© 2008 Universitair Ziekenhuis Gent1 State of the ART of the vitrification of human oocytes … an...
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Transcript of © 2008 Universitair Ziekenhuis Gent1 State of the ART of the vitrification of human oocytes … an...
© 2008 Universitair Ziekenhuis Gent 1
State of the ART of the vitrification of human oocytes … an embryologist view
Antalya 2011
Etienne Van den Abbeel
Department of Reproductive Medicine, University Hospital Gent, Belgium
22© 2008 Universitair Ziekenhuis Gent
Cryopreservation of human oocytes: why?
1. Fertility preservation for medical reasons
2. Fertility preservation for social reasons
3. Use of cryo-banked oocytes for egg donation
4. Avoids the production of supernumerary embryos in IVF
5. Accumulation of excess oocytes in IUI cycles
(Rienzi L ESHRE Stockholm 2011)
33© 2008 Universitair Ziekenhuis Gent
Cryopreservation and cryoprotectants and influence on oocyte constituents:
Zona pellucida
Cortical granules
Meiotic spindle
Chromosomes
Ca homeostasis
Mitochondria
SER
Transcriptome
Genome
Proteoom
Plasmamembrane
Cryopreservation of mammalian oocytes is a challenge
44© 2008 Universitair Ziekenhuis Gent
Efficient cryopreservation programmes?
Avoiding injuries
Dilemma
Which strategy is better for our patients: freezing or vitrification?
Vitrification is the best strategy?
55© 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)
66© 2008 Universitair Ziekenhuis Gent
Outline of the presentation
State of the ART of the technology
State of the ART of the efficiency
Slow freezing versus vitrification
Fresh oocytes versus vitrified oocytes
Open vitrification versus closed vitrification
77© 2008 Universitair Ziekenhuis Gent
Vitrification
State of the ART of the technology
Claims made for vitrification
Reduces the time of the cryopreservation procedure?
Flexibility
Eliminates the cost of expensive programmable freezing equipment?
No ice crystallization?
Very simple procedure?
88© 2008 Universitair Ziekenhuis Gent
Claims made for vitrification
No ice crystal formation?
Is vitrification a simple procedure?
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 tra
nsition curve
Ice phase
molecular structure of a viscous liquid and is not crystalline
1010© 2008 Universitair Ziekenhuis Gent
1997 Vajta et al
Minimal Volume Vitrification:
Challenge = avoiding IIF
Succesfull vitrification of human oocytes, embryos and blastocysts depends on a correct interplay between,
“sufficient” permeation of a “sufficient” high concentration of penetrating cryoprotectant
(equilibration step), “sufficient” dehydration by a non-penetrating cryoprotectant (vitrification step), a
“sufficient” high cooling rate direct contact with LN2 and small volumes) and a “sufficient” high warming
rate
1111© 2008 Universitair Ziekenhuis Gent
Basic principles of vitrification
Principle variables of vitrification
The effect of cooling and warming rates
Permeability of cells to water and CPA
CPA toxicity
1212© 2008 Universitair Ziekenhuis Gent
Vitrification
State of the ART of the efficiency
Slow freezing versus vitrification
Fresh oocytes versus vitrified
Open versus closed vitrification
1313© 2008 Universitair Ziekenhuis Gent
Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. (FS 2011)Cobo A, Diaz C.
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.
1414© 2008 Universitair Ziekenhuis Gent
1515© 2008 Universitair Ziekenhuis Gent
1616© 2008 Universitair Ziekenhuis Gent
Conclusions• More studies should be done!•The oocyte survival rate was higher in vitrified vs. slow-frozen oocytes (odds ratio [OR] 2.46, 95% confidence interval [CI] 1.82-3.32), although heterogeneity between studies was observed. • The fertilization rate was higher in vitrified vs. slow-frozen oocytes (OR 1.50, 95% CI 1.07-2.11). • Vitrification also resulted in a higher rate top-quality embryo (22.4% vs. 8.0%, OR 3.32, 95% CI 1.37-8.02) and embryo cleavage rate (day 2: 64.6% vs. 47.7%, OR 2.00, 95% CI 1.33-3.00; day 3: 53.0% vs. 33.3%, OR 2.25, 95% CI 1.32-3.85) as compared with slow freezing
•The rates of ongoing pregnancy, top-quality embryo, embryo cleavage, and fertilization did not differ between the vitrification and the fresh oocyte groups.
•A cautionnary note however
1717© 2008 Universitair Ziekenhuis Gent
Results some caution!
Substantial heterogeneity
Patient selection (good responders, Age effects (Ubaldi et al 2010), oocyte donors)
Warming and transfer policy
No uniform reporting of data and (or) study endpoints
Commercial bias?
Different devices and different media formulations used
Superiority of open devices?
1818© 2008 Universitair Ziekenhuis Gent
Vitrification of mature human oocytes: 2008-2010(Open VIT, open storage, open warming = cryo-TOP/cryo
Leaf)
Kuwayama et al; Antinori et al; Nagy et al; Chian et al; Rienzi et al; Cobo et al
Morphological survival: > 95% fully intact
Fertilisation: >90% 2PN
Pregnancy rates 40 to 65 %
Neonatal outcome reassuring sofar (Noyes et al, Wennerholm et al, Chian et al)
Long term consequences? CPA’sare not neutral
1919© 2008 Universitair Ziekenhuis Gent
Vitrification of oocytes
Is open vitrification a safe procedure?
Open devices
direct contact between samples and LN2
Long term LN2 storage (vapour storage) of apparently vitrified, minimal-volume (<1µl) samples
Spontaneous devitrification possible
Open versus closed vitrification and storage?
2020© 2008 Universitair Ziekenhuis Gent
Prospective randomised controlled trial (morphological survival): open (cryo-TOP) versus closed (closed VIT,
closed storage, open warming =CBS HSS) vitrification
Brussels: unpublished observations
Number of donors: 9 (144 oocytes)
Open Closed
Oocytes VIT 71 73
Oocytes warmed 64 73
Survival 83% 92% NS
2121© 2008 Universitair Ziekenhuis Gent
Prospective randomised controlled trial open (cryo TOP) versus closed (CBS HSS)
vitrification
Number of recipients: 23
Open Closed
Transfers 11 12
Embryos transferred 15 21
HCG 1 6
Embryos VIT 2 6
2222© 2008 Universitair Ziekenhuis Gent
Closed (CBS HSS) vitrification of oocytes: clinical results Number of donors 14
Number of recipients: 20
Number of oocytes warmed: 123
Number of oocytes survived (%): 111 (90.2)
% 2PN: 77.5
% GQ Embryos on Day 3: 61.6
Transfers: 20
Embryos transferred: 36
Clinical pregnancies (%): 10 (50)
Implantations (%): 12 (33.3)
Implantation rate per oocyte warmed: 13/123 (10.6%)
2323© 2008 Universitair Ziekenhuis Gent
Closed (vitrisafe) vitrification of oocytes: clinical results Pierre Van der Zwalmen (2010)
Gynecol Obstet Fertil 38 (541-546)
Number of oocytes warmed: 146
Number of oocytes survived (%): 137 (94%)
Patients: 22
Embryos transferred: 63
Clinical pregnancies (%): 9 (41)
% Implantations : 14%
Implantation rate per oocyte warmed: 6%
2424© 2008 Universitair Ziekenhuis Gent
Vitrification of oocytes
Closed vitrification is an efficient method for the vitrification of human oocytes
suporting the domininance of the warming rate over the cooling rate in vitrification
2525© 2008 Universitair Ziekenhuis Gent
General conclusions
Vitrification will it replace conventional freezing techniques?
• Recent published data of the vitrification of human oocytes,
indicate that vitrification works and produces better results than
conventional freezing. The efficiency of vitrification (FHB/oocyte
warmed) is 5-10% for oocytes,
• It has replaced conventional freezing
2626© 2008 Universitair Ziekenhuis Gent
General conclusions
Vitrification routine or experimental?
Technical challenges/technical proficiency
Development of more robust closed “true”vitrification
procedures
• Media and devices for vitrification: knowing and understanding what is important
• Interactions between cryoprotectants, cooling rates and warming rates on survival and developmental potential
Costs
2727© 2008 Universitair Ziekenhuis Gent
Vitrification of mature human oocytes 2011
Rienzi L, ESHRE 2011
Vitrification is an efficient method for cryopreservation of human MII phase oocytes. The overall efficiency was found to be consistently high in different centers. Female age and number of available oocytes are the most important predictive factors of success.
In any case however, oocyte cryopreservation cannot be considered a guarantee of life-long fertility.
Equal to fresh oocytes? ICSI required! ICSI done in fresh oocytes!
2828© 2008 Universitair Ziekenhuis Gent
Vitrification of mature human oocytes 2011
Noyes et al RBM online (RBM Online 19, 275-279)
...“Perhaps one hesitation regarding oocyte cryopreservation partially lies in the commercial interests of companies promoting premature elective fertility preservation. In addition, competing interests exist for those who manufacture oocyte storage containersand/ or oocyte cryopreservation culture media. Clearly, clinicians and embryologists need to be cognizant of these latter conflicts as more publications appear in the literature”...