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 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 -50°C/min RT -6°C LN2 (196°C) Slow cooling Programmed equipment (Planner) Equilibration with the cryoprotectants Drop in LN2 Time Seeding -2°C/min °C/min 1-3hr Time Vitrification Vitrification Dewar container Drop in LN2 5-10min

Concentration of cryoprotectants Permeable Low MW Non Permeable Low MW High MW Vitrification Ethylene glycol Ethylene glycol DMSO DMSO Erythritol Erythritol Intercellular cryoprotectant SucroseTrehalose Dehydration Ficoll, PEG Extracellular cryoprotectant M M 10 mg/ml Slow cooling 1,2 propanediol DMSOGlycerol SucroseTrehalose M M

-6°C -35°C - 0.3°C/min Equilibrium freezing Slow freezing Equilibrium freezing Rate of cooling Ice crystal Seeding LN2 cristallisation mechanical damage induce by ice crystal high Intracellular water efflux

Vitrification Rate of cooling LN2 RT 2.000°C °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 : °C/min –Ultra-rapid: °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 Straw EMGrids Cryoloop Hemi-Straw Cryotips 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. prevent intracellular ice formation and reduce the toxicity of high concentration of cryoprotectant and chilling damageThereby, prevent intracellular ice formation and reduce the toxicity of high concentration of cryoprotectant and chilling damage

BLASTOCYST VITRIFICATION

First pregnancy in 1985 Cohen J Slow Freezing protocols Seeding –6°C0.3°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 and per cent (p = 0.89), whereas hatching rates were and 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.ShrinkageBlast-Exp.B Vtirification cycles39 No.of thawed B-ExpB71 Viability, %53 (70.6%) Delivery /vitrif.21% Implantation18.4% VDZ Hum. Reprod 2002 Artificial Shrinkage No Art.ShrikageBlast-Exp.B Vtirification cycles39 No.of thawed B-ExpB108 Viability, %43.3% Delivery /vitrif.12.8% Implantation4.5%

Needle used shrinkage Laser pulse shrinkage Mukaida et al., 2006 Artificial Shrinkage

10%DMSO-10%EG 20%DMSO-20%EG 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 0,5M 4-5 min. PBS - 20% HSA PBS - 20% HSA Sucrose 0,25M 2 min. LN2 Culture for 4 h or 24 h in culture media before embryo transfer Cleavaged Stage Embryo/Blastocyst Thawing Procedure in Memorial Hospital IVF Labs Sucrose 0,125M 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 Stage 6-8 Cell Stage No.of thawing cycles 8152 Patients’age,♀ 30.2 ± ± 4.3 Survived / Vitrified rate (%) 78.1 % 73.4 % No.of transfer / ET 3.03 Β-hCG pregnancy rate 55.6 a % 40.8 b % Clinical pregnancy rate 40.7 a % 26.5 b % Implantation rate 18.5 a % 8.7 b % Comparation of Survival and Pregnancy Rates of 2 nd and 3 rd Day Cleavaged Stage Embryos Cryopreserved by Slow Freezing Memorial Hospital Results Chi-square test; p < 0.05

Slow Freezing (2-3 Day)Vitrification (3-4 Day) No.of thawing cycles Patients’age,♀ 30.7 ± ± 5.3 Survived / Vitrified rate (%) 76.6 % 85.2 % No.of transfer / ET Β-hCG pregnancy rate 50.7 a % 40.1 b % Clinical pregnancy rate 34.7 % 32.9 % Implantation rate 18.5 % 16.2 % Survival and Pregnancy Rates with Cleavaged Stage Embryos Cryopreserved by Either Slow Freezing As Compared with Vitrification Memorial Hospital Results Chi-square test; p < 0.05

VitrifiedFresh No.of thawing cycles Patients’age,♀ 30.4 ± ± 4.5 Survived / Vitrified rate (%) 755 / 851 (88.7) No.of transfer / ET Β-hCG pregnancy rate 56.3 % 67.5 % Clinical pregnancy rate 44.9 % 64.4 % Implantation rate 23.6 % 39.6 % Survival and Pregnancy Rates with Blastocysts Cryopreserved by Vitrification Memorial Hospital Results

No.of thawing cycles 38 Patients’age,♀ 32.4 ± 3.6 Survived / Vitrified rate (%) 85.2 No.of transfer / ET 2.6 ± 0.7 Β-hCG pregnancy rate 52.6 % Clinical pregnancy rate 42.1 % On-Going pregnancy 26.3 % Implantation rate 17.0 % Survival and Pregnancy Rates with Blastomere Biopsied Cleavaged Stage Embryos Cryopreserved by Vitrification Memorial Hospital Results

a d b a dc a,b; p<0.05 c,d; p<0.05 Cleavaged stage Embryos Cryopreservation Vs Blastocysts Vitrification

Evolution of the vitrification techniques Classical virification Ultra-rapid virification vitrification Vitrification + shrinkage Ultra-rapid Vitrification Ultra-rapid Vitrification + Zona hatching

Conclusions Primary Benefits of Ultra-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 °C/min Minimizes osmatic injuries Very simple protocols Eliminates the cost of expensive programmable equipment

Conclusions Variables of vitrification 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 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 REPRODUCTIV TECHNIQUESDIRECTOR: Prof. SEMRA KAHRAMAN MD. IVF CLINIC SEMRA KAHRAMAN MD. GÜVENÇ KARLIKAYA MD. HALE KARAGÖZOĞLU MD. AYNUR ERŞAHİN MD. MÜSTECEP KAVRUT MD. MUSTAFA ACET MD. NUR DOKUZEYLÜL MD. ŞEREF SARICA MD. CRYO / EMBRYO / ANDROLOGY CO CULTURE LABORATORY SEVIL UNAL Bio. HAKAN YELKE Bio. GÜNSELİ CENGİZ Bio. ZAFER ATAYURT Bio. YEŞİM KUMTEPE Bio. SEMRA MILIK Bio. ŞEBNEM ÜNVER Bio. ÖZLEM YUVACAN Bio. FERHAT CENGİZ Bio. SERKAN SELİMOĞLU Bio. ANDROLOGY Assoc. Prof. SEMİH ÖZKAN MD. PERINATOLOGY CİHANGİR YILANLIOĞLU MD. ALTUĞ SEMİZ MD. REPRODUCTIVE GENETICS FRANCESCO FIORENTINO PhD. GÜLAY ÖZGÖN MD. MOLECULAR GENETICS BAHAR İSMAİLOĞLU Bio. SELMA DEMİR FISH ÇAĞRI OĞUR Bio. ÇİĞDEM ÇINAR Bio. CYTOGENETICS ÖZLEM ÖNER Bio. ÖZLEM ÖNER Bio. ÇİLEM ASLAN Bio. RESEARCH AND DEVELOPMENT N.ZAFER CANDAN Mol. Bio. PUBLIC RELATIONS KÜBRA BURNAZ PATIENT RELATIONS ZEHRA ÖZKAL YASİN İZGİ I.T. DEPARTMENT AYHAN EMİNOĞLU SİBEL BEYAZAY İLETİŞİM YELİZ SOYDAN CEREN ERDEM NURSING YASEMİN GÜLER SAİME TEPEBAŞ SELVER ÇİÇEK CANAN YILMAZ HATİCE ALDEMİR DERYA SİVRİ SAFİYE SARIKOÇ HANDE MUTLU SEMA KANAT GÜLAY ERYİĞİT SEYHAN GÜNDÜZ INFORMATION DERYA ŞAHİN SEVİL TAULLAH AYŞEGÜL BEZK SEVDA UYANIK SELEN EMRE TURAN NURAN SEYVAN BÜŞRA DURMAZ GÜLŞEN TINKIR AYŞEN KALAYCI İLKAY AL ARCHIVE MUHAMMED KENAR RAMAZAN ÇALIK FINANCE AYŞEGÜL BEZK BETÜL YAVAŞ