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
