VITRIFICATION

Dr G A RAMA RAJU

Over viewComponents of

cryobiology

Slow freezing /vitrification

Key issues in

vitrification

sperm

EmbryoOocyt

e

Ovary /testis

Safety

New born

.

Prof Hans Tinneberg

Pioneers.

Nature

?

? A vitrified frog felt as a rock

A wood frog in winter - Alaska. Nature’s expt with VITRIFICATION

Cell injury.

1 Cell size shape ,permeability ,individual quality and sensitivity.2 cryoprotectant toxicity3 cooling and warming.

The land mark publication “Slow cooling” Trounson and Mohr 1983

A long gap…………………………………….

PLANER COMPUTERISED FREEZER

Vitrification a tipping point in ART

Gopubmed:

Father Basile.j.luyet

work between 1937-1958

1952 Time magazine

Innovator :

Spermatozoa : Early adopter

15

Early adopter: PARKES

Majority

Publication from different countries

CRYOPRESRVATIOON PROCESSES

•Add cryoprotectant

First Concept

•Cool cells to -196 & store

Second Concept

•Warm/Thaw

Third Concept

•Remove cryoprotectant

Fourth concept.

Disadvantage and benefits of vitrification

ResultsTimeCost

vitrification

Slow freezing

*

Variables Variable that effect vitrificationof vitrification that can profoundly influence its effectiveness

19

Summary of the primary benefits of vitrification

20Biology of Reproduction December 1, 2002 vol. 67 no. 6 1671-1680

Over view of current work.

DMSOPROPYLENE GLYCOLGLYCEROL

OTHER SACCHARIDESOSMOTIC ALTERATION

CPA

HEPES BUFFERED MEDIA

VITRIFICATION SOLUTIONS

Are all vitrification and warming solution same ?

Yes and No

MOPS

Sol-A: 7.5% of EG +

7.5% of DMSO

Sol-B: 15% of EG + 15% of

DMSO + 1M of SUCROSE + FICOLL (10 mg/ml)

ES: 7.5%

of EG

+ 7.5% of

DMSO

VS: 15% of EG + 15% of DMSO +

0.6M of SUCROSE

Sage Bio Pharma

V1:

BUFFER

V2: EG + Propanediol

V3: EG + Propanediol + FICOLL

Vitrolife

ES: 7.5%

of EG

+ 7.5% of 1,2

Propanediol

VS: 15% of EG + 15% of 1,2

Propanediol + 0.5M of SUCROSE

Medi Cult

Rama Raju et al

Composition of Vitrification Solutions

1M Sucrose

0.5M Sucrose

0.25M Sucrose

1M Sucrose

0.5M Sucrose

Sage Bio Pharma

W1:

Sucrose

W2: Sucrose

W3: Sucrose

Vitrolife

Medi Cult

0.125M Sucrose

1M Sucrose

0.5M Sucrose

0.25M Sucrose

Rama Raju et al

Composition of Warming Solutions

Carrier

Cryoloop,cryotip,pulled straw, electronic grid

Vitrification and slow freezing similar results

Japanese group

vajata

We are working on removal of water from

oocyte and working on that

Vitrification of 2 pronuclei

DAY 2 OR DAY 3 EMBRYO

33

Copyright restrictions may apply.

Devroey, P. et al. Hum Reprod Update 2009 15:391-408; doi:10.1093/humupd/dmp012

The odds of post-thawing survival rate of cleavage-stage embryos after vitrification or slow freezing

Our first vitrification twins

Blastocyst

38

Blastocyst expanded or hatched

39

Safety issues

Ultraviolet light to sterilize the liquid nitrogen

IVF children

Use of hospital services

Long hospitalizatio

n

Increase risk of infection epilepsy

Cerebral palsy

Sleep disturbances Convulsions Behavior

problems

Congenital malformation

Need for long tern follow up to look deeply

Safe?

cryopreservation children

Need for long tern follow up to look deeply

?

Perinatal outcomes in vitrified oocytes

Neonatal outcome of day 3 embryos

Perinatal outcome of vitrificaion blastocyst

acknowledgement Hans Tinneberg my mentor

summaryVisakhapatnam Beach .