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33 © 2008 Universitair Ziekenhuis Gent Introduction Cryopreservation of reproductive cells Stopping biological time -196°C Lethal intra-cellular ice formation Fate of cellular water Equilibrium (quasi-equilibrium) cooling Non-equilibrium cooling ( Freezing ) ( Vitrification)
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© 2008 Universitair Ziekenhuis Gent 1
QC/QA in cryopreservation laboratories within assisted reproduction units: future recommendations
Prof Dr Etienne Van den AbbeelDepartment of Reproductive Medicine, University Hospital Gent, BelgiumIstanbul 8 June 2013
22© 2008 Universitair Ziekenhuis Gent
IntroductionCryopreservation of human oocytes: why?
Legal
Ethical
Donor-acceptor programmes
Fertility preservation
Cancer
Non-medical reasons
Cryopreservation of human embryos: Why?
Increase efficiency of ARTTool to reduce multiple pregnanciesTransfer in natural cycle Fertility preservation
Efficient and safe cryopreservation procedures
33© 2008 Universitair Ziekenhuis Gent
Introduction
Cryopreservation of reproductive cells
Stopping biological time
-196°C
Lethal intra-cellular ice formation
Fate of cellular water
Equilibrium (quasi-equilibrium) cooling Non-equilibrium cooling ( Freezing ) ( Vitrification)
44© 2008 Universitair Ziekenhuis Gent
Dilemma: vitrification or freezing?
There are two circumstances in which vitrification methods should definitively be considered: where it is clear that extra-cellular ice is responsible for significant damage, and where the results of classical freezing methods are unsatisfactory (Pegg, 2005)
Classical freezing of oocytes is suboptimalClassical freezing of blastocysts is suboptimal
55© 2008 Universitair Ziekenhuis Gent
Introduction
Cryopreservation within assisted reproduction units: future recommendations
Vitrify …. But take care
66© 2008 Universitair Ziekenhuis Gent
Vitrification
Rationale for vitrification
Very simple procedure?
Reduces the time of the cryopreservation procedure?
Flexibility
No ice crystallization?
Eliminates the cost of expensive programmable freezing equipment?
More efficient?
77© 2008 Universitair Ziekenhuis Gent
Vitrification current status:
No standard procedure is available
Several roads can lead to Rome
The current plethora of protocols has to become canalized to a few of proven efficiency, which will greatly facilitate the comparison of data between centers as well as the troubleshooting of disappointing results within a center (Gosden, 2011)
88© 2008 Universitair Ziekenhuis Gent
Recommendations concerning vitrification now and in the future:
Closed vitrification techniques should be the gold standard
Closed vitrification methods should be robust, standardized and biologically safe
QC/QA in vitrification laboratories
Technical issues
99© 2008 Universitair Ziekenhuis Gent
T°
Concentration of solute
Th
Tg
Equilibrium Freezing Curve
Liquid phase
Glass phase
Molecular organization as in a crystal structure
Phase diagram
Glass transitio
n curve
Ice phase
molecular structure of a viscous liquid and is not crystalline
1010© 2008 Universitair Ziekenhuis Gent
1. Equilibrium Vitrification of reproductive cells (Rall et al 1985)
Probability of vitrification:
Cooling rates x [CPA]
2. “Minimal Volume open pulled straw Vitrification”(1997 Vajta et al)
Probability of vitrification:
Cooling rates x [CPA]
Sample Volume
1111© 2008 Universitair Ziekenhuis Gent
3. The effect of warming rates
Mouse model
Seki, Mazur ( 2009, 2010, 2012)
In minimal volume vitrification, the warming rate is dominant over the cooling rate
Cooling rate/warming rate x [CPA]
Sample volume
1212© 2008 Universitair Ziekenhuis Gent
4. The device: open versus closed vitrification
Human model
Cobo et al (2011) open devices better for minimal volume vitrification than closed devices because of very high cooling rates in open devices
Cobo et al (2013)
The warming rate is perhaps the best determinant factor for succes in minimal volume cooling…. This can be best achieved with open system?!
Open vitrification: cross contamination issues (Bielanksi et al, 2009)
Vapour storage, sterile liquid nitrogen
De Munck et al (2013)
Using a CBS HS closed VIT device a 90% survival rate can be achieved when human oocytes are immediately warmed in a large volume at 37°C
1313© 2008 Universitair Ziekenhuis Gent
The device and the warming rate: open versus closed vitrification
Vitrification Warming Storage
Cryo TOP open open open
Cryo Loop open open open
Flexipet open open open
Cryologic open open open
……
Cryo TIP closed closed closed
Cryopette closed closed closed
……
Rapid i closed open closed
CBS HS VIT closed open closed
Vitrisafe closed open closed
1414© 2008 Universitair Ziekenhuis Gent
Other variables of vitrification
CPA toxicity
Type and concentration of CPA
PG, EG, DMSO, Glyc ….
Temperature of exposure
Permeability of cells to water and CPA
Glyc<EG<DMSO<PG
Temperqture of exposure
Variability amongst oocytes and embryos
Oocytes<zygotes<embryos<blastocysts
1515© 2008 Universitair Ziekenhuis Gent
Take home message on technical issues
Minimal volume vitrification is non-equilibrium vitrification. It is a Vitrification method that not always leads to vitrification)
Succes of the vitrification method depends on a correct interplay between a “sufficient” high cooling rate, “sufficient” permeation of a sufficient high concentration of penetrating cryoprotectant, “sufficient” dehydration by a non-penetrating cryoprotectant, and a “sufficient” high warming
Standardization will be a challenge
1616© 2008 Universitair Ziekenhuis Gent
Recommendations concerning vitrification now and in the future:
Closed vitrification techniques should be the gold standard
Closed vitrification methods should be robust, standardized and biologically safe
QC/QA in vitrification laboratories
Clinical issues
Can it work?Does it work?Is it efficient?
1717© 2008 Universitair Ziekenhuis Gent
Vitrification: Evidence for practice
Slow freezing versus vitrification
Open versus closed vitrification
Closed versus closed vitrification
1818© 2008 Universitair Ziekenhuis Gent
Results from literature: some caution!
Different devices and different media formulations used
Oocyte collection cycle characteristics
Patient selection
Cryo policy (selection of oocytes/embryos before cryo)
Warming and transfer policy (selection oocytes/embryos after warming)
No uniform reporting of data and (or) study endpoints
Commercial bias?
1919© 2008 Universitair Ziekenhuis Gent
2020© 2008 Universitair Ziekenhuis Gent
Oktay Oktay et alet al., ., 2006/2008 2006/2008 (abstract)(abstract)
Variable Slow Freezing (2006) Vitrification (2006)
Age, mean 33.7 32.3
Fertilization rate 64.9 (2,478/3,818) 74.2 (637/859)
Clinical pregnancies per thawed oocyte
0.023 (153/6720) 0.045 (61/1354)
SLOW FREEZING VERSUS SLOW FREEZING VERSUS VITRIFICATION - oocytesVITRIFICATION - oocytes
Slow freezing/Vitrification (2008)
Clinical pregnancies per thawed oocyte
0.022 (314/14215)/0.058 (212/3672)
2121© 2008 Universitair Ziekenhuis Gent
Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. (Cobo et al, 2011)Grade A level of evidence?
OBJECTIVE: To perform a systematic review of the literature to identify randomized controlled trials assessing the efficacy of oocyte vitrification in terms of oocyte survival, fertilization, embryo development, and pregnancy rates.
DESIGN: Systematic review and meta-analysis of randomized controlled trials (>2500 papers and abstracts).
Five eligible studies were finally included.
They involved 4,282 vitrified oocytes, 3,524 fresh oocytes, and 361 slow-frozen oocytes between 2005 and 2009.
2222© 2008 Universitair Ziekenhuis Gent
2323© 2008 Universitair Ziekenhuis Gent
Discussion and critical points of vitrification of human oocytes
• More prospective studies should be done!
• Evident clinical heterogeneity was present• Statistical heterogeneity between studies especially for morphological survival
•The authors state that to obtain good results “open” vitrification should be used•The efficiency (implantation per oocyte warmed) of open vitrification of human oocytes from young donors is 10%• Unsufficient data for “older” patients
2424© 2008 Universitair Ziekenhuis Gent
Pelin Ci et (2013) Age-specific probability of live birth with oocyte cryopreservation: an individual patient data meta-analysis. Fertil Steril (Article in press)
Non-donor egg cycles
Original data from 10 studies including 2.265 cycles from 1.805 patients showed that live birth success rates declined with age regardless of the freezing technique (slow freezing vs vitrification). Women whose SF eggs were preserved before age 30 had a greater than 8.9% likelihood of implantation per embryo which declined to 4.3% for embryos from eggs frozen after 40. For vitrification cycles, implantation success declined from 13.2% for embryos from eggs frozen at 30 to 8.6% for embryos from eggs frozen at 40. So, estimated probabilities of live birth for vitrified oocytes were higher than those for slowly frozen
2525© 2008 Universitair Ziekenhuis Gent
Open versus closed vitrification
Stoop et al (2012) Cryo TOP vs CBS HS VIT
RCT, two devices, same vitrification and warming technology
144 oocytes, survival 83% versus 92% (NS)
Paffoni et al (2011) Cryo TOP vs Cryo TIP
Retrospective trial, two devices, different vitrification and warming technology
529 oocytes, survival 82.8 % versus 57.9 (p< 0.001)
Papatheodorou et al (2013) Open vitrisafe vs closed vitrisafe
Prospective randomised study, open vs closed same device, different Vitrification technology (CPA concentration)
1206 oocytes, survival 82.9% vs 91.0% (p< 0.05), no differences in pregnancy and implantation rates
2626© 2008 Universitair Ziekenhuis Gent
Closed versus closed vitrification
Closed (CBS HSS) vitrification of oocytes: Stoop et al (2012)
Number of oocytes warmed: 123
Number of oocytes survived (%): 111 (90.2)
Implantation rate per oocyte warmed: 13/123 (10.6%)
Closed (Vitrisafe) vitrification of oocytes: Van der Zwalmen et al (2010)
Number of oocytes warmed: 146
Number of oocytes survived (%): 137 (94%)
Implantation rate per oocyte warmed: 6%
Closed (cryo-TIP) vitrification of oocytes: Smith et al (2009)
Number of oocytes warmed: 349
Number of oocytes survived (%): 260 (75%)
Implantation rate per oocyte warmed: 5.2%
Closed (cryo-TIP) vitrification of oocytes: Paffoni et al (2011)
Number of oocytes warmed: 261
Number of oocytes survived (%): 151 (57.9%)
Implantation rate per oocyte warmed: 2.6%
2727© 2008 Universitair Ziekenhuis Gent
VITRIFICATION Day 3 embryos
2828© 2008 Universitair Ziekenhuis Gent
Cryopreservation of human embryos: freezing vs vitrification(review paper)
Kolibianakis et al (Current opinion in OB/GYN 21, 270-274, 2009)
Cryopreservation of human embryos by vitrification or slow freezing: which one is better?
Review to evaluate whether the published literature offers data to allow the clinician to choose the best between two cryopreservation methods
Vitrification as compared with slow freezing, appears to be better in terms of post-thawing survival rates for cleavage-stage embryos (odds ratio (OR): 6.35,95% CI: 1.14-35.26)
Postthawing blastocyst development of embryos cryopreserved in the cleavage stage is significantly higher with vitrification as compared with slow freezing (OR: 1.56, 95% CI: 1.07-2.27)
No significant difference in clinical pregnancy rates per transfer could be detected between the two cryo methods (OR: 1.66, 95% CI: 0.98-2.79).
2929© 2008 Universitair Ziekenhuis Gent
Cryopreservation of embryos: freezing versus vitrification
AbdelHafez et al, 2010 RBM-online
Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis
Locate randomized controlled trials comparing embryo cryopreservation methods
Conclusions: Vitrification is superior to slow freezing which in turn is superior to ultra-rapid freezing. However, more well designed and powered studies are needed to further corroborate these findings
3030© 2008 Universitair Ziekenhuis Gent
Results from literature: embryos (open vitrification)
El-Danasouri (2001)
Kuwayama (2005)
Mukaida (2007)
Desai (2007)
Balaban (2008)
Raju (2009)
Valojerdi (2009)
Clin P / ET Impl /E Transferred Impl / E Warmed
596/1849 575/3485 575/4242
(30.8%) (16.5%) (13.6%)
3131© 2008 Universitair Ziekenhuis Gent
Results from literature: embryos (closed vitrification)
Van Landuyt et al, 2010 (CBS HS VIT)
Clin P / ET Impl /E Transferred Impl / E Warmed
55/257 64/423 64/515
(21.4%) (15.1%) (12.4%)
3232© 2008 Universitair Ziekenhuis Gent
VITRIFICATION
BLASTOCYST
3333© 2008 Universitair Ziekenhuis Gent
Cryopreservation of human embryos: freezing vs vitrification(review paper)
Kolibianakis et al (Current opinion in OB/GYN 21, 270-274, 2009)
Cryopreservation of human embryos by vitrification or slow freezing: which one is better?
Review to evaluate whether the published literature offers data to allow the clinician to choose the best between two cryopreservation methods
Vitrification as compared with slow freezing, appears to be better in terms of post-thawing survival rates for blastocysts (OR:4.09, 95% CI:2.45-6.84)
No significant difference in clinical pregnancy rates per transfer could be detected between the two cryo methods (OR: 1.66, 95% CI: 0.98-2.79).
3434© 2008 Universitair Ziekenhuis Gent
Results from literature: blastocysts (open vitrification)
Choi (2000), Yokota (2001), Cho (2002), Reed (2002), Hiraoka (2004), Hu (2004), Stehlik (2005), Huang (2005), Kuwayama (2005), Mukaida (2007), Liebermann (2007), Son (2007), Van der Zwalmen (2007), Hiraoka (2008), Ebner (2009) Liebermann (2009), Rama Raju (2009)
Clin P / ET Impl /E Transferred Impl / E Warmed
4974/10197 3124/11117 3124/13629
(48.8%) (28.1%) (22.9%)
3535© 2008 Universitair Ziekenhuis Gent
Results from literature: blastocysts (closed vitrification)
Stachecki (2008), Van der Zwalmen (2009, 2013), Liebermann (2009), Van Landuyt (2011), De Croo (2013)
Clin P / ET Impl /E Transferred Impl / E Warmed
229/435 263/854 263/1004
(52.6%) (30.8%) (26.2%)
3636© 2008 Universitair Ziekenhuis Gent
Take home messages on clinical issuesGood survival, fertilization, embryo development and pregnancy rates can be obtained with closed and open vitrification of oocytes
Oocyte morphological survival rates are higher with vitrification as compared with classical freezing
The external validity of oocyte vitrification maybe limited to good responders or donors and there are insufficient data for other patient categories. Age might be a considerable factor
Vitrification provided a significant clinical breakthrough for the preservation of blastocysts
There is some debate as to the real benefit of VIT over slow freezing for D3 embryos
The long term safety of the technique remains to be confirmed
3737© 2008 Universitair Ziekenhuis Gent
CryopreservationHuman oocyte, embryo, blastocysts vitrification:
no standard procedure available
Future recommendations?
Standardization through automationTrue vitrification