Family Research Council

The Viability of Frozen Human Embryos: Lessons From Animal Research


In the long debate over the ethical permissibility of destroying embryonic human beings in research, it has sometimes been suggested that frozen embryos are somehow less human, or less alive, than are unfrozen embryos. For instance, in January 2000, Senator Arlen Specter of Pennsylvania introduced S. 2015, a bill providing for federal funding of human embryonic stem cell research. The bill provided, in pertinent part, that:

The human embryonic stem cells involved shall be derived only from embryos that otherwise would be discarded that have been donated from in-vitro fertilization clinics with the written informed consent of the progenitors.[1]

In a statement to the press, the senator said, "[T]he discarded embryos are not going to be used for human life. If there was any possibility, I would be the first to oppose their use for scientific research."[2]

Though his statement is ambiguous, it appears Sen. Specter meant to suggest that, once frozen, the embryo was either dead or could not be unfrozen, implanted, and brought to live birth. However, frozen embryos remain alive, and can be thawed, implanted and brought to a live, healthy birth. This has, in fact, been accomplished several times, both in the United States and elsewhere. For instance, on February 5, 2004, BBC News reported that an Israeli woman gave birth to twins from embryos frozen twelve years ago.[3]

While such reports conclusively rebut Specter's suggestion that frozen embryonic human beings cannot be brought to live birth, there is significant scientific research examining other animals--such as invertebrates, insects, frogs, and turtles--which reinforces the point. In this paper, we summarize research concerning how these creatures survive in low temperatures. Freezing places these creatures in suspended animation, but it does not kill them. We hope this will conclusively rebut any belief that frozen human embryos are either no longer alive or cannot be brought to a live birth. Frozen embryonic human beings, like the frozen animals discussed in this paper, can survive freezing. Indeed, whether frozen embryonic human beings will, as Specter put it, "be used for human life" (or, more accurately, whether they will be born alive) depends solely upon the will, good or ill, of the human beings into whose hands fate has placed them. Nothing about their dilemma robs them of the humanity they share with us.

Freeze Survival Techniques in Animals

Animals in their natural environments have to face many situations that, if not handled properly, will cause their deaths. One of these life threatening phenomena is cold weather. In the majority of cases, freezing of animal body tissues or organs is lethal. However, there are some species that can undergo some degree of freezing without damage. In order to prevent injuries due to low temperatures, two basic strategies have been developed:freeze-avoidance and freeze-tolerance. In this paper, I will indicate the mechanisms that are necessary to prevent damage by freezing. At the end, I will provide some data for freeze tolerance of intertidal marine invertebrates, insects, frogs, and turtles respectively.

Freeze-avoidance and Freeze-tolerance

Low temperatures are lethal to cold-blooded animals. These species can adopt two basic strategies to face temperatures below the freezing point of their body fluids (FP): freeze-avoidance and freeze-tolerance. Both strategies involve adaptations on behavioral, physiological, and biochemical levels. Freeze-avoidance is the safest way to prevent the lethal effects of freezing. The most effective strategy is to find a place for hibernation where the temperature does not fall bellow the freezing point (FP). However, some animals have developed another strategy. It enables them to keep their body fluids liquid even at temperatures far below FP.[4] This second strategy of freeze-avoidance is known as supercooling. "Supercooling (that is, remaining liquid below the FP) is a metastable state in which the probability of spontaneous freezing increases with decreasing temperature until it reaches 100 percent at the crystallization temperature (Tc; also known as the supercooling point)."[5] Supercooling is achieved by inhibition of ice nucleation and depression of FP. It is supported by body processes that:

1. eliminate potential ice nucleators[6]

2. add thermal antifreeze proteins to body fluids

3. accumulate high concentrations of cryoprotectants[7]

4. cause dehydration to reduce freezable water[8]

The contact between environmental ice and a supercooled animal is often lethal, because so-called "inoculative freezing"[9] occurs.

Freeze-tolerance is the most challenging hibernation strategy.[10] It is an ability to endure freezing stress without being injured.[11] Freeze-tolerant animals allow the extracellular water to freeze and only the cytoplasm remains liquid. It is generally accepted that intracellular freezing is lethal even for freeze-tolerant species.[12]

Because an animal in a frozen state is completely helpless, there must be good reasons why freeze-tolerance has been developed. There are at least three important advantages achieved by this hibernating strategy. The first advantage is the possibility of early spring emergence. Hibernating in less-protected hibernation sites, animals can detect the warmer spring temperatures sooner than those which hibernate in more protected places. This prolongs the time for their growing season. The second advantage has to do with the protection against predators. Turtles that hatch late in the season stay in place and first surface the following spring. But at that time the hatchlings are older and stronger, and thus, more prepared for facing the challenge of predators. The third main advantage of adopting the freeze tolerance is range extension. The species can penetrate into areas where freeze avoidance is not possible.[13]

Freeze tolerance requires several mechanisms for preventing possible freezing injuries:

1. Ice control mechanisms are of a great importance. A moderated extracellular ice formation must be induced to minimize physical damage by ice crystals. (Intracellular ice is lethal.)[14]

2. Cell-volume regulation must prevent shrinking below critical cell volume. This is necessary in order to be able to deal with the loss of intracellular water.

3. Mechanisms dealing with anoxia/ischemia[15] and metabolic arrest must sustain viability over long-term freezing. Otherwise the lack of oxygen for a longer period and the cessation of metabolism would be lethal.

4. Finally, the mechanisms that start spontaneous reactivation after thawing must be present.[16]

Freezing in general must be carefully controlled in order to ensure survival. Ice growth is initiated in two ways. It may be inoculated, i.e., body fluids start to freeze when brought in contact with environmental ice at or below FP. Or it may occur spontaneously in supercooled body fluids. The slower the freezing occurs the more time the cells have to prepare for freezing. The lower the temperature at which ice formation begins, the faster the ice will be formed, and thus less time will be available for implementing cryoprotective means.[17]

The animals that can survive freezing of their body fluids belong to various species. So far, freeze tolerance is observed with some insects (members of Coleoptera, Hymenoptera, Diptera, and Lepidoptera), marine invertebrates (bivalves--Mytilus edulis, Modiolus demissus, Cardium edule, Venus mercenaria; gastropods--Littorina littorea, Nassarius obsoletus, Acmea digitalis, Melampus bidentatus; and barnacles--Balanus balanoides), four species of land hibernating frogs (wood frog--Rana sylvatica, gray tree frog--Hyla versicolor, spring peeper--Hyla crucifer, and chorus frog--Pseudacris triseriata), reptiles (box turtles--Terrapene carolina, and painted turtles--Chrysemis picta, etc., garner snakes, some lizards).[18] I will introduce some information and data describing the freeze tolerance strategies of these animals, of which most have been collected in laboratory experiments.

A. Marine Invertebrates

In the intertidal zone, marine invertebrates can be exposed to sub-zero temperatures twice a day. Because of the environment, marine invertebrates cannot use dehydration to protect themselves from the impacts of freezing. The factors influencing freezing survival are: salinity, temperature, anaerobis,[19] seasonality and geographic variation, and microhabitat. Marine invertebrates use several strategies for freeze tolerance: ice-nucleating proteins (INP) that induce and control ice formation, thermal hysteresis proteins (antifreeze glycoproteins), metabolic depression and anaerobis, cryoprotectants, and change in the composition of membrane lipids.[20] The lower lethal temperatures are in the range of 14 to 5 degrees Farenheit (-10 to -15 degrees Celsius) and the highest acceptable amount of body water in the form of ice in some species is 65-80 percent.[21]

B. Insects

Examples of freeze tolerance in insects can be found at all stages of life. Some species[22] are even freeze-tolerant in more than one stage. Tolerance is highly developed in the Insecta, with common freezing survival of 14 to 5 degrees Farenheit (-10 to -15 degrees Celsius) temperatures. Some arctic species have lethal temperatures as low as -65 or even -92 degrees Farenheit (-55 and -70 degrees Celsius). There are several critical elements influencing freeze survival in insects. Ice-nucleating proteins help to initiate freezing at high subzero temperatures and thus control the slow reduction of cell volume and concentration of fluids. Thermal hysteresis proteins prevent recrystallization of intracellular ice. Polyhydric alcohols (sometimes sugars) are used as cryoprotectants. Dehydration does not occur, but freezable water is reduced by other means. Anoxia and metabolic depression contribute to long-term survival.[23]

C. Terrestrially Hibernating Frogs

The limits for frog survival are narrow but still sufficient for chosen hibernation sites. Frogs usually supercool to 28.4 or 26.6 degrees Fahrenheit (-2 or -3 degrees Celsius) and can survive freezing at 21.2 to 17.6 degrees Farenheit (-6 to -8 degrees Celsius). It is important to note that the survivable temperatures vary with individual species and location. However, it seems that survival ranges are well matched to the needs of the species. Long-term survival is sufficient--animals of all four species that have been studied survived three days frozen at 26.6 degrees Farenheit (-3 degrees Celsius). When frozen, frogs have stiffened limbs, ice crystals under the skin and interspersed with skeletal muscles, and ice filling the abdominal cavity and surrounding all organs. There is no breathing, no heartbeat, and no bleeding when the aorta is severed. Organs such as the liver and the heart are pale, because of blood withdrawal. The heart is the first organ to start functioning again when thawed. Frogs, in order to secure freeze tolerance, also use cryoprotectants (glucose, glycerol, etc.). They might die from dehydration if hibernating in direct contact with air because so-called "freeze drying" can occur.[24] Metabolism during freezing is stopped (no breathing, heartbeat, and blood flow) and the survival depends on anaerobic potential and use of endogenous energy (creatine phosphate) and fuel (glycerol, possibly amino acids). Freeze-tolerance potential declines after emerging from hibernating sites. The frog species H. versicolor can tolerate 35 percent of body fluids frozen at 21.2 degrees Farenheit (-6 degrees Celsius); R. sylvatica can tolerate 48 percent at 24.8 F (-4 degrees Celsius).[25]

D. Turtles

The above-mentioned species of turtles remain in their nest after hatching. This may cause the hatchlings to be exposed to sub-zero temperatures. Many experiments were made to determine the characteristics and limits of freeze tolerance of turtles. Turtles before freezing in laboratory conditions usually supercool. When freezing is just about to occur, the body temperature rises slightly below the FP and controlled ice formation starts.[26] Chrysemys picta can tolerate 45-55 percent of body fluids being frozen; Trachemys scripta elegans can tolerate a maximum of 49 percent of body water being frozen. As with the frogs, the freezing tolerance might vary not only with species, but also with the location of a given population.

Conclusion

It is difficult to determine exact data for freezing tolerance in individual animal species. These data depend on the way the laboratory experiments are conducted. For example, the lowest survivable temperature (or the length of time animals can be exposed to freezing) depends on how fast the temperature was lowered during the experiments.[27] And further doubts concerning the exactness of the laboratory experiments might be raised. However, there is no doubt that the above discussed species really can survive freezing. Before laboratory experiments began, these species had been observed surviving freezing temperatures in their natural environments.

BIBLIOGRAPHY

Bailey R. M. (1949). "Temperature toleration of garter snakes in hibernation." Ecology, Vol. 30, No. 2.

Block W. (1991). "To fr eeze or not to freeze? Invertebrate survival of sub-zero temperatures." Functional Ecology, Vol. 5.

Churchill T. A., Storey K. B. (1992). "Responses to freezing exposure of hatchling turtles Trachemys Scripta Elegans: Factors influencing the development of freeze tolerance by reptiles." Journal of Experimental Biology, Vol. 167.

Churchill T. A., Storey K. B. (1992a). "Natural freezing survival by painted turtles Chrysemys picta marginata and C. picta bellii." American Journal of Physiology, Vol. 262.

Claussen D. L., Constanzo J. P. (1990). "A simple model for estimating the ice content of freezing ectotherms." Journal of Thermal Biology, Vol. 15, No. 3/4.

Claussen D. L., Zani P. A. (1991). "Allometry of cooling, supercooling, and freezing in the freeze-tolerant turtle Chrysemys picta." American Journal of Physiology, Vol. 261.

Costanzo J. P., Iverson J. B., Wright M. F., Lee R. E. (1995). "Cold hardiness and overwintering strategies of hatchlings in an assemblage of northern turtles." Ecology, Vol. 76, No. 6.

Diamond J. M. (1989). "Resurrection of frozen animals." Nature, Vol. 339, Issue 6225.

Layne J. R. (1992). "Postfreeze survival and muscle function in the leopard frog (Rana pipiens) and the wood frog (Rana sylvatica)." Journal of Thermal Biology, Vol. 17, No. 2.

Layne J. R., Lee R. E. (1987). "Freeze tolerance and the dynamics of ice formation in wood frogs (Rana sylvatica) from southern Ohio." Canadian Journal of Zoology, Vol. 65.

Layne J. R., Lee R. E., Huang J. L. (1990). "Inoculation freezing at high subzero temperatures in a freeze-tolerant frog (Rana sylvatica) and insect (Eurosta solidaginis)." Canadian Journal of Zoology, Vol. 68.

Mazur P. (1984). "Freezing of living cells: mechanisms and implications." American Journal of Physiology, Vol. 247.

Packard G. C., Packard M. J. (1990). "Patterns of survival at subzero temperatures by hatchling painted turtles and snapping turtles." Journal of Experimental Zoology, Vol. 254.

Packard G. C., Packard M. J., Ruble K. A. (1993). "Hatchling snapping turtles overwintering in natural nests are inoculated by ice in frozen soil." Journal of Thermal Biology, Vol. 18, No. 4.

Storey K. B., Storey J. M. (1988). "Freeze tolerance in animals." Physiological Reviews, Vol. 68, No. 1.

Storey K. B., Storey J. M. (1996). "Natural freezing survival in animals." Annual Review of Ecology and Systematics, Vol. 27.

1. SEC. 498C. RESEARCH ON HUMAN EMBRYONIC STEM CELL...(b) SOURCES OF EMBRYONIC CELLS (1).

2. Maggie Fox, "Cells debate centers on whether embryos are people," Reuters, April 26, 2000.

3. British Broadcasting Channel "Twins Born from 12-Year-Old Embryos" February 5, 2004: http://news.bbc.co.uk/1/hi/health/3459515.stm

4. Some arctic insects can supercool to -560F (-500C). Cf. Storey K. B., Storey J. M. (1996), Natural freezing survival in Animals, Annual Review of Ecology and Systematics, Vol. 27: 366.

5. Storey K. B., Storey J. M. (1996). "Natural freezing survival in animals," Annual Review of Ecology and Systematics, Vol. 27: 367.

6. Substances that support creation of ice.

7. Substances that protect the cells from freezing (e.g. glycerol, glucose, etc.)

8. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in Animals," Physiological Reviews, Vol. 68, No. 1: 31.

9. Inoculation refers to the fact that the ice formation is initiated by the external ice. If a supercooled animal is inoculated by environmental ice, the ice formation is usually rapid and thus lethal. However, at high sub-zero temperatures inoculation might be used as a means to initiate freezing. Cf. Layne J. R., Lee R. E., Huang J. L. (1990), "Inoculation freezing at high subzero temperatures in a freeze-tolerant frog (Rana sylvatica) and insect (Eurosta solidaginis)," Canadian Journal of Zoology, Vol. 68.

10. Cf. Storey K. B., Storey J. M. (1996), "Natural freezing survival in animals," Annual Review of Ecology and Systematics, Vol. 27: 368.

11. Cf. Ibid., 369.

12. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in animals," Physiological Reviews, Vol. 68, No. 1, 28

13. Cf. Storey K. B., Storey J. M. (1996), "Natural freezing survival in animals," Annual Review of Ecology and Systematics, Vol. 27: 369-370.

14. See endnote 9.

15. anoxia--total deprivation of oxygen; ischemia--a lack of blood supply in an organ or tissue

16. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in animals," Physiological Reviews, Vol. 68, No.1: 28-29

17. Cf. Storey K. B., Storey J. M. (1996), "Natural freezing survival in animals," Annual Review of Ecology and Systematics, Vol. 27: 374.

18. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in animals," Physiological Reviews, Vol. 68, No. 1; and Storey K. B., Storey J. M. (1996), "Natural freezing survival in animals," Annual Review of Ecology and Systematics, Vol. 27

19. An ability to survive without breathing when access to oxygen is impossible.

20. A group of organic compounds consisting of the fats and other substances of similar properties: they are insoluble in water, soluble in fat solvents and alcohol, and greasy to the touch, and are important constituents of living cells.

21. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in animals," Physiological Reviews, Vol. 68, No. 1.

22. This is true for insects whose development extends beyond one year. Cf. Storey K. B., Storey J. M. (1988), "Freeze tolerance in animals," Physiological Reviews, Vol. 68, No. 1.

23. Cf. Ibid.

24. "Freeze drying" is connected with the fact that water evaporates even when frozen. Direct contact with air supports this evaporation. Frogs' whole-body dehydration is lethal.

25. Cf. Ibid.

26. Controlled by cyoprotectants as glucose, glycerol, lactate, glycogen, etc.

27. Cf. Layne J. R. (1992), "Postfreeze survival and muscle function in the leopard frog (Rana Pipiens) and the wood frog (Rana Sylvatica)," Journal of Thermal Biology, Vol. 17, No. 2: 123.