embryo freezing and blastocysts vitrification mol. bio.zafer nihat candan memorial hospital, art...
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EMBRYO FREEZING AND BLASTOCYSTS VITRIFICATION
Mol. Bio.Zafer Nihat Candan
Memorial Hospital, ART & Reproductive Genetic Center, Istanbul, TURKEY
CRYOPRESERVATION OF LIFE
• To enable to interfere biological clock and to stop it for a while.
• Basically, cryopreservation enable to storage the gamet cells, embryos and somatic cells by arresting or slowing metabolic activities until the subsequent thawing procedure.
HISTORY OF EMBRYO CRYOPRESERVATION
• Cryopreservation era arised with the accidentally understanding of the value of cryoprotectants in 1949 by Christopher Polge.
• By the early 1970s, Wilmut and Whittingham developed independent methods for freezing mouse embryos in DMSO.
• By the 1980s, the freezing of human embryos emerged as a common procedure in the treatment of infertile couples.
• First successfull pregnacy with the frozen-thawed cleavaged stage embryo transfer was reported by Trounson and Mohr in 1983.
• Two year later, Cohen published new procedure for human blastocyst stage embryo freezing.
• In 1985, new approach, termed vitrification, in which highly concentrated crypective agents were used, was successfully applied on mouse embryos.
WHY CRYOPRESERVE HUMAN EMBRYOS?
• Reduced multiple pregnancy in any one treatment cycle and provides storage of good quality surplus embryos for later use.
• Preserved embryos in initial attempt obviate the need for repeated surgery and high hormone induction to obtain eggs in subsequent trial.
• Thaw cycle is cost-effective, aproximately 1/5 of that of normal IVF cycle.
• Freezing provides tha chance to detect infectious disease and genetic abnormalities, by offering extended time for proper screening and analysis.
• Cyropreservation of embryos, which would otherwise be discarded, allow parents to have additional child if they wish.
• Overian hyperstimulated syndrome can be prevented by freezing all embryos instead of transfering.
WHY CRYOPRESERVE HUMAN EMBRYOS?
SLOW FREEZING
VITRIFICATION
Two procedures have been used so far for gamet and embryo cryopreservation
SLOW FREEZING
Expensive equipment Higher incidence of
intracellular ice formation Low concentration of
cryopropectant, less toxic Not cost-effective ??
VITRIFICATION
No ice crystal
Absence of mechanical injuriy
Possible osmotic stress
Liquid Phase Solid phase
Amorphous state/ Vitrified state /Glassy state
Vitrification is the solidification of a solution at low temperature without ice crystal formation
CRYOPROTECTANT TOXICITY??
Use of high concentration of cryoprotectant provides:
liquid/solid transition temperature
high viscosity
impossibility for the molecules to re-arrange
High speed of cooling, which results from
low volume of cryoprotectant (~1-2µl)
type of carrier
How to avoid ice crystal formation?
Amorphous
state
SLOW FREEZING VERSUS
VITRIFICATION
-35°C-35°C
-50°C/min
RTRT
-6°C-6°C
LN2LN2 (196(196°C°C))
Slow cooling Programmed equipment (Planner)
Equilibration with the cryoprotectantsEquilibration with the cryoprotectants
Drop in LN2
Time
Seeding-2°C/min
- 0.3 °C/min
1-3hr Time
Vitrification Vitrification Dewar container
Drop in LN2
5-10min
Concentration of cryoprotectantConcentration of cryoprotectantss
PermeablePermeable
Low MWLow MW
Non PermeableNon Permeable
Low MWLow MW
High MWHigh MW
VitrificationVitrification
Ethylene glycol Ethylene glycol
DMSODMSO
ErythritolErythritol
Intercellular
cryoprotectant
SucroseSucrose
TrehaloseTrehalose
Dehydration
Ficoll, Ficoll, PEGPEG
Extracellular
cryoprotectant
10-20 M
0.5-0.75 M
10 mg/ml
Slow coolingSlow cooling
11,,2 propanediol2 propanediol
DMSODMSO
GlycerolGlycerol
SucroseSucrose
TrehaloseTrehalose
0.2-1 M
1- 1.5 M
-6°C
-35°C
CP + CP + salt salt ++proteins proteins
11
1 1 Osmolarity increase outsideOsmolarity increase outside
2 2 Efflux Efflux of H20 of H20
33 Osmolarity increase insideOsmolarity increase inside
Solution
CP + salt
2233
- 0.3°C/min
Equilibrium freezingEquilibrium freezing
Slow freezing EquiliSlow freezing Equilibbrium freezingrium freezing
Rate of coolingRate of cooling
Ice crystal
Seeding
LN2
cristallisationmechanical damage induce by ice crystal high
Intracellular water efflux
VitrificationVitrification
Rate of coolingRate of cooling
LN2
RT
2.000°C -20.000°C/min
High conc. Cryprotectants
High cooling rate
High viscosity
amorphous state (vitrified state)
mechanical damage induce by ice crystal low
Cooling speed of vitrification
• Directly, depending on the type of used carriers;
– , Rapid : 2.500 °C/min
– Ultra-rapid: 20.000 °C/min
Conventional vitrification in straw Rapid
An Optimum Rate of Cooling
This results from the balance of two phenomena;
– At rates of cooling slower than the optimum, cell death is due to the long periods of exposure to hypertonic conditisions
– At the rates of cooling faster than the optimum, cell death is associated with intra cellular ice formation
Special Carriers of Ultra-Vitrification
Open Pulled StrawEMGridsCryoloopHemi-StrawCryotips
Open Pulled Straw
EMGrids
Hemi-Straw
Cryoloop
Advantages of Ultrarapid Vitrification
• Increase the cooling and warming rates.
• Direct contact between a small volume of vitrification solution and LN2.
• Thereby, prprevent intracellular ice formation event intracellular ice formation and reduce the toxicity of high concentration and reduce the toxicity of high concentration of cryoprotectant and chilling damageof cryoprotectant and chilling damage
BLASTOCYST VITRIFICATION
First pregnancy in 1985First pregnancy in 1985Cohen JCohen J
Slow Freezing protocolsSlow Freezing protocolsSeeding –6°C 0.3°C/min -37°C
Glycerol 10%
Regarding the poor results with slow blastocyst freezing, many groups report to switch back to day 1 and day 2-3 freeze/thaw programs
How can we improve the results of blastocyst freezing?
move to vitrification procedure? Pantos et al.,2001
The survival rates of blastocysts cryopreserved by vitrification and slow-freezing were 68.33 and 65.52 per cent (p = 0.89), whereas hatching rates were 51.22 and 44.74 per cent, respectively (p = 0.64).Therefore, vitrification of blastocyst-stage-embryos may be a useful, economic method for freezing the excess blastocysts in some centers where blastocysts are routinely transferred. (Wanvisa et al.,2003)Stehlik et al.,2005
Important parameters of blastocysts vitrification
• Blastocyst quality
• Day of vitrification, 5 to 6
• Artificial shrinkage application
blastocoel could be a source of ice crystal
===> reduction in the survival rates
Art.ShrinkageArt.Shrinkage BBlastlast-E-Exp.xp.BB Vtirification cyclesVtirification cycles 3939No.of thawed B-ExpBNo.of thawed B-ExpB 7171Viability, %Viability, % 53 (70.6%)53 (70.6%)Delivery /vitrif.Delivery /vitrif. 21%21%ImplantationImplantation 18.4%18.4%
VDZ Hum. Reprod 2002
Artificial Shrinkage
No Art.ShrikageNo Art.Shrikage BBlastlast-E-Exp.xp.BB Vtirification cyclesVtirification cycles 3939No.of thawed B-ExpBNo.of thawed B-ExpB 108108Viability, %Viability, % 43.3%43.3%Delivery /vitrif.Delivery /vitrif. 12.812.8%%ImplantationImplantation 4.54.5%%
Needle used shrinkage
Laser pulse shrinkage
Mukaida et al., 2006
Artificial Shrinkage
10%DMSO-10%EG10%DMSO-10%EG20%DMSO-20%EG20%DMSO-20%EGFicoll 400 MW – 0,65M suc.Ficoll 400 MW – 0,65M suc.
2 min-4min 40 sec.
Cleavaged Stage Embryo/Blastocyst Vitrification System in Memorial Hospital IVF Labs In-house made solution
equilibration with the cryoprotectant solution - vitrification
Direct Plunge into LN2
Artificial Laser Shrinkage Hemi-straw system
Thawing is carried out 4h or 24 h before transfer at RT (22-25°C)
Sucrose 0,5MSucrose 0,5M
4-5 min.
PBS - PBS - 20% 20% HSAHSA
Sucrose 0,25MSucrose 0,25M
2 min.
LN2
Culture for 4 h or 24 h in Culture for 4 h or 24 h in culture media culture media before embryo transferbefore embryo transfer
Cleavaged Stage Embryo/Blastocyst Thawing Procedure in Memorial Hospital IVF Labs
Sucrose 0,Sucrose 0,1125M25M
2 min.
1 min.
Artificial Shrinkage
Before laser shot
After laser shot,
3-5min
After laser shot, 30s, 1min
Efflux of blastocoel solution
5hr incubation
Thawed Blastocysts
3-4 Cell Stage3-4 Cell Stage 6-8 Cell Stage6-8 Cell Stage
No.of thawing cyclesNo.of thawing cycles 8181 5252
Patients’age,Patients’age,♀♀ 30.2 30.2 ±± 4.1 4.1 32.1 32.1 ±± 4.3 4.3
Survived / Vitrified rate (%)Survived / Vitrified rate (%) 78.1 %78.1 % 73.4 %73.4 %
No.of transfer / ETNo.of transfer / ET 3.03.0 33
ΒΒ-hCG pregnancy rate-hCG pregnancy rate 55.6 55.6 aa % % 40.840.8bb % %
Clinical pregnancy rateClinical pregnancy rate 40.7 40.7 aa % % 26.526.5bb % %
Implantation rateImplantation rate 18.5 18.5 aa % % 8.78.7bb % %
Comparation of Survival and Pregnancy Rates of 2nd and 3rd Day Cleavaged Stage Embryos Cryopreserved by Slow
Freezing
Memorial Hospital ResultsChi-square test; p < 0.05
Slow Freezing Slow Freezing
(2-3 Day)
Vitrification Vitrification
(3-4 Day)(3-4 Day)
No.of thawing cyclesNo.of thawing cycles 138138 7676
Patients’age,Patients’age,♀♀ 30.7 30.7 ±± 4.2 4.2 31.7 31.7 ±± 5.3 5.3
Survived / Vitrified rate (%)Survived / Vitrified rate (%) 76.6 %76.6 % 85.2 %85.2 %
No.of transfer / ETNo.of transfer / ET 3.03.0 2.82.8
ΒΒ-hCG pregnancy rate-hCG pregnancy rate 50.7 50.7 aa % % 40.140.1bb % %
Clinical pregnancy rateClinical pregnancy rate 34.7 %34.7 % 32.9 %32.9 %
Implantation rateImplantation rate 18.5 %18.5 % 16.2 %16.2 %
Survival and Pregnancy Rates with Cleavaged Stage Embryos Cryopreserved by Either Slow Freezing As
Compared with Vitrification
Memorial Hospital ResultsChi-square test; p < 0.05
VitrifiedVitrified FreshFresh
No.of thawing cyclesNo.of thawing cycles 167167 416416
Patients’age,Patients’age,♀♀ 30.4 30.4 ±± 5.2 5.2 30.1 30.1 ±± 4.5 4.5
Survived / Vitrified rate (%)Survived / Vitrified rate (%) 755 / 851 (88.7)755 / 851 (88.7)
No.of transfer / ETNo.of transfer / ET 2.62.6 2.32.3
ΒΒ-hCG pregnancy rate-hCG pregnancy rate 56.3 %56.3 % 67.5 %67.5 %
Clinical pregnancy rateClinical pregnancy rate 44.9 %44.9 % 64.4 %64.4 %
Implantation rateImplantation rate 23.6 %23.6 % 39.6 %39.6 %
Survival and Pregnancy Rates with Blastocysts Cryopreserved by Vitrification
Memorial Hospital Results
No.of thawing cyclesNo.of thawing cycles 3838
Patients’age,Patients’age,♀♀ 32.4 32.4 ±± 3.6 3.6
Survived / Vitrified rate (%)Survived / Vitrified rate (%) 85.285.2
No.of transfer / ETNo.of transfer / ET 2.6 2.6 ±± 0.7 0.7
ΒΒ-hCG pregnancy rate-hCG pregnancy rate 52.6 %52.6 %
Clinical pregnancy rateClinical pregnancy rate 42.1 %42.1 %
On-Going pregnancyOn-Going pregnancy 26.3 %26.3 %
Implantation rateImplantation rate 17.0 %17.0 %
Survival and Pregnancy Rates with Blastomere Biopsied Cleavaged Stage Embryos Cryopreserved by Vitrification
Memorial Hospital Results
76,6
85,2 88,7
34,7 32,9
44,9
0
10
20
30
40
50
60
70
80
90
Survival Rate Clinical Pregnancy Rate
Slow Freezing(Cleavaged stage)
Vitrification(Cleavaged Stage)
Vitrification(Blastocyst Stage)
a
d
b
a
d c
a,b; p<0.05
c,d; p<0.05
Cleavaged stage Embryos Cryopreservation Vs Blastocysts Vitrification
Evolution of the vitrification techniques
0
5
10
15
20
25
30
35
40
Ongoing pregnancyImplantation rate
Classical virificationClassical virification Ultra-rapid virificationUltra-rapid virification
vitrification
Vitrification+
shrinkage
Ultra-rapidVitrification
Ultra-rapidVitrification
+Zona hatching
Conclusions Conclusions
Primary Benefits of Primary Benefits of Ultra-rapid vitrificationUltra-rapid vitrification
•Utilizes higher concentration of cryoprotectant that allows shorter exposure times to the cryoprotectant
• Rapid vitrification/warming; reduce the cryopreservation procedure up to 10 min.
• Loading embryos in carrier in a small volume of cryoprotectants provides a significant increase in the cooling rate from 20.000-30.000 °C/min
• Minimizes osmatic injuries
• Very simple protocols
• Eliminates the cost of expensive programmable equipment
Conclusions Conclusions
Variables of Variables of vitrificationvitrification• Type and concentration of cryoprotectants, even all cryoprotectants are toxic
• Temperature of vitrification solution at exposure
• Lenght of time embryos are exposed to the final cryoprotectant before plunging into liquid nitrogen
• Variability in the volume of cryoprotectant solution surrounding the embryos
• Technical proficiency of the embryologists
• Direct contact of the LN2 and the CP medium containing the embryos, which may be a source of contamination,
• Sealed (high security straw) use
• Sterile LN2
• Storage invapour LN2
Can be solutions to avoid contamination
Conclusions Conclusions
• Which cryopreservation technic is being used dependening on the strategy of embryo transfer day.
• Slow freezing comparing the vitrification is more effective for 2nd day embryos regarding the survival and pregnancy rates.
• On the other hand blastocysts should be vitrified unless the new slow cooling technique is adapted.
• Son slayt olarak isimler ve toplu fotoğraf konabilir
ASSISTED REPRODUCTIVASSISTED REPRODUCTIVTECHNIQUESTECHNIQUESDIRECTOR:DIRECTOR:
Prof. SEMRA KAHRAMAN MD.Prof. SEMRA KAHRAMAN MD.
IVF CLINICIVF CLINICSEMRA KAHRAMAN MD.SEMRA KAHRAMAN MD.
GÜVENÇ KARLIKAYA MD. GÜVENÇ KARLIKAYA MD. HALE KARAGÖZOĞLU MD.HALE KARAGÖZOĞLU MD.
AYNUR ERŞAHİN MD.AYNUR ERŞAHİN MD.MÜSTECEP KAVRUT MD.MÜSTECEP KAVRUT MD.
MUSTAFA ACET MD.MUSTAFA ACET MD.NUR DOKUZEYLÜL MD.NUR DOKUZEYLÜL MD.
ŞEREF SARICA MD.ŞEREF SARICA MD.CRYO / EMBRYO / ANDROLOGYCRYO / EMBRYO / ANDROLOGY
CO CULTURE LABORATORYCO CULTURE LABORATORYSEVIL UNAL Bio. SEVIL UNAL Bio.
HAKAN YELKE Bio. HAKAN YELKE Bio. GÜNSELİ CENGİZ Bio. GÜNSELİ CENGİZ Bio. ZAFER ATAYURT Bio.ZAFER ATAYURT Bio.YEŞİM KUMTEPE Bio. YEŞİM KUMTEPE Bio.
SEMRA MILIK Bio. SEMRA MILIK Bio. ŞEBNEM ÜNVER Bio. ŞEBNEM ÜNVER Bio.
ÖZLEM YUVACAN Bio. ÖZLEM YUVACAN Bio. FERHAT CENGİZ Bio. FERHAT CENGİZ Bio.
SERKAN SELİMOĞLU Bio. SERKAN SELİMOĞLU Bio. ANDROLOGYANDROLOGY
Assoc. Prof. SEMİH ÖZKAN MD.Assoc. Prof. SEMİH ÖZKAN MD.
PERINATOLOGYPERINATOLOGYCİHANGİR YILANLIOĞLU MD.CİHANGİR YILANLIOĞLU MD.
ALTUĞ SEMİZ MD.ALTUĞ SEMİZ MD.
REPRODUCTIVE GENETICSREPRODUCTIVE GENETICSFRANCESCO FIORENTINOFRANCESCO FIORENTINOPhD. GÜLAY ÖZGÖN MD.PhD. GÜLAY ÖZGÖN MD.
MOLECULAR GENETICSMOLECULAR GENETICSBAHAR İSMAİLOĞLU Bio.BAHAR İSMAİLOĞLU Bio.
SELMA DEMİRSELMA DEMİR
FISHFISH ÇAĞRI OĞUR Bio.ÇAĞRI OĞUR Bio.
ÇİĞDEM ÇINAR Bio.ÇİĞDEM ÇINAR Bio.
CYTOGENETICSCYTOGENETICS ÖZLEM ÖNER Bio.ÖZLEM ÖNER Bio.ÇİLEM ASLAN Bio.ÇİLEM ASLAN Bio.
RESEARCH ANDRESEARCH ANDDEVELOPMENTDEVELOPMENT
N.ZAFER CANDAN Mol. Bio. N.ZAFER CANDAN Mol. Bio.
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