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Damia Castello, an Embryologist from IVI Spain presented on vitrification of embryos post bipsy with cryotop method at the RI Istanbul Conference and Workshop on the 23rd and 24th January 2014. He covered the following: - Injuries during cryopreservation - Strategies to minimize injuries - Cryopreservation procedures - Vitrification & Cryotop vitrification method - Vitrification Protocol Embryo - Vitrification in PGD blastocysts
Citation preview
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•"
www.dibimed.com Dibimed
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!!