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Findikli-UARM Talk-1

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Establishing successful cryopreservation
programme in the clinic:
Laboratory aspects
Necati FINDIKLI, Ph.D.
Scientific Director
Bahceci Women’s Health Group, Turkey
Bahceci Women’s Health Group
Istanbul-Turkey (2)
Diyarbakır-Turkey
Famagusta-N.Cyprus
Erbil-N.Iraq
Baghdad-Iraq
Pristina-Kosovo
Sarajevo-Bosnia
Baku-Azerbaijan
Ankara-Turkey
Nothing to disclose…
First IVF Baby from frozen embryos “Zoe Leyland” was born in Melbourne, in
March, 28th, 1984.
Dr. Alan Trounson
Dr. Carl Wood
Since then, the approach has become the integral part of every IVF centre.
Used mainly in cases with good quality surplus embryos and in cases with
severe OHSS.
Oocyte Cryopreservation
• 1978 - First IVF birth, in 1978 (UK)
• 1982 - First birth from frozen oocyte (Australia)
• Late 1980s : successful pregnancies
by slow freeze – rapid thaw protocols
• There was a lack of progress due to
technical concerns & low success rates
•
•
•
•
Due to:
Extraordinary volume and poor membrane conductivity
instability of microtubules & microfilaments
Zona hardening
Oocyte Cryopreservation
•
Early 2000s:
• Enforcing effect of legislative restrictions
• Introduction of vitrification
• Use of ICSI after warming of oocytes
has improved the outcome and oocyte cryopreservation has found wider
applications.
•
In 2013: ASRM
four RCTs:
•
•
•
•
has lifted the experimental label on oocyte cryopreservation following
Cobo et al., 2008
Cobo et al., 2010
Rienzi et al., 2010
Permegiani et al., 2011
(Studies stated that vitrified/warmed oocytes can result in similar fertilization and
pregnancy rates compared to fresh oocytes)
How to minimize the risk of OHSS?
Addition of antagonist (to prevent premature LH surge)
Triggering oocyte final maturation by a GnRH agonist
Resulted in “Luteal-phase defect”
Can it be corrected by deferring ET by vitrification?
subsequent warming and ET
(Humaidan et al. 2005; Kolibiniakis et al., 2005; Griesinger et al. 2007;Blockeel et al. 2015)
In freeze-all cycles, the primary aim is to improve the
endometrial conditions & receptivity”
“Segmentation of ovarian stimulation”
Cycle
Day 2/3
OPU
In Vitro Emb. Culture
COH
Cycle
Day 2/3
Risk of OHSS
OPU
COH
Embryo Transfer
In Vitro Emb. Culture
Frozen Embryo Transfer
Embryo Freezing
Elevated P levels
(Devroey et al. 2011)
(Roque et al. 2013)
(Blockeel et al. 2015)
• Treatment protocol-related reasons
•
Save the surplus embryos from wastage
•
Reduce the risk of OHSS during COH
•
Improve the endometrial receptivity
• Procedure/patient-related reasons
• Save time for embryo manipulation
•
Create a psychosocial comforting period between OPU – ET
• Clinical outcome-related reasons
•
Minimize the risk of multiple pregnancies
•
Maximize the PR by embryo accumulation
•
Increase the cumulative PR rate
Establishing a successful cryo program
Pressure from the clinic
Technical Problems
Education/Experience
Extra time and
space requirements
Variable results
Gossips/Rumors
Cryopreservation –Main steps
 Exposure to Cryoprotectant
 Cooling (slow/rapid) -196 °C
 Storage at low temperature
 Thawing/warming by gradual rehydration
 Dilution and removal of Cryoprotectant
 Recovery and return to a physiological environment
Types of Cryo Protocols
13
Types of Cryo Protocols
Wong et al ., 2014
14
Physical & Chemical Factors
Physical and chemical factors of cryopreservation with potential censequences for
biological structures
Physical Factors:
• Fluctuation of osmotic pressure
• Mechanical forces of ice crystals
• Electrical tension between crystals
• Increased hydrostatic pressure
Chemical Factors:
• Redistribution of the ionic components
• Changes in pH
• Phase transition in biopolymers
• Redox transformation
• Specific (toxic) and non-specific effects of cryoprotectants
• Free radicals
(Kopeika et al. 2015)
Cryoinjury (Cryodamage)
Main elements responsible for cryoinjury:
•
•
•
•
Solution effect
Intracellular ice formation
Minimal cell volume
Phase transition of membranes
Main factors to be considered
1.
2.
3.
4.
5.
6.
7.
Initial quality&type of the material,
The conditions under which the material is obtained,
Cryoprotectants/commercial kits to be used,
Length of croyoprotectant exposure,
The type of carrier/loading device,
Rate of cooling,
Rate of warming.
Gamete /embryo quality
All gametes/embryos are equal, but some are “more equal” than the others
• Modifications in protocols and handling should be needed based on the
“nature” of the gamete/embryo (high or poor quality)
• Cryopreservation media should be improved in order to minimize the
stress (antioxidants, free radical chelators)
Gamete/embryo quality
Gamete/embryo culture conditions
Gamete/embryo culture conditions
What happens if the culture is not optimal?
• Low cleavage rates
• Fragmantation and unequal cleavage
• Low blastocyst development (# and quality)
• Low viability
• Low cryotolerance
Effect of Temperature on oocytes

Changes in temperature cause defects in meiotic spindle.

Even after temperature is stabilized, deficiencies in the
alignment of chromosomes during division causes aneuploidy.

Changes in temperature also cause defects in cell membrane.

Molecular transport is blocked and metabolic reactions are
severely affected.
Types of Cryoprotectants
Membrane-permeating CPs:
Membrane non- permeating CPs:
(Intracellular)
(Extracellular)

Proplene glycol (PG)

Dimethylsulfoxide (DMSO)

Glycerol(Gly)

Ethylene glycol (EG)

1,2 Propanediol (PROH)

Butylene glycol(BG)
 Ficoll

Methanol (Met)
 Dextran (Dex)
 Sucrose
 Trehalose
 Lactose
Sugars
 Raffinose
 D-mannitol
 Polyvinylpyrrolidine (PVP)
Polymers
 Polyethylene glycol (PEG)
23
Vitrification
Slow Freezing
Exposure to Cryoprotectant
Warming
•
•
•
•
1.0 M Sucrose
Thawing
Solution
0.5 M Sucrose
Dilution
Solution
No Sucrose
Washing
Solution
Type of Carriers/Loading Device
Open Pulled Straw
Cryoloop
Cryoleaf
Vitriplug
Cryotop
…
Open Pulled Straw
Cryoloop
Cryotop
Kitazato
Cryotip
Cryolock
CryoBioSystem HSV straw
Rapid-i,RapidStraw
Cryotop® SC
…
26
ALPHA Consensus meeting on cryopreservation
ALPHA Consensus meeting on cryopreservation:KPIs
ALPHA Consensus meeting on cryopreservation:KPIs
Continuous QA/QC
Monitorization in the lab
Procedures
Treshold level %
Oocyte Cryopreservation
Oocytes with normal morphology
…
Fertilization rate
…
Degeneration rate after ICSI
…
Cleavage rate on day 2
…
Cleavage rate on day 3
…
% of Grade I embryos on day 3
…
Blastocyst development rate
…
Embryo Cryopreservation
Survival rate after vitrification
…
Cleavage rate after warming
…
What is the best strategy?
• Embryo selection vs. pooling
• Timing of embryo cryopreservation
• Subsequent ET or extended culture after warming
Day2/3 cryo
Warming
Day 2/3 ET
Warming
24 h culture
Warming
Extended culture
Warming
<Day 5/6 cryo
Num. of embryos to be frozen
Embryo selection capacity
Cycle cancellation rate
Day 3/4 ET
Day 5 ET
Day 5 ET
Cleavage-stage
Blastocyst-stage
High
Lower
Lower
High
Low
Higher
(Wong et al. 2014)
What is the best strategy?
“Clinical implementation”
Day2/3 cryo
Warming
Day 2/3 ET
Warming
24 h culture
Warming
Day 3/4 ET
Extended culture
Warming
<Day 5/6 cryo
Day 5 ET
Day 5 ET
Bahceci Fulya IVF Centre
2012
2013
2014
2015
2016
Num. of total OPU cycles
2284
2680
3149
3733
4169
Freeze-all cycles (%)
37.0
42.9
56.4
70.5
81.2
What is the best strategy?
“Optimal strategy”
Embryos with
expansion degree
>3CC
Grade 1-2 embryos
<20% fragmentation
The rest –culture to blastocyst stage
Freezing
Day2/3 cryo
<Day 5/6 cryo
Re-freeze the
embryos with >3CC
Grade 1-2 embryos
<20% fragmentation
Culture to blastocyst stage
Warming
Warming
FET
Freeze-all for poor responders?
“Minimizing the drop out rate”
OPU Total Cryo
COH
OPU Total Cryo
COH
Thaw ET
FET
COH
OPU
Or in combination with Fresh cycle
(Cobo et al. 2012)
Cryopreservation in PGS V2
COH
OPU
Total
Cryo
after
Day 5/6 Biopsy
Genetic
Result
Abnormal
FET
Normal
Thaw
ET
Cryopreservation for pET
COH
OPU Total Cryo
FET
Thaw
ET
(Ruiz-Alonso et al. 2013)
Endometrial Receptivity Array (ERA)
(Findikli et al. unpublished)
Financial aspects
•
•
•
•
•
Cost of cryopreservation/warming materials,
Cost of storage tanks & LN2,
Cost of additional biologists/technicians,
Cost of endometrial preparation or NC monitoring.
…
Oocyte freezing
• Fertility preservation in cancer patients
• Oocyte cryopreservation for medical reasons
•
Endometriosis, autoimmune diseases etc.
• Oocyte Donation
• Elective oocyte cryopreservation (social freezing)
•
Age-related fertility decline
• “Emergency" oocyte cryopreservation
Argyle et al., 2016
Social oocyte freezing
Concerns
• Procedure-associated risks?
• Proper consenting?
• 1468 women undergoing elective oocyte cryopreservation for non-oncologic reason
• 137 returned to use them – pregnancy rates were found to be age-dependent
• Optimal number of stored MII oocytes should be at least 8 – 10.
Cobo et al., 2015
Unknown Effects of Cryopreservation
Unknown Effects of Cryopreservation
Unknown Effects of Cryopreservation
44
‘Oocyte vitrification does not increase adverse obstetric and perinatal outcomes in
children conceived with vitrified oocytes’
‘Highly successful cryopreservation of all embryo developmental stages is possible’
‘There are no variables clearly exerting a negative effect on the survival and delivery rates.’
46
47
• Although current literature favors freeze all approach, we
still need strong evidence (Grade A) such as improved
live birth rates from properly planned RCTs or large
observational studies.
• The main hurdle which limits the wider application of
this strategy is the existance of considerable differences
in cryopreservation and FET strategies in clinics and labs.
• Once the standards are established, it will soon be an
integral part of an IVF clinic.
• In order to use the strategy in its optimal terms:
• A Clinic must:
• Have an exceptional postwarming survival rates (at least >85%)
• Have a flexible and patient-friendly clinical workup
• A Laboratory must:
•
•
•
•
Have a dedicated laboratory area
Have good quality materials for cryopreservation
Have predetermined gamete/embryo selection methodology
Have experienced full-time staff (For freezing/warming only)
Success in Assisted Reproductive Techniques depends
• Not only on:
– Luck
– Sophisticated infrastructure and technological
equipments
• But mainly on:
–
–
–
–
Well-educated and experienced staff,
Harmonized and consistent team work,
Good corporate organization,
Appropriate total quality management policies
Can we have an exceptional Cryo programme?
• The answer can probably be yes,
“..but only if you have a proper QA/QC program and
handle every case individually”
Bahçeci Women’s Health Group- IVF Laboratories
Dr. Necati FINDIKLI (Director)
Embryology & Andrology & R&D & Genetics Laboratories
Turan AKSOY
Hakan YÜCETAŞ
Asuman İŞLER GÜLEL
İbrahim Orçun OLCAY
Bilgen ÖZTÜRK
Elif YALÇIN
Sinan YILDIZ
Erkan ALPARSLAN
Büşra DEMİRARSLAN
Berkay AKÇAY
Eyüp ELİFOĞLU
Dilek CENGİZ ÇELİK
Osman CİNGÖZ
Çisem LİMANDAL
Müge YILMAZ
Önder ÇOBAN
Münevver ÇOBAN SERDAROĞULLARI
Cihan GÖKTAŞ
Oğuzhan HACIFAZLIOĞLU
Meral GÜLTOMRUK
Betül ÇAPAR
Çağrı OĞUR
Oya ŞAHİN
Serkan KARKİLİ
Selman YILDIZ
Gürkan YURTTAŞ
Asuman ARSLAN
Mevlüde GÜL
Sıla TEMİZ
Burak DOĞAN
Kağan BAKAN
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