© 2008 Universitair Ziekenhuis Gent1 QC/QA in cryopreservation laboratories within assisted reproduction units: future recommendations Prof Dr Etienne

© 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


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)


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


Introduction

Cryopreservation within assisted reproduction units: future recommendations

Vitrify …. But take care


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?


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)


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


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


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


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


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


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


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


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


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?


Vitrification: Evidence for practice

Slow freezing versus vitrification

Open versus closed vitrification

Closed versus closed vitrification


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?



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)


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.



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


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


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


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 survived (%): 137 (94%)

Implantation rate per oocyte warmed: 6%

Closed (cryo-TIP) vitrification of oocytes: Smith et al (2009)


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 survived (%): 151 (57.9%)

Implantation rate per oocyte warmed: 2.6%


VITRIFICATION Day 3 embryos


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).


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


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%)


Results from literature: embryos (closed vitrification)

Van Landuyt et al, 2010 (CBS HS VIT)


55/257 64/423 64/515

(21.4%) (15.1%) (12.4%)


VITRIFICATION

BLASTOCYST


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).


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)


4974/10197 3124/11117 3124/13629

(48.8%) (28.1%) (22.9%)


Results from literature: blastocysts (closed vitrification)

Stachecki (2008), Van der Zwalmen (2009, 2013), Liebermann (2009), Van Landuyt (2011), De Croo (2013)


229/435 263/854 263/1004

(52.6%) (30.8%) (26.2%)


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


CryopreservationHuman oocyte, embryo, blastocysts vitrification:

no standard procedure available

Future recommendations?

Standardization through automationTrue vitrification

Documents

© 2008 Universitair Ziekenhuis Gent1 QC/QA in cryopreservation laboratories within assisted reproduction units: future recommendations Prof Dr Etienne