128
Human Embryo Cryopreservation—Methods, Timing, and other Considerations for Optimizing an Embryo Cryopreservation Program Amy E. T. Sparks, PhD, HCLD1
Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa Semin Reprod Med 2015;33:128–144
Abstract Keywords
► ► ► ► ► ►
cryopreservation embryo blastocyst pronuclear stage vitrification slow freezing
Address for correspondence Amy E. T. Sparks, PhD, HCLD, Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, 40014 PFP, Iowa City, IA 52242 (e-mail: [email protected]).
The contribution of embryo cryopreservation to the birth rate per in vitro fertilization cycle has escalated from a rare subsidy to a vital tool that is called upon to augment the cycle outcome. Embryology laboratories must identify the embryo stage, quality criteria and methodology that will optimize their ability to preserve each embryo’s reproductive potential. This chapter reviews the principles of cryopreservation, outcomes based on embryo stage and cryopreservation method and benchmarks that may be employed by the laboratory to measure the performance of their embryo cryopreservation program.
Since the birth of the first child following in vitro fertilization (IVF) in 1978, there have been a remarkable number of advances in assisted reproductive technologies. Improved control and monitoring of ovarian stimulation, the use of micromanipulation methods to assist fertilization, and optimization of embryo culture conditions often result in a large number of embryos. Extended embryo culture gives embryologists the opportunity to view morphologic markers that have been shown to be indicative of an embryo’s reproductive potential.1–4 As our ability to select good quality embryos for transfer has improved, attention has shifted from merely achieving pregnancies and live births to optimizing outcomes by achieving singleton pregnancies and deliveries.5–7 To achieve this level of optimization, the IVF center must have an embryo cryopreservation program that can preserve embryos for future pregnancy attempts. When good-prognosis patients have multiple good quality embryos, clinicians should have confidence in their cryopreservation program and use it as a tool to reduce multiple births by encouraging patients to transfer a single embryo.8–11 Embryo cryopreservation is also needed when embryo transfer is not desired due to risk of ovarian hyperstimulation, suspected uterine asynchrony, fertility preservation is desired, or when awaiting preimplantation genetic test results.12–15 Improvements in assisted reproductive technologies have led to increased
Issue Theme Best Practices in In Vitro Fertilization; Guest Editor, Bradley J. Van Voorhis, MD
demand for embryo cryopreservation methods to keep pace as it is a crucial element of a successful IVF program. Cryopreservation requires embryos, to be exposed to nonphysiologic ultralow temperatures to achieve “cryogenic suspension of life” and cryoprotectants to avoid the phase transition of water to ice during cooling. Now, more than ever before, embryologists are challenged to fulfill the patients’ and clinicians’ expectation that the reproductive potential of each cryopreserved embryo will be maintained despite being subjected to the additional stress of cooling and warming. This article aims to review the principles and challenges of cryopreservation, current cryopreservation methods, and outcomes based on the embryo stage of cryopreservation, and discuss strategies that may be practiced to optimize an embryo cryopreservation program.
Principles of Cryopreservation Regardless of the cryopreservation method used, the goal is the same—to suspend embryos in time by cooling embryos from ambient, room temperature (20°C) to 196°C. This requires embryos to be exposed to an environment that they have no intrinsic ability to survive, putting them at risk for a variety of types of damage or “cryoinjury” during temperature and phase transitions. More detailed reviews of the principles
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546826. ISSN 1526-8004.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
1 Department of Obstetrics and Gynecology, Carver College of
Human Embryo Cryopreservation
þ15 to 5°C Chilling injury is defined as permanent damage that occurs before the cells are exposed to freezing temperatures. As embryos are cooled to 5°C, there is a phase transition of phospholipids from liquid to gel phase, impacting the cell membrane lipid bilayer’s fluidity, permeability.19–21 This damage is irreversible and it presents a significant challenge when cryopreserving lipid-rich embryos from some mammals. Chilling injury also may damage cytoplasmic lipid droplets, microtubules, and oocyte meiotic spindles.22,23 Mammalian embryos’ resistance to chilling injury varies with development so cryopreservation protocols must be tailored to the embryo stage.19,24
5 to 80°C Ice crystals, intracellular and/or extracellular, form when temperatures reach 5 to 80°C. The damage caused by ice crystals can be mechanical or chemical.25 In 1972 Mazur et al proposed what is now known as the “two-factor” hypothesis for cell injury at this temperature range.26 If cells are cooled slowly, formation of extracellular ice causes the solutes in the medium surrounding the embryo to become hypertonic. The advantage of slow cooling is that the hypertonic solution coaxes water out of the cell, minimizing intracellular ice formation. However, prolonged exposure to high concentrations of electrolytes can cause cell injury by the “solution effect,” destabilizing proteins and damaging the cell membrane.27 Rapid cooling may be equally detrimental as water cannot exit the cell fast enough, leading to formation of lethal intracellular ice.22
50 to 150°C Solutions may fracture at 50 and 150°C. It has been suggested that large cells or cell groups such as oocytes and embryos could be injured by fractured solutions, damaging the zona pellucida or cytoplasm.28 This remains a hypothetical risk and the frequency of injury due to solution fracture has not been reported.
150 to 196°C Embryos are stored in nitrogen vapors (190°C) or, more typically, in liquid nitrogen (196°C). Patients often ask whether prolonged storage leads to additional injury. To date, there is no evidence of damage during storage for a duration relative to a human lifetime. Accidental warming is a common cause of cryoinjury, but if storage temperatures are maintained, the embryos are essentially suspended in time as chemical reactions are halted at 120°C and storage at 196° C prohibits thermally driven reactions.29 Background radiation had been proposed as a concern, but the accumulation of direct damage from background ionizing radiation would require centuries of storage.30 Storage in liquid nitrogen poses a risk for transmission of infectious agents because the liquid is not sterile and most microorganisms can survive storage in liquid nitrogen.31 There have been no reported
129
cases of viral transmission from cryopreserved human embryos and two cases of bovine viral diarrhea virus transmission after cryopreserved embryo transfer in cattle.32,33 Though these reported infections cannot be attributed specifically to embryo cryopreservation and storage, Bielanski et al demonstrated under experimental conditions that concerns about disease transmission are justified and should be taken into consideration when selecting devices for cryopreservation and storage equipment.34,35
Warming from 190 to 20°C During warming the risks of injury are similar to those incurred during cooling. The effects of warming depend on whether intracellular ice formed or the cell was dehydrated during cooling. If intracellular ice forms, rapid thawing may rescue the cell by preventing small intracellular ice crystals from recrystallizing into bigger, harmful ice crystals. Apart from maintaining the plasma membrane that encloses cytoplasm with a seemingly viable morphology, cryopreservation techniques must protect embryos from injury that may not be apparent upon morphologic evaluation, including damage to intracellular organelles, the cytoskeleton and cell junctions.36–39 Despite all these threats to embryo’s health and survival, with the aid of cryoprotectants and established cryopreservation techniques, they have a remarkable ability to repair or bypass the damage and continue to develop after thawing.40,41
Cryoprotectants Cryoprotectants, defined by Karow as “any additive which can be provided to cells before freezing and yields a higher postthaw survival than can be obtained in its absence,” are essential for cryopreservation of cells.42 There are two classes of cryoprotective agents (CPAs): (1) permeating agents have small molecules that cross the cell’s membrane, displace intracellular water and balance intracellular solutes and (2) nonpermeating, large-molecule CPAs that maintain an extracellular osmotic gradient that aids in further cell dehydration. The examples of permeating CPAs include 1,2-propanedial (PROH), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol. Sugars serve as the nonpermeating CPAs, with lowmolecular-weight disaccharides such as sucrose and trehalose typically selected for this role. The combination of the two types of CPAs creates an antifreeze cocktail that reduces intracellular ice formation by removing water from inside the cell and, once inside the cell, depresses the freezing point of water remaining in the cell. The nonpermeating CPAs create an osmotic gradient that helps restrict water movement across the cell membrane, preventing osmotic shock when CPAs are removed during thawing. In order for a cell to survive cryopreservation water and permeating CPAs must be able to move across the cell membrane. When embryos are placed in a hyperosmotic solution with CPAs, the osmotic gradient draws highly permeable intracellular water out of the cell, causing the cell to shrink and permeating CPAs to slowly diffuse into the cell. Several factors must be considered to optimize the exchange of water for CPAs: (1) the ratio of cell membrane surface area to water volume, (2) Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
of cryopreservation in reproductive medicine have been previously published.16,17 The type of damage that occurs during cooling can be divided into four temperature ranges.18
Sparks
Human Embryo Cryopreservation
Sparks
the stage-specific permeability for water, and (3) the stagespecific membrane permeability by CPAs changes as the embryo develops with oocytes to early-stage embryos having lower permeability to CPAs than morulae and blastocysts.23,43,44 Water and permeating CPAs cross the cell membrane by two pathways, simple diffusion through the lipid bilayer that is highly dependent on temperature and through temperature-independent, hydrophilic channels formed by proteins called aquaporins.45 A recent study reported that there is a change in the type and amount of aquaporins expressed with each stage of development and these changes may contribute to the stage-specific membrane permeability to CPAs.46 A visual depiction of the channel types, membrane permeability, and change in cell size with each development stage was published in the proceedings of the Alpha consensus meeting on cryopreservation and this has been reproduced in ►Fig. 1.47
Methods There are two general methods used for embryo cryopreservation: slow freezing and rapid freezing to achieve vitrification.
Details of each cryopreservation method have been described by others.48–50 Both methods use permeating and nonpermeating CPAs that are removed in a stepwise fashion to permit controlled embryo rehydration during warming. The main differences between the methods include the duration of exposure to CPAs, concentration of CPAs, the cooling rate, and warming rate.
Slow Freezing The process of slow freezing, also known as equilibrium freezing, is designed to maintain a balance between intracellular ice formation and osmotic damage using low or nontoxic concentrations of CPAs.51–53 Prior to freezing, embryos are equilibrated in a hyperosmotic solution containing 1 to 2 M permeating CPA (PROH or DMSO for pronuclear and cleavagestage embryos, glycerol for blastocysts) with the CPA introduced in a step-wise fashion or a single step over the course of 10 to 20 minutes. Cell shrinkage is observed while the water leaves the cell and is slowly replaced by the penetrating CPA. Once equilibrium is achieved, the cell volume is restored. The embryos are moved to a final solution that has a combination of 1 to 2 M permeating and 0.2 to 0.3 M nonpermeating CPAs
Fig. 1 Pathways for the movement of water and CPAs across the plasma membrane. The movement of water becomes more efficient with embryo development to the blastocyst stage, as the Arrhenius activation energy declines. (a) Movement of CPAs and water through the plasma membrane is via simple diffusion in the early developmental stages (A1) and via channel diffusion (aquaporins) by the blastocyst stage (A2). (b) Movement of water and CPAs by simple diffusion or via channels also depends on the type of CPA. (c) The efficiency of dehydration and CPA uptake also is influenced by cell size, increasing with increasing surface/volume ration (cell size highlighted). CPAs, cryoprotective agents; DMSO, dimethyl sulfoxide; EG, ethylene glycol; GL, glycerol; H 2O, water. (Used with permission from Alpha Scientists in Reproductive Medicine. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online 2012;25:146–167). Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
130
prior to being loaded into 0.25 mL straws or cryovials and placed in a controlled-rate freezer. The embryos are cooled at a rate of 1 to 2°C/min to 6 to 8°C, which is slightly above the solution’s melting point. The CPA concentrations used for slow cooling are insufficient to prevent intracellular ice formation alone so the concentration is increased during the cooling process by induction of ice formation. Extracellular ice formation is manually induced (seeding) by touching the vial or straw with precooled forceps as far away from the embryos as possible. As water transitions to ice, extracellular CPAs and other solutes become more concentrated, creating another osmotic gradient that drives more water out of the embryo and permits additional CPA to enter the cell. The temperature of the controlled-rate chamber is maintained for an additional 10 minutes to allow the embryos to reequilibrate and then the temperature is very slowly (0.3°C/min) lowered to below 30°C. At this point the intracellular CPA is sufficient to prevent additional intracellular ice formation and the cell is plunged into liquid nitrogen. The optimal thawing rate depends on the temperature that cooling was terminated. Embryos that had cooling terminated at a high temperature (30 to 40°C) will have more water and require faster warming rates (200–350°C/min) than when cooling is terminated at 80°C and more gradual rehydration (25°C/min) is required. Rehydration is controlled by placing the embryos in a concentration of nonpermeating CPA (typically sucrose) that is twofold higher than the concentration of the final cryopreservation solution. This reduces the degree of osmotic imbalance across the cell’s membrane, permitting sufficient time for the penetrating CPAs to diffuse out of the cell and slowing the rate that highly permeable water returns to the cell.54
Vitrification “Vitrification” is a term used to describe the transformation of a substance into glass, but this term is commonly used to describe the methods used to achieve this transformation. Similar to slow freezing, cell dehydration is achieved by permeating CPAs, but unlike slow cooling, there is no attempt to maintain equilibrium on either side of the cell membrane, the time provided for dehydration is brief, the concentration of CPAs is higher, and the rate of cooling is radically higher. How do cells survive? The strategy of vitrification is to use ultrarapid cooling to transform liquid into glass by a dramatic increase in viscosity and minimize the time the sample is exposed to the temperature ranges associated with chilling injury and ice crystal formation.55 Embryo vitrification protocols are much simpler than slow freezing and there is no need for a controlled-rate freezer or other expensive equipment. Permeating CPAs (commonly a combination of EG and sucrose along with PROH, glycerol, or DMSO) are introduced with a two-step approach, exposing embryos to 50% concentration of the final CPA concentration for 5 to 15 minutes to permit intracellular water to exit the cell and CPA permeation to establish an equilibrium and limiting exposure to the final 30 to 40% solution of CPAs to the minimum amount of time (50% intact blastomeres) and 50% of all thawed embryos will have 100% of the blastomeres intact.48 A retrospective analysis more than 800 single embryo transfers (SETs) in which 36 of embryos were cryopreserved at the four-cell stage on day 2 revealed similar implantation rates (fetal heartbeat/embryo) for fourcell–stage embryos with 100% blastomere survival (26%, n ¼ 615) and 75% blastomere survival (27.5%; n ¼ 131) compared with embryos that were 50% intact (n ¼ 85).78 No comparison was made to fresh outcomes in the original article, but it was stated in later review that implantation rates for the 100 and 75% intact four-cell stage embryos (26%) for patients in the same age group (
Human Embryo Cryopreservation—Methods, Timing, and other Considerations for Optimizing an Embryo Cryopreservation Program Amy E. T. Sparks, PhD, HCLD1
Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa Semin Reprod Med 2015;33:128–144
Abstract Keywords
► ► ► ► ► ►
cryopreservation embryo blastocyst pronuclear stage vitrification slow freezing
Address for correspondence Amy E. T. Sparks, PhD, HCLD, Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, 40014 PFP, Iowa City, IA 52242 (e-mail: [email protected]).
The contribution of embryo cryopreservation to the birth rate per in vitro fertilization cycle has escalated from a rare subsidy to a vital tool that is called upon to augment the cycle outcome. Embryology laboratories must identify the embryo stage, quality criteria and methodology that will optimize their ability to preserve each embryo’s reproductive potential. This chapter reviews the principles of cryopreservation, outcomes based on embryo stage and cryopreservation method and benchmarks that may be employed by the laboratory to measure the performance of their embryo cryopreservation program.
Since the birth of the first child following in vitro fertilization (IVF) in 1978, there have been a remarkable number of advances in assisted reproductive technologies. Improved control and monitoring of ovarian stimulation, the use of micromanipulation methods to assist fertilization, and optimization of embryo culture conditions often result in a large number of embryos. Extended embryo culture gives embryologists the opportunity to view morphologic markers that have been shown to be indicative of an embryo’s reproductive potential.1–4 As our ability to select good quality embryos for transfer has improved, attention has shifted from merely achieving pregnancies and live births to optimizing outcomes by achieving singleton pregnancies and deliveries.5–7 To achieve this level of optimization, the IVF center must have an embryo cryopreservation program that can preserve embryos for future pregnancy attempts. When good-prognosis patients have multiple good quality embryos, clinicians should have confidence in their cryopreservation program and use it as a tool to reduce multiple births by encouraging patients to transfer a single embryo.8–11 Embryo cryopreservation is also needed when embryo transfer is not desired due to risk of ovarian hyperstimulation, suspected uterine asynchrony, fertility preservation is desired, or when awaiting preimplantation genetic test results.12–15 Improvements in assisted reproductive technologies have led to increased
Issue Theme Best Practices in In Vitro Fertilization; Guest Editor, Bradley J. Van Voorhis, MD
demand for embryo cryopreservation methods to keep pace as it is a crucial element of a successful IVF program. Cryopreservation requires embryos, to be exposed to nonphysiologic ultralow temperatures to achieve “cryogenic suspension of life” and cryoprotectants to avoid the phase transition of water to ice during cooling. Now, more than ever before, embryologists are challenged to fulfill the patients’ and clinicians’ expectation that the reproductive potential of each cryopreserved embryo will be maintained despite being subjected to the additional stress of cooling and warming. This article aims to review the principles and challenges of cryopreservation, current cryopreservation methods, and outcomes based on the embryo stage of cryopreservation, and discuss strategies that may be practiced to optimize an embryo cryopreservation program.
Principles of Cryopreservation Regardless of the cryopreservation method used, the goal is the same—to suspend embryos in time by cooling embryos from ambient, room temperature (20°C) to 196°C. This requires embryos to be exposed to an environment that they have no intrinsic ability to survive, putting them at risk for a variety of types of damage or “cryoinjury” during temperature and phase transitions. More detailed reviews of the principles
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546826. ISSN 1526-8004.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
1 Department of Obstetrics and Gynecology, Carver College of
Human Embryo Cryopreservation
þ15 to 5°C Chilling injury is defined as permanent damage that occurs before the cells are exposed to freezing temperatures. As embryos are cooled to 5°C, there is a phase transition of phospholipids from liquid to gel phase, impacting the cell membrane lipid bilayer’s fluidity, permeability.19–21 This damage is irreversible and it presents a significant challenge when cryopreserving lipid-rich embryos from some mammals. Chilling injury also may damage cytoplasmic lipid droplets, microtubules, and oocyte meiotic spindles.22,23 Mammalian embryos’ resistance to chilling injury varies with development so cryopreservation protocols must be tailored to the embryo stage.19,24
5 to 80°C Ice crystals, intracellular and/or extracellular, form when temperatures reach 5 to 80°C. The damage caused by ice crystals can be mechanical or chemical.25 In 1972 Mazur et al proposed what is now known as the “two-factor” hypothesis for cell injury at this temperature range.26 If cells are cooled slowly, formation of extracellular ice causes the solutes in the medium surrounding the embryo to become hypertonic. The advantage of slow cooling is that the hypertonic solution coaxes water out of the cell, minimizing intracellular ice formation. However, prolonged exposure to high concentrations of electrolytes can cause cell injury by the “solution effect,” destabilizing proteins and damaging the cell membrane.27 Rapid cooling may be equally detrimental as water cannot exit the cell fast enough, leading to formation of lethal intracellular ice.22
50 to 150°C Solutions may fracture at 50 and 150°C. It has been suggested that large cells or cell groups such as oocytes and embryos could be injured by fractured solutions, damaging the zona pellucida or cytoplasm.28 This remains a hypothetical risk and the frequency of injury due to solution fracture has not been reported.
150 to 196°C Embryos are stored in nitrogen vapors (190°C) or, more typically, in liquid nitrogen (196°C). Patients often ask whether prolonged storage leads to additional injury. To date, there is no evidence of damage during storage for a duration relative to a human lifetime. Accidental warming is a common cause of cryoinjury, but if storage temperatures are maintained, the embryos are essentially suspended in time as chemical reactions are halted at 120°C and storage at 196° C prohibits thermally driven reactions.29 Background radiation had been proposed as a concern, but the accumulation of direct damage from background ionizing radiation would require centuries of storage.30 Storage in liquid nitrogen poses a risk for transmission of infectious agents because the liquid is not sterile and most microorganisms can survive storage in liquid nitrogen.31 There have been no reported
129
cases of viral transmission from cryopreserved human embryos and two cases of bovine viral diarrhea virus transmission after cryopreserved embryo transfer in cattle.32,33 Though these reported infections cannot be attributed specifically to embryo cryopreservation and storage, Bielanski et al demonstrated under experimental conditions that concerns about disease transmission are justified and should be taken into consideration when selecting devices for cryopreservation and storage equipment.34,35
Warming from 190 to 20°C During warming the risks of injury are similar to those incurred during cooling. The effects of warming depend on whether intracellular ice formed or the cell was dehydrated during cooling. If intracellular ice forms, rapid thawing may rescue the cell by preventing small intracellular ice crystals from recrystallizing into bigger, harmful ice crystals. Apart from maintaining the plasma membrane that encloses cytoplasm with a seemingly viable morphology, cryopreservation techniques must protect embryos from injury that may not be apparent upon morphologic evaluation, including damage to intracellular organelles, the cytoskeleton and cell junctions.36–39 Despite all these threats to embryo’s health and survival, with the aid of cryoprotectants and established cryopreservation techniques, they have a remarkable ability to repair or bypass the damage and continue to develop after thawing.40,41
Cryoprotectants Cryoprotectants, defined by Karow as “any additive which can be provided to cells before freezing and yields a higher postthaw survival than can be obtained in its absence,” are essential for cryopreservation of cells.42 There are two classes of cryoprotective agents (CPAs): (1) permeating agents have small molecules that cross the cell’s membrane, displace intracellular water and balance intracellular solutes and (2) nonpermeating, large-molecule CPAs that maintain an extracellular osmotic gradient that aids in further cell dehydration. The examples of permeating CPAs include 1,2-propanedial (PROH), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol. Sugars serve as the nonpermeating CPAs, with lowmolecular-weight disaccharides such as sucrose and trehalose typically selected for this role. The combination of the two types of CPAs creates an antifreeze cocktail that reduces intracellular ice formation by removing water from inside the cell and, once inside the cell, depresses the freezing point of water remaining in the cell. The nonpermeating CPAs create an osmotic gradient that helps restrict water movement across the cell membrane, preventing osmotic shock when CPAs are removed during thawing. In order for a cell to survive cryopreservation water and permeating CPAs must be able to move across the cell membrane. When embryos are placed in a hyperosmotic solution with CPAs, the osmotic gradient draws highly permeable intracellular water out of the cell, causing the cell to shrink and permeating CPAs to slowly diffuse into the cell. Several factors must be considered to optimize the exchange of water for CPAs: (1) the ratio of cell membrane surface area to water volume, (2) Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
of cryopreservation in reproductive medicine have been previously published.16,17 The type of damage that occurs during cooling can be divided into four temperature ranges.18
Sparks
Human Embryo Cryopreservation
Sparks
the stage-specific permeability for water, and (3) the stagespecific membrane permeability by CPAs changes as the embryo develops with oocytes to early-stage embryos having lower permeability to CPAs than morulae and blastocysts.23,43,44 Water and permeating CPAs cross the cell membrane by two pathways, simple diffusion through the lipid bilayer that is highly dependent on temperature and through temperature-independent, hydrophilic channels formed by proteins called aquaporins.45 A recent study reported that there is a change in the type and amount of aquaporins expressed with each stage of development and these changes may contribute to the stage-specific membrane permeability to CPAs.46 A visual depiction of the channel types, membrane permeability, and change in cell size with each development stage was published in the proceedings of the Alpha consensus meeting on cryopreservation and this has been reproduced in ►Fig. 1.47
Methods There are two general methods used for embryo cryopreservation: slow freezing and rapid freezing to achieve vitrification.
Details of each cryopreservation method have been described by others.48–50 Both methods use permeating and nonpermeating CPAs that are removed in a stepwise fashion to permit controlled embryo rehydration during warming. The main differences between the methods include the duration of exposure to CPAs, concentration of CPAs, the cooling rate, and warming rate.
Slow Freezing The process of slow freezing, also known as equilibrium freezing, is designed to maintain a balance between intracellular ice formation and osmotic damage using low or nontoxic concentrations of CPAs.51–53 Prior to freezing, embryos are equilibrated in a hyperosmotic solution containing 1 to 2 M permeating CPA (PROH or DMSO for pronuclear and cleavagestage embryos, glycerol for blastocysts) with the CPA introduced in a step-wise fashion or a single step over the course of 10 to 20 minutes. Cell shrinkage is observed while the water leaves the cell and is slowly replaced by the penetrating CPA. Once equilibrium is achieved, the cell volume is restored. The embryos are moved to a final solution that has a combination of 1 to 2 M permeating and 0.2 to 0.3 M nonpermeating CPAs
Fig. 1 Pathways for the movement of water and CPAs across the plasma membrane. The movement of water becomes more efficient with embryo development to the blastocyst stage, as the Arrhenius activation energy declines. (a) Movement of CPAs and water through the plasma membrane is via simple diffusion in the early developmental stages (A1) and via channel diffusion (aquaporins) by the blastocyst stage (A2). (b) Movement of water and CPAs by simple diffusion or via channels also depends on the type of CPA. (c) The efficiency of dehydration and CPA uptake also is influenced by cell size, increasing with increasing surface/volume ration (cell size highlighted). CPAs, cryoprotective agents; DMSO, dimethyl sulfoxide; EG, ethylene glycol; GL, glycerol; H 2O, water. (Used with permission from Alpha Scientists in Reproductive Medicine. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online 2012;25:146–167). Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
130
prior to being loaded into 0.25 mL straws or cryovials and placed in a controlled-rate freezer. The embryos are cooled at a rate of 1 to 2°C/min to 6 to 8°C, which is slightly above the solution’s melting point. The CPA concentrations used for slow cooling are insufficient to prevent intracellular ice formation alone so the concentration is increased during the cooling process by induction of ice formation. Extracellular ice formation is manually induced (seeding) by touching the vial or straw with precooled forceps as far away from the embryos as possible. As water transitions to ice, extracellular CPAs and other solutes become more concentrated, creating another osmotic gradient that drives more water out of the embryo and permits additional CPA to enter the cell. The temperature of the controlled-rate chamber is maintained for an additional 10 minutes to allow the embryos to reequilibrate and then the temperature is very slowly (0.3°C/min) lowered to below 30°C. At this point the intracellular CPA is sufficient to prevent additional intracellular ice formation and the cell is plunged into liquid nitrogen. The optimal thawing rate depends on the temperature that cooling was terminated. Embryos that had cooling terminated at a high temperature (30 to 40°C) will have more water and require faster warming rates (200–350°C/min) than when cooling is terminated at 80°C and more gradual rehydration (25°C/min) is required. Rehydration is controlled by placing the embryos in a concentration of nonpermeating CPA (typically sucrose) that is twofold higher than the concentration of the final cryopreservation solution. This reduces the degree of osmotic imbalance across the cell’s membrane, permitting sufficient time for the penetrating CPAs to diffuse out of the cell and slowing the rate that highly permeable water returns to the cell.54
Vitrification “Vitrification” is a term used to describe the transformation of a substance into glass, but this term is commonly used to describe the methods used to achieve this transformation. Similar to slow freezing, cell dehydration is achieved by permeating CPAs, but unlike slow cooling, there is no attempt to maintain equilibrium on either side of the cell membrane, the time provided for dehydration is brief, the concentration of CPAs is higher, and the rate of cooling is radically higher. How do cells survive? The strategy of vitrification is to use ultrarapid cooling to transform liquid into glass by a dramatic increase in viscosity and minimize the time the sample is exposed to the temperature ranges associated with chilling injury and ice crystal formation.55 Embryo vitrification protocols are much simpler than slow freezing and there is no need for a controlled-rate freezer or other expensive equipment. Permeating CPAs (commonly a combination of EG and sucrose along with PROH, glycerol, or DMSO) are introduced with a two-step approach, exposing embryos to 50% concentration of the final CPA concentration for 5 to 15 minutes to permit intracellular water to exit the cell and CPA permeation to establish an equilibrium and limiting exposure to the final 30 to 40% solution of CPAs to the minimum amount of time (50% intact blastomeres) and 50% of all thawed embryos will have 100% of the blastomeres intact.48 A retrospective analysis more than 800 single embryo transfers (SETs) in which 36 of embryos were cryopreserved at the four-cell stage on day 2 revealed similar implantation rates (fetal heartbeat/embryo) for fourcell–stage embryos with 100% blastomere survival (26%, n ¼ 615) and 75% blastomere survival (27.5%; n ¼ 131) compared with embryos that were 50% intact (n ¼ 85).78 No comparison was made to fresh outcomes in the original article, but it was stated in later review that implantation rates for the 100 and 75% intact four-cell stage embryos (26%) for patients in the same age group (
