In 1972, David Whittingham, Stanley Leibo, and Peter Mazur published the paper, “Survival of Mouse Embryos Frozen to -196 ° and -269 °C,” hereafter, “Survival of Mouse Embryos,” in the journal Science. The study marked one of the first times that researchers had successfully cryopreserved, or preserved and stored by freezing, a mammalian embryo and later transferred that embryo to a live mouse who gave birth to viable offspring. Previously, scientists had only been successful cryopreserving single cells, like red blood cells. Mammalian embryos, on the other hand, were more difficult to cryopreserve because they are more complex and therefore more easily weakened or destroyed by the formation of ice within its cells. Whittingham, Leibo, and Mazur’s work provided a successful model for mammalian embryo cryopreservation, a technology that later expanded to cryopreserve more complex embryos, such as human embryos.

Peter Mazur was a researcher in the US who developed new ways of preserving biological material by freezing it, a process called cryopreservation. If done correctly, cryopreservation enables scientists to store or study biological material for an extended period of time. If done incorrectly, cryopreservation can easily harm or destroy biological material. Mazur worked to find the best ways to cryopreserve different cells, embryos, and organs in order to minimize the damage caused by freezing. Throughout the 1960s and 1970s, Mazur and his colleagues published a series of papers that ultimately led to the discovery of previously unexplored factors that can cause harm to cells during the cryopreservation process. He called that discovery the two-factor hypothesis. That same year, Mazur also contributed to one of the first successful attempts at cryopreserving viable mouse embryos. Mazur’s work to improve the cryopreservation process helped to establish foundational knowledge that was later used in many different fields, such as reproductive health and conservation.

Stanley Paul Leibo studied the cryopreservation of embryos in the US in the twentieth century. Cryopreservation is a method of preserving biological material through freezing. Early in his career, Leibo collaborated with other scientists to study why cells were oftentimes injured during freezing. Later, Leibo and his team accomplished one of the first successful births using previously-frozen mammalian embryos. Leibo continued evolving simpler and more reliable methods of cryopreservation and embryo transfer for many different species over the course of his career, such as the development of a one-step procedure of transferring fertilized embryos between cattle. Leibo’s work to develop simple and reliable ways to cryopreserve cells and embryos enabled its use in a wider scope of research, including agriculture, reproductive medicine, and conservation.

In 1972, Peter Mazur, Stanley Leibo, and Ernest Chu published, “A Two-Factor Hypothesis of Freezing Injury: Evidence from Chinese Hamster Tissue-culture Cells,” hereafter, “A Two-Factor Hypothesis of Freezing Injury,” in the journal, Experimental Cell Research. In the article, the authors uncover that exposure to high salt concentrations and the formation of ice crystals within cells are two factors that can harm cells during cryopreservation. Cryopreservation is the freezing of cells to preserve them for storage, study, or later use. Mazur originally suggested the two factors in a 1970 paper, but that article was based on evidence from simple yeast cells. By using hamster cells in 1972, Mazur, Leibo, and Chu confirmed that Mazur’s two-factor hypothesis applied to more complex mammalian cells. The article dispelled the widely accepted notion that rapid cooling rates were safest for all cells, and instead showed that each kind of cell had a different optimal cooling rate depending on the solution in which it froze.

In Jeter v. Mayo, the Court of Appeals of Arizona in 2005 held that a cryopreserved, three-day-old pre-embryo is not a person for purposes of Arizona's wrongful death statutes, and that the Arizona Legislature was best suited to decide whether to expand the law to include cryopreserved pre-embryos. The Court of Appeals affirmed a decision by the Maricopa County Superior Court to dismiss a couple's wrongful death claim after the Mayo Clinic (Mayo) allegedly lost or destroyed several of their cryopreserved pre-embryos. In reaching its decision, the Court of Appeals explored ethical and legal issues relating to cryopreserved pre-embryos, including prior case law, the principles of statutory construction, and the Arizona Legislature's role in balancing the societal interests involved.

In the 1949 article “Revival of Spermatozoa after Dehydration and Vitrification at Low Temperatures,” researchers Christopher Polge, Audrey Ursula Smith, and Alan Sterling Parkes demonstrated that glycerol prevents cells from dying while being frozen. Polge and his colleagues discussed several procedures in which they had treated sperm cells from various species with glycerol, froze those cells, and then observed the physiological effects that freezing had on the treated sperm. The researchers concluded that glycerol safely preserves sperm samples from a variety of species. Polge, Smith, and Parkes’s 1949 article detailed one of the first successful uses of a chemical medium to preserve viable cells in a frozen state, a process that eventually enabled the first vertebrate embryo to be successfully conceived using frozen sperm.

The San Diego Zoo Institute for Conservation Research (SDZICR) in San Diego, California, is a research organization that works to generate, use, and share information for the conservation of wildlife and their habitats. In 1975, Kurt Benirschke, a researcher at the University of California, San Diego (UCSD) who studied human and animal reproduction, and Charles Bieler, the director of the San Diego Zoo, collaborated to form the Center for Reproduction of Endangered Species (CRES). In 2009, the San Diego Zoo announced the creation of SDZICR, which expanded and replaced CRES, to provide central organization and management of scientific programs at the San Diego Zoo. By 2004, Allison Alberts was the director of research and for more than a decade oversaw the SDZICR's many research initiatives, including the collection and storage of genetic and reproductive information of rare and endangered animal and plant species.

In 1952, researchers Christopher Polge and Lionel Edward Aston Rowson, who worked at the Animal Research Center in Cambridge, England, detailed several experiments on protocols for freezing bull semen for use in the artificial insemination of cows. Freezing sperm extends the life of a viable sperm sample and allows it to be used at later times, such as in artificial insemination. The researchers examined the effects of freezing conditions on bull sperm and how well they produce fertilized embryos once thawed. Polge and Rowson concluded that bull sperm can retain its fertility throughout the freezing process and that frozen bull sperm can yield pregnancy rates of up to seventy-nine percent. Polge and Rowson provided the first conclusive evidence that frozen mammalian sperm, once thawed, can produce viable pregnancies.

Twentieth-century researcher Ernest John Christopher Polge studied the reproductive processes of livestock and determined a method to successfully freeze, thaw, and utilize viable sperm cells to produce offspring in animals. In 1949, Polge identified glycerol as a cryoprotectant, or a medium that enables cells to freeze without damaging their cellular components or functions. Several years later, Polge used glycerol in a freezing process called vitrification, which enabled him to freeze poultry sperm, thaw that sperm, and use it to fertilize vertebrate embryos. He later adapted those methods to be applied to several other species including goats, cows, and pigs, which enabled farmers to fertilize livestock with sperm or embryos after long-term storage. Additionally, Polge's development of methods to freeze and store living samples has equipped reproductive health researchers and medical professionals with the abilities to mass collect and store human sperm.

In a dispute over the allocation of cryopreserved preembryos, the Supreme Court of Washington resolved the case of David J. Litowitz v. Becky M. Litowitz (2002) by reaching a decision that neither party wanted. David Litowitz sought to find adoptive parents for two cryopreserved preembryos created during his marriage to Becky Litowitz when the couple was attempting to have children using in vitro fertilization (IVF). Becky sought to implant the preembryos in a surrogate in an effort to parent a child. In June 2002, the court instead determined that the preembryos should have been destroyed. The court focused on the former couple s written consent agreement signed at the time of their participation in the IVF program, which stated that the preembryos would be destroyed after five years of storage.