Vitrification of Embryos

BASAK BALABANAmerican Hospital of IstanbulAssisted Reproduction Unit

AMERICANHOSPITAL

TJOD Meeting, Antalya Turkey 2013

Cryopreservation Cryopreservation TechniquesTechniques

• Slow conventional freezing

• Traditional Vitrification

• Ultrarapid vitrification

What is vitrification?

• Vitrification is the reversible transition of a liquid into an amorphous non-crystalline glass

• Slow freezing uses low concentrations of CPAs (~ 10%), cooling rates of ≤1˚C/min, and warming rates of ~250 ˚C/min

• Vitrification uses high con.of CPAs (30-40%), use of saccharides as supplements,cooling rates >1000 ˚C/min(usually >10.000 ˚C/min) , and very rapid warming rates

Vitrification solutions

DMSO+Acetamide+ propylene glycol

Ethylene glycol+ Ficoll+Sucrose

Ethylene glycol+ DMSO

Ethylene glycol+ glycerol

Slow Freezing solutions

DMSO /1-2 PROH + Sucrose

Glycerol+

Sucrose

Kasai et al. RBM Online 2004

Base medium+

Cryoprotectant

Problems Associated with Traditional Vitrification Procedures

• High levels of cryoprotectants are toxic to embryos • (4-10 M compared to 0.5-1.0M)

• Procedure must be performed at 4oC• Technically demanding

Advantages of Ultra-Rapid Vitrification

• Increases in cooling rates alleviates toxicity of high levels of cryoprotectants

• Can be performed at room temperature or 37oC

Variables in Vitrification

• Cooling &warming rates

• Concentration of the cryoprotectant:

• Sample size and carrier systems

• To minimize the volume of the vitrification solution special carriers are used for vitrification process

** Open pulled straws ** Flexipet- denuding pipette ** Microdrops ** Electron-microscopic copper grids ** Hemistraw system ** small nylon coils or nylon mash ** Cryotop,cryotip ** Cryoloop

Concerns with Regards to Sterility of Liquid N2 storage

• No publication since 1985 had shown cross contamination with viruses, after screening over 450 publications

Tedder et al., 1995Hepatitis B transmission

Bielanski et al., 2000Viral contamination

Bielanski et al., 2003Microbial contamination, no viral cross contamination

Kyuma et al., 2003No microbial or viral cross contamination

Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBRwere NOT feasible

Cryopreservation of human embryos by vitrification or slow freezing: A systematic review and meta-analysis

Pubmed search: 873, only 4 included!!,Primary outcome: Postthaw survival rate,Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR

Loutradi et al., F&S 2008

0.001 0.01 0.1 10 100 1000

Vit.Slow

Cryopreservation of cleavage stage embryos by vitrification vs. slow freezing??Which one is better?

Efstratios et al.,Cur.Op.Obs&Gynec. 2009

Li and Rama Raju found no stat sig. dif for CPR

Survival

Blastocyst development

**Biopsied embs.

0.002 0.1

Slow

10 500

Vit.

**4 Vit& SF, 2 UF& SF Desai et al.,RBM Online 2010

Results

• Current meta-analysis indicate that embryo vitrification is superior to slow freezing based on direct comparison of embryo survival and CPR

• OPR, IR were also higher • Within the limited data set available, using

both direct and indirect evidence, UF appeared to be inferior to SF as well as V.

• Further randomized trials that examine neonatal outcomes & congenital anomalies are necessary to judge the efficacy and safety of vitrification

Desai et al., RBM Online 2010

Outcome of vitrified cleavage-stage embryos: 1872 cycles

Cobo et al., F&S 2012

A randomized controlled study on human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation

• To compare the blastocyst development between embryos that were cryopreserved by either slow freezing or vitrification on day 3

• To determine whether slow freezing and vitrification have different effects on human cleavage stage embryo metabolism

Human Reproduction 2008Balaban B¹, Urman B¹, Ata B, Isiklar A¹, Larman MG², Hamilton B² and Gardner DK²

Embryo development & Metabolic Analysis

Slow Slow FreezingFreezing

VitrificationVitrification 95%CI of 95%CI of differencedifference

P valueP value

Cryosurvival (%)Cryosurvival (%) 206/232206/232

(88.7)(88.7)

222/234222/234

(94.8)(94.8)

+1 – 11%+1 – 11% 0.020.02

Embs. with 100% Embs. with 100% blastomere blastomere survival (%)survival (%)

106/206106/206

(51.4)(51.4)

173/222173/222

(77.9)(77.9)

+18 - %35+18 - %35 0.000.00

Blastocyst Blastocyst formation (%)formation (%)

102/206102/206

(49.5)(49.5)

134/222134/222

(60.3)(60.3)

+1 – 20%+1 – 20% 0.020.02

Blasts.≥ 3AA (%)Blasts.≥ 3AA (%) 43/10243/102

(42.1)(42.1)

70/13470/134

(52.2)(52.2)

- 2.7 – 22.8%- 2.7 – 22.8% 0.120.12

Hatching Hatching blastocysts (%)blastocysts (%)

22/10222/102

(21.5)(21.5)

42/13442/134

(31.3)(31.3)

- 1.4 – 20.9%- 1.4 – 20.9% 0.090.09

Balaban & Gardner et al., HR 2008

0

5

10

15

20

25

Slow Freezing Vitrification

*

Pyr

uva

te u

pta

ke

(pm

ol/e

mb

ryo/

h)

Clinical Outcome with VitrificationNo.of patient’s warmedNo.of patient’s warmed 7373

No.of embryos vitrified(Mean)No.of embryos vitrified(Mean) 314 (4.2)314 (4.2)

No.of embryos warmedNo.of embryos warmed 241(3.3)241(3.3)

Cryosurvival (%) 222222 (92.1)(92.1)

No.of embs. with 100%survivalNo.of embs. with 100%survival 160(72.1)160(72.1)

No.of morula on day 4 (day of ET)No.of morula on day 4 (day of ET) 138(62.1)138(62.1)

Balaban & Gardner et al., HR 2008

No.of ET (Mean)No.of ET (Mean) 168(2.3)168(2.3)

No.of morula ET (%)No.of morula ET (%) 146(86.9)146(86.9)

CPR(%)CPR(%) 36(49.3)36(49.3)

IR(%)IR(%) 50(29.7)50(29.7)

OPR(%)OPR(%) 33(45.2)33(45.2)

MPR(%)MPR(%) 13(36.1)(1-triplet, 13(36.1)(1-triplet, 12 twins)12 twins)

Abortion(%)Abortion(%) 3(8.3)3(8.3)

DelivDeliveeries(%)ries(%) 8(24.2:8/33)(2-8(24.2:8/33)(2-twins, 6 singleton)twins, 6 singleton)

Neonatal Outcome of Vitrified Cleavage Stage Embryos

Rama Raju et al. F&S 2009No.sig. dif. for neonatal parameters:Mean gestational age, birth weights for singleton & MPR, PR induced complications,Incidence of birth defects ( major & minor malformations)

Perinatal &neonatal outcomes of 494 babies from 972 vitrified day 3 ET

Shi et al., F&S 2012

**

Cryopreservation of blastocysts by vitrification or slow freezing: A systematic review and meta-analysis

Pubmed search: 873, only 4 included!!,Primary outcome: Postthaw survival rate,Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR

Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBRwere NOT feasible

Loutradi et al., F&S 2008

Cryopreservation of blastocysts by vitrification vs. Slow freezing??Which one is better?

Efstratios et al.,Cur.Op.Obs&Gynec. 2009

Only Bernal compared CPR, no sig. diff.

Cryosurvival

Youssry et al.,RBM Online 2008>10.000 blasts. vitrified

Outcome of vitrified blastocysts: 1278 cycles; donor &infertile patients

Cobo et al., F&S 2012

Van Landuyt et al.,HR 2011, 759 IVF/ICSI cycles,Survival day 5: 79.3%,day 6:70.1%SET OPR:14.2%, DET OPR: 20.5%,IR day 5:14.3%, day 6:13.7%

ET rate: proportion of thawed/warmed blasts. that are sufficient quality to transfer

Takahashi et al.,F&S 2005Liebermann et al., F&S 2006 also reported no adverse effect

Children born after vitrification of blastocysts: Systematic review

No control group in any of the studies??? Wennerholm et al.,HR 2009

Vitrification results with higher cryosurvival rates for biopsied human embryos

Poor cryosurvival rates (approx. 30%) and clinical outcome reported after conventional slow freezingof biopsied cleavage stage embryos ( Joris et al., HR 1999, Magli et al., HR 1999)

Zheng et al.,HR 2005

Escriba et al., F&S 2008 had shown similar cumulative OPR/OPU for PDG and non-PGD group with blastocyst vitrification

Flexibility of re-cryopreserving cells by vitrification method

• It’s presumed that refrozen & thawed embryos using conventional methods results with detrimental cryoinjury

• Chang C . RBM Online 2008 Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification.

• Kumasako et al., F&S 2009 The efficacy of the transfer of twice frozen thawed embryos with the vitrification method

• Peng et al.,RBM Online 2011 Live birth after transfer of a twice vitrified warmed blastocyst that had undergone trophoectoderm biopsy

• James et al., RBM Online 2012 Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates

Concerns regarding Vitrification• Majority of the articles published on the clinical efficieny of vitrification

for human cells utilized open carriers. And so LN2 still remains to be a potential source of contamination since the technique is based on direct contact between the vitrification solution containing cryoprotectant agents and LN2. So from a clinical point of view:

*** Closed systems to avoid contamination are suggested, especially based on the new regulations of EUTCD. Few randomized clinical trials had shown similar efficiency..(Kuwayama RBM 2005-embryos, Van Landuyt HR 2011-blasts.,Stoop RBM 2012-oocytes)

*** Storage of cells in the vapour phase of N2 instead of LN2(Cobo F&S 2010)

*** Sterilization of LN2 (Parmegiani RBM, HR 2011)

• Safety of vitrification solutions with high concentrations of cryoprotectants??Low toxicity vitrification solutions must be designed in the future

• Genetical structure of the vitrified cell?? Chromosal abnormalities, gene expressions ...... More comparitive studies with fresh cells are needed to prove the safety of the technique

RESULTS• Vitrification protocols are now starting to

enter the mainstream of human ART• Vitrification as a cryopreservation method

has many primary advantages and benefits based on the published data

• It’s likely that in near future vitrification will become the most suitable method for cryopreservation of any cells and perhaps tissues when concerns regarding safety are improved by scientifically properly designed studies