Robert B. Shabanowitz, PhD
In 1978, the first child from the advanced assisted reproductive technology of in vitro fertilization was born. Five years later, identical twins were born after transfer of an embryo that had previously been frozen and thawed. In 1988, a pregnancy was reported from an egg that had undergone assisted fertilization by placing spermatozoa directly into the perivitelline space using micromanipulation. The very rapid progress of assisted reproductive technologies in humans was possible because of the immense foundation of basic research on the events surrounding fertilization and early development. These studies have been in progress for decades.
Freezing living cells dates back to at least 1776 (1) when Lazzaro Spallanzani "froze" stallion sperm in snow and later noted the recovery of sperm motility upon warming. Interest and experimentation in cryopreservation (cryo- Gr., cold, frost) continued, and in the late 1940's, M.C. Chang (2) reported on the birth of a litter of rabbits from embryos that had previously been stored at 0�C. In 1949, the successful cryopreservation of human sperm cells was reported by Polge et al. (3). However, it was the collaborative efforts of Whittingham, Mazur and Leibo (4), in the early 1970's that culminated in the successful freezing of mouse embryos to the temperature of liquid nitrogen (-196�C) with subsequent high survival rates following controlled thawing. During the ensuing years, live offspring from previously frozen-thawed embryos have been produced in cows, rabbits, sheep, rats, goats, horses, antelopes and baboons. Cryopreservation, therefore has become a well established and important element in the animal husbandry industry (for reviews see 5-7).
The need for human embryo cryopreservation arose as a direct consequence of the rapid development and success of human in vitro fertilization (IVF). An increase in the number of eggs recovered after improved methods of superovulation, and higher fertilization and cleavage rates have resulted in a greater number of viable embryos available for transfer. Since replacement of more than 3-4 embryos increases the risks associated with multiple gestation, human embryo cryostorage became an ethical necessity in order to provide an acceptable method for the preservation of extra embryos. In humans, the first births (identical twin girls) from a previously frozen-thawed embryo occurred on December 26, 1983 in the Netherlands (8).
The success of human embryo freezing, as judged by the ensuing clinical pregnancy rate, depends upon a variety of factors. Rates as high as 23% have been reported using methods for freezing early embryos. However, overall success with freezing is probably much closer to 8-15% (9). The replacement of frozen-thawed embryos represents an extension of the original stimulated cycle of IVF, and therefore allows extra embryos to be replaced during a non-stimulated cycle. Together, fresh embryo transfer (ET) and later ET of frozen-thawed embryos increases the overall success rate for a single cycle of IVF. The success of human embryo cryopreservation has been so rapid, that in June 1990, the ethics committee of the American Fertility Society moved embryo cryopreservation from the status of a clinical experiment to a valid clinical technique (10).
I. Principles of cell freezing\thawing
Although it may seem an anomaly, successful freezing of cells actually involves the preferential formation of ice crystals in the extracellular medium, with minimal ice formation occurring within the cell; excess intracellular ice formation is one cause of cell death. With a precisely controlled freezing process, instead of being frozen, cells are slowly dehydrated. This process involves slow, gradual, controlled cooling at rates of approximately -0.3°C. per minute, and requires the use of special programmable cell freezing equipment. Depending upon the method, once the temperature of the medium has reached -35°C. to -110°C., the embryos (which have been placed into small plastic straws or glass vials) are then plunged into liquid nitrogen for long term storage. This entire process may require 3-6 hours to complete.
Simple buffered salt media, such as phosphate buffered saline, are generally used for cryopreservation. During cell freezing, the cells and medium are slowly super-cooled (cooled to temperatures below the freezing point, but remain a liquid) before the initiation of extracellular ice formation by a process called seeding. Seeding is generally performed at -5°C. to -7°C., and is accomplished by touching the straw or ampule, at a site distant from the embryos, with a pair of tweezers pre-cooled in liquid nitrogen. Manual seeding (heterogenous nucleation) at a precise temperature and location is important to prevent spontaneous (homogenous nucleation), uncontrolled freezing of the medium, which can damage and kill the embryos. Freezing of the extracellular medium removes water in the form of ice crystals, leaving behind a more concentrated salt solution.
Since ice crystals are unable to cross the cell membrane, the cytoplasm remains supercooled, and water leaves the cell due to the osmotic force of the more concentrated extracellular medium. As the temperature continues to fall, a continuing cycle of extracellular ice formation and concomitant solute concentration followed by the osmotic loss of intracellular water gradually dehydrates the cell. Successful dehydration, therefore, precludes cellular damage by preventing the formation of large intracellular ice crystals. Freezing protocols are generally classified as rapid if the final temperature prior to plunging directly into liquid nitrogen is from -30 to -40°C. and slow if the embryos are cooled to -80 to -110°C. prior to plunging. Liquid nitrogen has a temperature of -196°C., and at this very low temperature, essentially all metabolic processes cease, and embryos can probably be stored for hundreds of years without damage. In most programs, ethical and legal issues usually limit storage to 6-10 years.
Cryosurvival is also dependent upon controlled thawing, which allows for a gradual rehydration of cells as they are warmed. In order to prevent recrystallization of water within a cell with resultant cell damage, successful cryopreservation requires a proper match between the freeze and thaw programs. Thawing rates depend upon how cells were initially frozen. If cells were plunged into liquid nitrogen at temperatures of -30 to -40°C. (rapid freeze), a rapid thaw is required, typically by direct immersion of the straw or ampule into a +37°C. water bath; this represents warming rates in excess of +200°C/minute. A slow thaw is required if plunging occurred at -80°C. or lower (slow freeze), and requires the use of a programmable cell freezer. In this method, the cell freezer is pre- cooled, usually to about -80 C. and the straws or ampules containing the embryos are transferred from storage in liquid nitrogen directly into the freezer, allowed to equilibrate a short time and then slowly warmed at controlled rates of approximately +8.0°C./minute to about 4°C.
Cell freezing requires the use of a cryoprotectant and cells can not survive the freeze-thaw process without these special additives. Cryoprotectants are divided into two major groups, permeating and nonpermeating. The most widely used permeating cryoprotectants are propanediol, dimethylsulfoxide (DMSO), and glycerol. Sucrose is the most widely used nonpermeating cryprotectant, and mostly serves for osmotic control. There are several important properties of cryoprotectants. Cryoprotectants:
- lower the freezing point, and therefore allow the embryos and freezing medium to be supercooled to a specific sub-zero temperature before seeding,
- protect the cell membrane from freeze-related injury
- decrease the deleterious effects of high salt concentrations as cells dehydrate during the freezing process (solution effects)
The choice of cryoprotectant depends upon its colligative properties, potential toxicity and permeability characteristics as well as the specific stage of embryo development at the time of freezing. Different cryoprotectants are necessary because the physicochemical properties of growing embryos change over time, and each unique cryoprotectant suits the specific requirements for each specific embryonic stage. The most commonly used cryoprotectants are propanediol (11) (1.5 molar) for pronuclear (zygotes) and 2-4 cell embryos, DMSO (12) (1.5 molar) for 8-16 cell embryos and glycerol (13) (8% v/v) for expanded blastocysts.
Cryoprotectants are added to the freezing medium just prior to freezing. To prevent osmotic damage, embryos are transferred stepwise through increasing concentrations of cryoprotectant in order to gradually reach the final concentration of cryoprotectant required. Addition of cryoprotectants is usually performed at room temperature. The embryos are then loaded into glass ampules or plastic straws prior to being placed into a programmable cell freezer and frozen at controlled rates.
Cryoprotectants must also must be removed after thawing embryos. This is typically accomplished by transfer of the embryos through stepwise dilutions of the cryoprotectant. However, due to osmotic effects, the cells will swell and shrink during each dilution step, and this can damage the embryo. An impermeant solute, such as sucrose, however, can also be used in certain one-step dilution procedures. These special thaw solutions do not contain the permeating cryoprotectant. The presence of extracellular sucrose in the thaw medium acts as a counter solute to prevent the rapid influx of water into the cells while at the same time, the permeating cryoprotectant is effectively removed from the cell as it moves down its concentration gradient. When embryos are placed into these one-step thaw solutions, the cells rapidly shrink as water moves out. Shrinking, however, is much less deleterious to cells than swelling. Expansion to normal cellular volumes occurs once the embryos are transferred to in vitro culture medium.
Not all of the individual cells of early embryos survive the freeze-thaw process. Generally one expects at least half the original blastomeres (individual cells of an early embryo) to survive, and pregnancies can ensue from embryos that have even lost 50% of their original blastomeres. Prior to embryo transfer, a short in vitro incubation period of 2-24 hours may sometimes be used to allow for further observation and evaluation of embryo cryosurvival. Embryo survival can then be evaluated by the ability to commence divisions.
The methods described so far for embryo freezing require expensive programmable cell freezers. In another form of cryopreservation, called vitrification, very high concentrations of cryoprotectants (e.g. 3-4 molar DMSO) are used to permeate embryos. After permeation, the embryos are plunged directly into liquid nitrogen, thereby eliminating the need for a programmable freezer. This ultra-fast freezing results in the formation of a non-crystalline, glass-like solid within cells, and the damaging effects of intracellular ice formation are therefore precluded. Although pregnancies from previously vitrified human embryos have been reported (14), further studies are required to improve this technique and make it more widely accepted.
IV. Timing embryo transfer
The correct timing for embryo transfer after thawing is a critical factor. Attachment and implantation of the embryo normally occurs early during the second week following fertilization. Therefore, careful monitoring of the ovulatory cycle near the expected time of ovulation is necessary to ensure that a thawed embryo can be replaced at a period of time in which that particular cell stage would normally be present within the reproductive tract. There is a window of time during which the embryo may be replaced, generally 24-32 hours. Embryonic age is generally calculated as the time from egg collection to the time of freezing. The endometrium is considered in synchrony with the embryo when the age of the endometrium after ovulation equals the age of the in vitro developed embryo. Optimal time for replacement of the embryos has been shown to be at synchrony or up to twelve hours early (negative synchrony). Replacement of embryos into an endometrium that is older in comparison to the embryonic age (positive synchrony) has been shown to be less successful.
Replacement of thawed embryos requires specific calculation of when ovulation occurs. This is the critical factor that determines how to synchronize for frozen-thawed embryo age and uterine age (15). Ovulation can be monitored by daily ultrasounds, the rise in basal body temperature, blood or urine luteinizing hormone concentrations, or a combination of these. Additionally, a urine specimen can be used to determine the presence of pregnanediol glucuronide, which will indicate whether or not ovulation has actually occurred. Optimal time for frozen embryo transfer can also be determined to coincide with optimal implantation as calculated by monitoring the rise in progesterone following ovulation. In some programs, controlled stimulated cycles are used, which precludes monitoring the exact time of ovulation (16). Depending on the age of embryos when they were frozen, embryo transfer can be anywhere from two to six days after ovulation.
In a recent report (9), the pregnancy rate per transfer of frozen-thawed embryos was 17.4% for zygotes, 12.5% for embryos, and 4.3% for blastocysts. Freezing human oocytes (nonfertilized eggs) has also been performed, and a twin pregnancy was reported after thawing, insemination and replacement (17). The overall success of freezing oocytes, however, is very poor. This may be due to the sensitivity and easy disruption of the mitotic spindle, which is arrested at metaphase II, by the freeze-thaw process. Development of successful oocyte freezing would help establish oocyte banking as an accepted means to store female gametes. Extensive research in this area, therefore, continues to be important.
The cryopreservation of human embryos has become an important element in active IVF-ET programs. Without the extensive background provided by researchers in the field of animal husbandry, the rapid development of successful freezing of human embryos would not have been possible. Success with human embryo cryopreservation continues to improve with time as we learn the requirements specific for human embryos.
2. Chang, MC: Normal development of fertilized rabbit ova stored at low temperature for several days. Nature 159:602, 1947.
3. Polge, C, Smith, AU, Parkes, AS: Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666, 1949.
4. Whittingham DG, Leibo SP, Mazur P: Survival of mouse embryos frozen to -196 and -269�C. Science 178:411, 1972.
5. Ashwood-Smith, MJ: The cryopreservation of human embryos. Human Reprod. 1:319, 1986.
6. Friedler S, Giudice LC, Lamb EJ: Cryopreservation of embryos and ova. Fert. Steril. 49:743, 1988.
7. Leibo SP: Cryobiology: preservation of mammalian embryos. Basic Life Sciences 37:251, 1986.
8. Zeilmaker GH, Alberda A, van Gent I, Rijkmans CMPM, Drogendijk AC: Two pregnancies following transfer of intact frozen-thawed embryos. Fert. Steril. 42:293, 1984.
9. Fugger EF: Clinical status of human embryo cryopreservation in the United States of America. Fert. Steril. 52:986, 1989.
10. The cryopreservation of fertilizing eggs and preembryos. Fert. Steril. 53 (Supl.):58S, 1990.
11. Testart J, Lassalle B, Belaish-Allart J, Hazout A, Forman R, Rainhorn JD, Frydman R: High pregnancy rate after early human embryo freezing. Fert. Steril. 46:268, 1986.
12. Mohr LR, Trounson A, Freeman L: Deep-freezing and transfer of human embryos. J. InVitro Fertil. Embryo Transfer 2:1, 1985.
13. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB: Pregnancies following the frozen storage of expanding human blastocysts. J. InVitro Fertil. Embryo Transfer 2:59, 1985.
14. Gordts S, Roziers P, Campo R, Noto V: Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fert. Steril. 53:469, 1990.
15. Cohen J, DeVane GW, Elsner CW, Kort HI, Massey JB, Norbury SE: Cryopreserved zygotes and embryos and endocrinologic factors in the replacement cycle. Fert. Steril. 50:61, 1988.
16. Schmidt CL, de Ziegler D, Gagliardi CL, Mellon RW, Taney FH, Kuhar MJ, Colon JM, Weiss G: Transfer of cryopreserved-thawed embryos: the natural cycle versus controlled preparation of the endometrium with gonadotropin-releasing hormone agonist and exogenous estradiol and progesterone (GEEP). Fert. Steril. 52:609, 1989.
17. Chen C: Pregnancy after human oocyte cryopreservation. The Lancet, April 19: 884, 1986.
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