Damià Castelló

www.dibimed.com

Vitrification of embryos postbiopsy with

Cryotop Method

Injuries during cryopreservation

Influenced by

Species Stage

Mechanism

Ice crystal Toxic Osmotic Chilling etc.

Factors

SizeShape Permeability Quality Sensitivity

Strategies to minimize injuries

o Slow freezing:

An attempt to maintain a balance between various sources of injury byusing low CPA concentration and controlled ice formation.

o Vitrification:

Radical elimination of ice formation, and to reduce toxic and osmotic damage.

Cryopreservation procedures

Slow Freezing

Transformation of a liquid in solid with formation of ice. It is essential that the ice does not form inside the cell:

o Use of CPAs at low concentration

(1,5M). oCooling rate (0,3ºC/min).

oProgrammable freezers.

Vitrification

Transformation of a liquid in very viscous solid with no ice:

oHigh CPAs concentration (3-6M).

o Very high cooling rates: 15.000 a 30.000ºC/min.

oDirect immersion into liquid Nitrogen.

Vitrification

Solidification of water or water based solutions without ice crystal formation.

It is facilitated by:

High CPA concentration (3-6M)

High cooling rates:15.000 to 30.000 ºC/min

Direct immersion into LN

Cryotop Vitrification MethodWhat is?It is a MVC device, consisting of a fine polypropilene strip, which allow the oocytes to be vitrified within very low amount of volume (0,1µl).

Vitrification Kit Thawing Kit

o Valid for all stages of development, since Oocyte to Blastocyst.o Cryotop SC allows vitrification in closed system for storageo Vitrification Media is synthetic (free of albumin) to reduce risks.o Threalose (endotoxin free) replaced Sucrose for more safety.o Vitrification up to 16 Oocytes, PN, Embryos or Blastocysts in 20 min.o Thawing up 16 Oocytes, PN, Embryos or Blastocysts in 20 min.

KITAZATO CRYOTOP VITRIFICATION:

Protocol

Vitrification Protocol Embryo

Vitrification Protocol Embryo

Vitrification in PGD blastocysts

Our experience in IVI:

December 2011 to August 2013.

81 pacients.

257 Blastocysts.

•When we biopsy Trophoectoderm?

Assisted Hatching

Vitrification Trophoectoderm Biopsy

Survival:

Biopsy doesn’t afect survival after thawing using Cryotop Method.

Survival Not Survival Survival Rate

Euploid 54 4 95.2%

Aneuploid 217 10 92.6%

Total 271 14 94.8%

Clinical Outcomes:

n %

Patients Not analysed Blastocysts 8 9.9

Patients Not Transfered Blastocyst 27 33.3

Patients Transfered Blastocyst 36 14Clinical Pregnancy Rate/cycle 30.8Clinical Pregnancy Rate/transfer 69.4

Implantation rate 62.0

A total of 25 pregnantpatients:

Live Born + Ongoing 86.9%

n

Deliveries 6

Ongoing 14

Miscarriage 3

Not call response 2

Results

Outcomes of vitrified earlycleavage-stage and blastocyst-stage embryos in a cryopreservati on program: evaluation of 3,150warmi ng cyclesAlla Cobo. Ph 0 . l\1arill J o s d f tos Santos. Ph 0 ,. Oa'llia Castel o. Ph O.. Pi ar Garniz. Ph.D.•Pi arCampos. M.L.T., a..,d J01:e Remoh1, M.O.

0 2 0 3 0 5 0 6

n (o/o)

147497

n (%)

1 ,7253,491

n (o/o)

6751 079

n (%)

603952

No. of V11dm11•19 cyclP\No. of warmed embryos

,.,

. . INo. of c>mluyc>'> r!'p l.1u•d lm r .11

+SD):>SO (1 9 - 0.8)"

3,057 ( I 8 ± 0 .6)"

(1.5 - 0.6)0lrnpldnl.itm1

r. i t"CPFVcycle

76 177.2)'59 (l0 .1)r

1.058(34.6)b.c

708 ( 1 0)

306 (34 9i•.<751 (41 fiJ

44129.9) 571 (33.1)ar; er

. ' I...r 11<,e ..uridqp rr1h>

I r; 03 I ) 123 (17.3)

56 (18 9) 73 (29)DfVcycle LBfVcyde

44 (29.9) 570 ( 'l.D)677 (39.2;•

235 •..:M 8)

178 <29.515 l 1.h.3>° l 1 4 \40

6.o.t>190 <3z.s 1•"

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Oocyte Vitrification in ART

100%

80%

60%

40%

20%

0%

CRYOTOP Survival rates published worldwide

124Oocytes

Rienzi 2010

3.286Oocytes

Cobo 2010

153Oocytes

Nagy 2009

3.491Embryos

Cobo 2012

298Embryos

Tseng- Kailing 2010

2.031Blastocysts

Cobo 2012

2.543Embryos

Wenhau Shi 2012

192Oocytes

Krinos 2011

442Blastocysts

Dandan Zhu 211

186Oocytes

Ching- Chien

Chang 2013

Thank you!!