis
Heterogenous
curve (Tm)
nucleation area
curve (Th)
Homogenous
nucleation
- 40°C
Cooling rate
Ice phase
-100°C
Glass phase
No crystal
1013 poise
curve (Tg)
transition
-120°C
Glass
Concentration of solute

9. PRONUCLEAR, CLEAVAGE STAGE and BLASTOCYST VITRIFICATION HISTORYEG
Vitrification
DMSO
Vitrification
15% EG, 15% DMSO +
M sucrose
Vitrification
16% PROH, 16% EG M sucrose
1998
Mukaida et al.
Cleavage stage 79%
2001
El-Danasouri and Selman
Cleavage stage 49.3%
2002
Cho et al.
Blastocyst
2005
Rama Raju et al.
Cleavage stage 95.3%
1999
Hsieh et al.
Cleavage stage 62.5%
2000
Yokota et al.
Blastocyst
2003
Venderzwallen et al.
2005
Kuwayama et al.
Cleavage stage 98%
2006
Mukaiada at al.
Artificial shrinkage of blastocyst
2007
Desai et al.
Cleavage stage 85%
2009
Ebner et al.
Closed system
2010
Kahraman and Candan
Day 4 embryos 85.9%
Biopsied day %
2015
Decrock et al.
Cleavage stage vitrification
RCT
2008
Balaban et al.
RCT
Cleavage stage 94.8%

10. Minimum Volume – Open System Approach
Concentration of cryoprotectant in VS 30% (EG and DMSO)
Volume of 0.1 µL
Cooling rate of approximately 23,000ºC/min
Osmolarity of VS ≈8.000 to mOsm/L
Kuwayama et al., 2005, 2007
Ana Cobo, 2008

11. Open vs closed system Efficiency and safety
To avoid possible contamination sterile nitrogen can be used (Vajta et al., 1998, Parmegiani et al., 2009).
In alternative, specific devices able to avoid direct contact of the samples with the nitrogen during vitrification (closed system) and/or storage are available (Kuleshova and Shaw 2000, Isachenko et al., 2006, Abdelhafez et al., 2011, Vanderzwalmen et al., 2009, Parmegiani 2011).

12. The revolution of vitrification approach – in the lab

13. Vitrification has changed the structure of the lab
- Incredible improvements in survival rate (also for oocytes and biopsied embryos) - New timings of cryopreservation (according to cell developmental stage) - New flexibility of the lab.
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14. Slow-freezing vs vitrification: clinical pregnancy and survival rates
RCTs
Cohort studies
A typical laboratory could improve a ≈60% embryo survival rate using slow-freezing to somewhere between 74% and 99% by using vitrification 
Rienzi et al., Hum Reprod update, 2016

15. Vitrification is successful for biopsied embryos
Joris et al., Hum Reprod. 1999
Reduced survival after human embryo biopsy and subsequent cryopreservation.
Jericho et al, Hum Reprod. 2003
A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos.
Escribá et al., Fertil Steril. 2008
Vitrification of preimplantation genetically diagnosed human blastocysts and its contribution to the cumulative ongoing pregnancy rate per cycle by using a closed device.
Zhang et al., Reprod Biomed Online. 2009
Vitrification of biopsied embryos at cleavage, morula and blastocyst stage.
Chang et al., Hum Reprod. 2013
Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases.
Schoolcraft et al., Fertil Steril. 2011
Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients.

16. Vitrification is successful for all stages of development: including oocytes

17. It has been difficult to overcome the intrinsic sensitivity of the cell
zona pellucida
hardening
Cytoplasmic and cytoskeleton damage
Oocyte ageing
membrane permeability
Meiotic spindle depolymerization
Polar body degeneration/fusion
Impact on oocyte physiology

18. Oocyte vitrification validation
2008: Efficiency in donation program not compromised with vitrification
(Cobo et al., 2008; Nagy et al., 2008)
2008: The clinical pregnancy rate double with the introduction of vitrification
(Tulandi, 2008; Cao et al., 2009; Smith et al., 2010 (RCT))
2010: Prospective randomized study with own sibling oocytes demonstrates the lab
efficiency of the technique (RCT) (Rienzi et al., 2010)
2010: Cumulative ongoing pregnancy rate with oocyte vitrification in a standard infertility
program (Ubaldi et al., 2010)
2010: Prospective randomized study with donor oocytes demonstrates clinical
efficiency of the technique (RCT) (Cobo et al., 2011)
Prospective randomized study comparing slow freezing cs vitrification (RCT)
(Smith et al., 2010)
2011: Efficiency of oocytes vitrification in the infertile population (RCT)
(Parmegiani et al., 2011)
2012: Multicentric longitudinal cohort study to confirm reproducibility (Rienzi et al., 2012)
Meta-analysis of randomized controlled studies (Oktay et al. 2006, Cobo et al., 2011; Cil 2013, Glujovky et al., 2014, Potdar et al 2014)

19. Pregnancy rate: Vitrification vs slow freezing

20. Oocyte survival: Vitrification vs slow freezing
Rienzi et al., Hum Reprod update, 2016

21. RCT: cryo-banked oocytes
Vitrification has almost allowed overpassing the gap in lives birth between fresh and cryopreserved oocytes in good prognosis patients.

22. Oocyte cryopreservation applications in ART
Oocyte cryopreservation is an emerging discipline that has already a key place for different applications:
Fertility preservation for medical reasons
Fertility preservation for social reasons
Use of cryo-banked oocytes for egg donation
Avoids the production of supernumerary embryos in IVF
Accumulation of excess oocytes in IUI cycles
Accumulation of oocytes in case of poor response
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23. Medical indications
In ITALY:
women between years at risk of premature ovarian failure (ISTAT 2012)
women suffering endometriosis
(Benaglia et al, 2010)
2.500 women <40 years with diagnosis of breast cancer
Oktay et al., 2010

24. Clinical live births in cancer patients

25. Personal considerations
The issue of persevering oocytes instead of embryos is of considerable importance.
Oocyte-cryopreservation gives women a reproductive autonomy. Instead, the use of male-partner/donor sperm to create embryos introduces several ethical, moral and legal concerns.
For this reason, we emphasize that there are scientific, moral and ethical reasons to promote oocyte (and NOT embryo) cryopreservation as gold standard in female onco-fertility (women and post-puberal girls, when possible before oncological treatment).
Rienzi and Ubaldi, 2015 

26. Cryopreservation can be personalized for each embryonic stage
Vitrification can be applied for all stages of development: at the right moment
before oocyte aging
before
singamy
when viability
is assessed
according to the expansion
EGA
Cryopreservation can be personalized for each embryonic stage

27. The revolution of vitrification approach – clinical implications

28. © Merck KGaA Darmstadt/Germany
Efficient cryopreservation program for oocytes and embryos
Enhances cumulative live birth rate per oocyte retrieval cycle
Allows systematic application of elective single embryo transfer policy
Provides the opportunity to perform cycle segmentation
Extends time for embryo evaluation
Permits fertility preservation for medical and non-medical indications
Enables egg banking for donation and/or for oocyte accumulation
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29. Elective SET is equally effective (cumulatively) but significanlty safer than double ET
meta-analysis of individual patient data from 8 RCTs
CUMULATIVE LIVE BIRTH RATE
RISK OF MULTIPLE BIRTH
eSET (n = 683)
Double ET (n = 684)
Now what is the right number of embryos to transfer. Today we know!! This number is ONE single embryo per transfer:
Why? Because we have cumulatively the same livebirth rate… BUT it is more safe!!!! Less risk multiple pregnancy.. Essential to be guarranted to the patients. Essential if we want healphy child
Adapted from McLernon, et al. BMJ., 2010
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30. Contribution of cryo-cycle to LBR
It has been estimated that in Europe in 2011, cryopreservation contributed to the overall live birth rate by 4% (raising from 19.7% with only fresh cycles to 24.0% including cryo-cycles) (Kupka et al., 2016).
In countries where cryopreservation is systematically applied, the rise is even more significant: Finland +13.4%, Switzerland +10.2% and Australia and New Zealand +13,5% (Kupka et al., 2016, Macaldowie et al., 2012).
These data reflect the improvements obtained in embryo culture, cryopreservation and/or the more conservative embryo transfer policy adopted.

31. Cycle segmentation SEGMENT A SEGMENT B GnRH AGONIST TRIGGERING
Lower extrauterine pregnancies
(Shapiro et al., 2012; Li et al., 2015)
Improved LBR (Roque et al., 2013)
GnRH AGONIST TRIGGERING
in a
GnRH ANTAGONIST
CYCLE
Almost no ovarian hyperstimulation sindrome
(Youssef et al., 2014 )
SEGMENT A
SEGMENT B
What can we infer from these evidences?
The best stimulation should be performed to fully exploit ovarian reserve WITHOUT exposing the patient to hyperstimulation risks..
This can be done by agonist triggering and cryopreservation of all oocytes/embryos/blastocyst. Today possible with vitrification approach
Transfer in a receptive endometrium.. To be improved by new approaches of endometrial receptivity assessment, lower risks of extrauterine pregnancies has been reported
Embryo transfer in a
receptive endometrium
Devroey, et al. Human Reproduction, 2011.
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32. Cycle segmentation for PGD-A in AMA
Elective single embryo transfer policy combined with enhanced embryo selection and vitrification is a realistic option also in poor prognosis patients of advanced maternal age.
First evidence – RCT – too low numbers but a beginning! To be continued!!
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33. Oocyte accumulation for poor-responder patients
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34. The future is already here

35. Standardization of the technique
Unlike slow freezing, vitrification does not need a freezing machine to provide specific cooling parameters. The technique is totally performed manually and is thus operator-dependent.
Vitrification effectiveness may thus be highly variable and depending on the laboratory protocols and experience. The heterogeneity of methods applied cause also serious difficulties with transportation of vitrified samples between laboratories using different cryoprotectants mixture and/or vitrification carriers.
A radical process of standardization is necessary.

36. Semi-automated vitrification is already available

37. Conclusions
Cryopreservation is essential in ART and should be optimized in each single IVF laboratory.
According to the updated evidences, vitrification is the best strategy for all stages of development and allows:
Increased cumulative live birth, reduced multiple pregnancy and ovarian hyperstimulation syndrome rates;
Female fertility preservation and cryo-egg donation programs;
New perspectives for embryo evaluation are imaginable;
If standardized and/or automated its consistency and efficiency could be assured.
Validation is necessary to asses the efficiency and the power of the new technologies
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