© 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)


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


Efficient cryopreservation programmes?

Avoiding injuries

Dilemma

Which strategy is better for our patients: freezing or vitrification?

Vitrification is the best strategy?


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)


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


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?


Claims made for vitrification

No ice crystal formation?

Is vitrification a simple procedure?


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


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


Basic principles of vitrification

Principle variables of vitrification

The effect of cooling and warming rates

Permeability of cells to water and CPA

CPA toxicity


Vitrification

State of the ART of the efficiency

Slow freezing versus vitrification

Fresh oocytes versus vitrified

Open versus closed vitrification


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.




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


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?


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


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?


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


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


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%)


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%


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


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


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


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!


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”...

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