EMBRYO FREEZING AND BLASTOCYSTS VITRIFICATION Khalid Mohammed Karam Obstetrics, Gyne. & ART specialist (PhD)

CRYOPRESERVATION OF LIFE To enable and interfere the biological clock and 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.

WHY CRYOPRESERVE HUMAN EMBRYOS? 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. Ovarian hyperstimulated syndrome can be prevented by freezing all embryos instead of transfering.

Two procedures have been used so far for gamete and embryo cryopreservation SLOW FREEZING VITRIFICATION

SLOW FREEZING Expensive equipment Higher incidence of intracellular ice formation Low concentration of cryopropectant, less toxic cost-effective

VITRIFICATION Vitrification is the solidification of a solution at low temperature without ice crystal formation Liquid Phase Solid phase Amorphous state/ Vitrified state /Glassy state No ice crystal Processus par lequel un liquide se solidifie sans formation de cristaux de glace Possible osmotic stress Absence of mechanical injury CRYOPROTECTANT TOXICITY??

How to avoid ice crystal formation? 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 Amorphous state On essaye d’éviter la formation de cristaux par: Concentration élevée en CP diminution du point de fusion augementation de la viscosité Vitesse de Refroidissement très rapide qui est influencée par la taille de l’échantillon à congelé et par le type de support des ovocytes ou des embryons.

SLOW FREEZING VERSUS VITRIFICATION

Slow cooling Programmed equipment (Planner) -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 - 0.3 °C/min 1-3hr Time Vitrification Dewar container Drop in LN2 5-10min One of the main difference concerns the concentartion of CP that is very high in the vitrificcation step as compared to the slow freezing procedure.

Concentration of cryoprotectants Slow cooling 1,2 propanediol DMSO Glycerol Sucrose Trehalose 0.2-1 M 1- 1.5 M Vitrification Ethylene glycol DMSO Erythritol Intercellular cryoprotectant Sucrose Trehalose Dehydration Ficoll, PEG Extracellular 10-20 M 0.5-0.75 M 10 mg/ml Permeable Low MW Non Permeable High MW

Rate of cooling Slow freezing Equilibrium freezing - 0.3°C/min Seeding Slow freezing Equilibrium freezing -6°C - 0.3°C/min Intracellular water efflux -35°C Ice crystal The first strategy to prevent ice formation was to adopt a long slow-cooling stage. The traditional slow-rate freezing can be regarded as an equilibrum freezing. In fact we for each apparition of a new extracellular ice crystal we observed an increase of the extracellular osmolarity followed by an efflux of the water and a reduction of the volume of the cell with as consequence an increasing in the intracellular osmolarity. LN2 Equilibrium freezing cristallisation mechanical damage induce by ice crystal high

Rate of cooling Vitrification High conc. Cryprotectants 2.000°C -20.000°C/min RT High conc. Cryprotectants High cooling rate High viscosity The vitrification technique is a radical change from the conventional equilibrum cooling methods Vitrification is a process which, by combining the use of concentrated solution with rapid cooling, avoids the formation of ice.The sample is solidified by an extreme elevation in viscosity and reach low T° in a glassy state, which has the molecular structure of a viscous liquid and is not crystalline. Under such rapid cooling, water molecules don’t have time to arrange themselves into a crystalline lattice structure. The advantage of vitrification techniques, is that damaging intracellular ice is not formed and effects of extarcellular ice formation, ice crystal growth and thawing are avoided. LN2 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 Cryoloop Open Pulled Straw EMGrids Cryoloop Hemi-Straw Cryotips Hemi-Straw EMGrids Open Pulled Straw

Advantages of Ultrarapid Vitrification Increase the cooling and warming rates. Direct contact between a small volume of vitrification solution and LN2. Thereby, 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°C 0.3°C/min -37°C Glycerol 10% Since the first human pregnancy resulting from a frozen blastocyst in 1985 (Cohen), a standardized slow freezing technique has become an integral part in most ART laboratories.

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

Artificial Shrinkage Art.Shrinkage Blast-Exp.B Vtirification cycles 39 No.of thawed B-ExpB 71 Viability, % 53 (70.6%) Delivery /vitrif. 21% Implantation 18.4% No Art.Shrikage Blast-Exp.B Vtirification cycles 39 No.of thawed B-ExpB 108 Viability, % 43.3% Delivery /vitrif. 12.8% Implantation 4.5% VDZ Hum. Reprod 2002

Cleavaged Stage Embryo/Blastocyst Vitrification System in Memorial Hospital IVF Labs In-house made solution equilibration with the cryoprotectant solution - vitrification 20%DMSO-20%EG Ficoll 400 MW – 0,65M suc. 10%DMSO-10%EG 2 min-4min 40 sec. Hemi-straw system Direct Plunge into LN2 Artificial Laser Shrinkage

Thawing is carried out 4h or 24 h before transfer at RT (22-25°C) Cleavaged Stage Embryo/Blastocyst Thawing Procedure in Memorial Hospital IVF Labs Thawing is carried out 4h or 24 h before transfer at RT (22-25°C) LN2 Sucrose 0,25M 2 min. Sucrose 0,125M 2 min. 4-5 min. Sucrose 0,5M PBS - 20% HSA 1 min. Culture for 4 h or 24 h in culture media before embryo transfer

Artificial Shrinkage

Before laser shot After laser shot, 30s, 1min After laser shot, 3-5min Efflux of blastocoel solution After laser shot, 3-5min

Thawed Blastocysts 5hr incubation

Primary Benefits of Ultra-rapid vitrification 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 20.000-30.000 °C/min Minimizes osmatic injuries Very simple protocols Eliminates the cost of expensive programmable equipment

Variables of vitrification 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.