The ART of cryopreservation and its changing landscape

The purpose of this review is to educate the reader on the role that cryopreservation has played and continues to play in the ever-evolving field of assisted reproductive technologies, specifically in clinical human fertility treatment. We discuss the science behind the cryopreservation methods and investigated some of the major considerations that any clinic or cryobank faces in terms of risks and liabilities, physical challenges that accompany the constantly growing collection of cryopreserved specimens, and what this means on the ethical and legal front. Finally, we take a glimpse in the future to explore what may be on the horizon for the preservation of gametes and reproductive tissues. The purpose of this review is to educate the reader on the role that cryopreservation has played and continues to play in the ever-evolving field of assisted reproductive technologies, specifically in clinical human fertility treatment. We discuss the science behind the cryopreservation methods and investigated some of the major considerations that any clinic or cryobank faces in terms of risks and liabilities, physical challenges that accompany the constantly growing collection of cryopreserved specimens, and what this means on the ethical and legal front. Finally, we take a glimpse in the future to explore what may be on the horizon for the preservation of gametes and reproductive tissues. DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/34574 DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/34574 Cryobiology is the science related to the suspension and preservation of the function, growth, and development of tissue and cells at cold temperatures for later use, with little or no decrease in efficacy. While water exchanges and enzyme reactions are stopped, cells can be held in suspended animation for the long term (1Leibo SP. Fundamental cryobiology for clinical embryologists. In: Cryobiology and cryopreservation of human gametes and embryos. Brussels, Belgium 2004;9-16.Google Scholar). Several great scientists have worked to achieve our current knowledge of how tissues and cells can be cryopreserved with apparently little damage. The serendipitous discovery of the properties of glycerol (2Polge C. Smith A.U. Parkes A.S. Revival of spermatozoa after vitrification and dehydration at low temperatures.Nature. 1949; 164: 666Google Scholar) as an excellent cryoprotectant for sperm cells led eventually to the first human pregnancy from a frozen oocyte (3Chen C. Pregnancy after human oocyte cryopreservation.Lancet. 1986; 1: 884-886Google Scholar) using dimethyl sulfoxide (DMSO), another cryoprotectant. It is interesting that the often-quoted first reported human birth from a frozen embryo by Zeilmaker et al. (4Zeilmaker G.H. Alberda A.T. van Gent I. Rijkmans C.M. Drogendijk A.C. Two pregnancies following transfer of intact frozen-thawed embryos.Fertil Steril. 1984; 42: 293-296Google Scholar) may have actually been second to that of Mukerji et al. (5Kumar A.T. Historical notes: architect of India's first test tube baby: Dr. Subhas Mukerji (16 January 1931 to 19 July 1981).Curr Sci. 1997; 72: 526-531Google Scholar) by almost 6 years, October 3, 1978. In fact, the case of Mukerji et al (5Kumar A.T. Historical notes: architect of India's first test tube baby: Dr. Subhas Mukerji (16 January 1931 to 19 July 1981).Curr Sci. 1997; 72: 526-531Google Scholar). was only 2 months and 9 days after the birth of the world's first in vitro fertilization (IVF) infant, Louise Brown, reported by Steptoe and Edwards (July 25, 1978). The work by Mukerji et al (5Kumar A.T. Historical notes: architect of India's first test tube baby: Dr. Subhas Mukerji (16 January 1931 to 19 July 1981).Curr Sci. 1997; 72: 526-531Google Scholar), reported in a scientific journal of low impact, combined with the fact that the parents of the infant were not willing to have their names released to the public, contributed to the lack of acknowledgment of this breakthrough. Zeilmaker et al. (4Zeilmaker G.H. Alberda A.T. van Gent I. Rijkmans C.M. Drogendijk A.C. Two pregnancies following transfer of intact frozen-thawed embryos.Fertil Steril. 1984; 42: 293-296Google Scholar) had the foresight (6Shapiro B.S. Daneshmand S.T. Garner F.C. Aguirre M. Hudson C. Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders.Fertil Steril. 2011; 96: 344-348Google Scholar) to recognize that the hormone milieu of a stimulated IVF patient may negatively affect uterine receptivity and, thus, sought to cryopreserve embryos for future transfer, avoiding the unphysiologically stimulated endometrium. There are several factors that determine cell survival from cryopreservation: the osmotic response of the cell when suspended in a solution of a permeating cryoprotectant; concentration of the extracellular solution as the temperature is reduced and as ice forms and grows in the extracellular matrix; osmotic loss of water from the cell in response to increasing concentration of the extracellular solution as water is removed in the form of ice; rate at which the specimen is cooled to subzero temperatures, primarily from approximately −5°C to −35°C; subzero temperature at which the cell is stored and its handling during storage; rate at which the cryopreserved sample is warmed and thawed; and osmotic response of the cell when it is removed from the cryoprotectant solution (1Leibo SP. Fundamental cryobiology for clinical embryologists. In: Cryobiology and cryopreservation of human gametes and embryos. Brussels, Belgium 2004;9-16.Google Scholar). Most cells reside in isotonic solutions of approximately 300 mOsmol, but during cryopreservation, they are placed into hypertonic solutions to get the high concentration of cryoprotectants into the cell and remove water from inside the cell, thereby preventing damaging ice crystals from forming. Certain molecules, called cryoprotectants (or antifreeze agents), can increase the survival of cells by their addition to the media before or during cooling. The most common ones are glycerol, DMSO, ethylene glycol, propylene glycol, and methanol. The actual mechanism of their actions is not fully understood but may be related to their ability to lower the freezing point of solutions (sometimes as low as −50°C) and reduce ice formation by hydrogen bonding with water molecules to prevent ice crystal formation (7Towey J.J. Dougan L. Structural examination of the impact of glycerol on water structure.J Phys Chem B. 2012; 116: 1633-1641Google Scholar). Because these molecules are usually less permeable than water, removal of cryoprotectants during warming must be performed gradually to prevent cell bursting. For this reason, cryoprotectants are usually removed by a stepwise dilution and/or using a nonpermeating solute such as sucrose to act as an osmotic buffer to lessen the chance of osmotic shock (8Renard J.P. Heyman Y. Leymonie P. Plat J.C. Sucrose dilution: a technique for field transfer of bovine embryos frozen in the straw.Theriogenology. 1983; 19: 145Google Scholar). The addition of some cryoprotectants (e.g., sucrose) that are not penetrating the cells helps to stabilize cellular membranes (9Yong K.W. Laouar L. Elliott J.A.W. Jomha N.M. Review of non-permeating cryoprotectants as supplements for vitrification of mammalian tissues.Cryobiology. 2020; 96: 1-11Google Scholar). There also is a special class of nonpermeating cryoprotectants called ice blockers. This includes polyglycerol and polyvinyl alcohol, which help prevent ice crystal growth during warming (10Robles V. Valcarce D.G. Riesco M.F. The use of antifreeze proteins in the cryopreservation of gametes and embryos.Biomolecules. 2019; 9: 181Google Scholar). It is important to note that the base solution into which cryoprotectants are dissolved is also significant in cell survival (11Stachecki J.J. Cohen J. Willadsen S.M. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.Cryobiology. 1998; 37: 346-354Google Scholar). Two types of cooling methods are typically used: slow cooling and rapid cooling, or vitrification. To be precise though, it should be noted that vitrification is the endpoint one hopes to achieve through ultrarapid cooling. In fact, the traditional methods of slow cooling in mice and humans probably resulted in intracellular vitrification. The pioneering work by Rall and Fahy (12Rall W.F. Fahy G.M. Ice-free cryopreservation of mouse embryos at-196 degrees C by vitrification.Nature. 1985; 313: 573-575Google Scholar) was instrumental in the development of ultrarapid cooling (nonequilibrium cooling) in human embryos and oocytes. The most common method for the vitrification of blastocysts is based loosely on the method of Vanderzwalmen et al. (13Vanderzwalmen P. Bertin G. Debauche Ch Standaert V. Bollen N. van Roosendaal E. et al.Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing.Hum Reprod. 2003; 18: 1504-1511Google Scholar), whereas the most common oocyte vitrification method is based on Katayama et al. (14Katayama K.P. Stehlik J. Kuwayama M. Kato O. Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy.Fertil Steril. 2003; 80: 223-224Google Scholar, 15Kuwayama M. Vajta G. Kato O. Leibo S.P. Highly efficient vitrification method for cryopreservation of human oocytes.Reprod Biomed Online. 2005; 11: 300-308Google Scholar). They used what became known as the Cryotop method with 15% DMSO and 15% ethylene glycol and 0.5 M of sucrose. During these days, both oocytes and embryos are routinely vitrified using these methods, and few improvements have been made to these techniques since 2003. Regardless of what technique is used, the success of cryopreservation and warming is ultimately dependent on proper training, implementation, and execution of the chosen protocol to ensure that cell survival is optimized (16Practice Committees of the American Society for Reproductive Medicine, Society of Reproductive Biologists and TechnologistsA review of best practices of rapid-cooling vitrification for oocytes and embryos: a committee opinion.Fertil Steril. 2021; 115: 305-310Google Scholar). Cryobiology already forms the backbone for several treatments in human medicine, such as frozen-thawed hepatocyte liver infusion for liver disease (17Gramignoli R. Dorko K. Tahan V. Skvorak K.J. Ellis E. Jorns C. et al.Hypothermic storage of human hepatocytes for transplantation.Cell Transplant. 2014; 23: 1143-1151Google Scholar) and frozen pancreatic islet cell transplantation for the treatment of complicated type 1 diabetes (18Kojayan G.G. Alexander M. Imagawa D.K. Lakey J.R.T. Systematic review of islet cryopreservation.Islets. 2018; 10: 40-49Google Scholar). In the field of reproductive medicine, cryobiology is used for cryopreserving semen, ova, embryos, and gonadal tissues. Fertility preservation with oocyte or embryo cryopreservation has become increasingly popular over the last decade. Reasons people elect to undergo this procedure include social indications such as delayed childbearing, infertility diagnosis, medical conditions such as diminished ovarian reserve and gender dysphoria, or for newly diagnosed oncologic diseases that require gonadotoxic therapies, with the most common indication being for elective fertility preservation (19Kawwass J.F. Shandley L.M. Boulet S.L. Hipp H.S. Oncologic oocyte cryopreservation: national comparison of fertility preservation between women with and without cancer.J Assist Reprod Genet. 2020; 37: 883-890Google Scholar). Sperm banking before cancer treatment is the most common indication for sperm cryopreservation (20Teloken I.B. Petracco A. Fay A.P. Teloken C. Badalotti M. Human sperm cryopreservation prior cancer treatment: follow up of 480 males for 23 years.Am Soc Clin Oncol. 2020; Google Scholar), although there has been a trend toward social sperm banking recently because of advanced paternal age being associated with an increased genetic risk in offspring (21Pennings G. Couture V. Ombelet W. Social sperm freezing.Hum Reprod. 2021; 36: 833-839Google Scholar). In 2012, the American Society for Reproductive Medicine (ASRM) removed the experimental label for oocyte cryopreservation (22Practice Committees of the American Society for Reproductive Medicine, Society for Assisted Reproductive TechnologyMature oocyte cryopreservation: a guideline.Fertil Steril. 2013; 99: 37-43Google Scholar), and since that time, the total number of oocyte cryopreservation cycles has significantly increased. Kawwass et al. (19Kawwass J.F. Shandley L.M. Boulet S.L. Hipp H.S. Oncologic oocyte cryopreservation: national comparison of fertility preservation between women with and without cancer.J Assist Reprod Genet. 2020; 37: 883-890Google Scholar), using the Society for Assisted Reproductive Technology (SART) Clinic Outcome Reporting System, found that the absolute number of oocyte cryopreservation cycles increased from 2,719 in 2012 to 13,824 in 2018 with oocyte thaw cycles increasing at a slower rate from 348–1,810 during this time span. They also found the duration of months from oocyte cryopreservation to thaw increased over this time span. In 2012, the mean number of months from oocyte cryopreservation to thaw was 15.7 months compared with 29.4 months in 2018. Of those that underwent oocyte thaw, almost half had supernumerary embryos cryopreserved (19Kawwass J.F. Shandley L.M. Boulet S.L. Hipp H.S. Oncologic oocyte cryopreservation: national comparison of fertility preservation between women with and without cancer.J Assist Reprod Genet. 2020; 37: 883-890Google Scholar). The same trend was also observed when examining embryo cryopreservation and utilization. Christianson et al. (23Christianson M.S. Stern J.E. Sun F. Zhang H. Styer A.K. Vitek W. et al.Embryo cryopreservation and utilization in the United States from 2004–2013.F S Rep. 2020; 1: 71-77Google Scholar), using the SART Clinic Outcome Reporting System from 2004 to 2013, observed that the number of freeze all cycles increased during from 7.9% in 2004 to 40.7% in 2013, and in fresh ART cycles in which all embryos were cryopreserved, the mean number of embryos cryopreserved was 6.3 per patient. Because of the increase in freeze all cycles, the number of embryos cryopreserved per year increased from 158,383 in 2004 to 303,203 in 2013, and over the 10-year span, 1,954,548 embryos were cryopreserved with less than half, 717,345 embryos, transferred (23Christianson M.S. Stern J.E. Sun F. Zhang H. Styer A.K. Vitek W. et al.Embryo cryopreservation and utilization in the United States from 2004–2013.F S Rep. 2020; 1: 71-77Google Scholar). Christianson et al. (23Christianson M.S. Stern J.E. Sun F. Zhang H. Styer A.K. Vitek W. et al.Embryo cryopreservation and utilization in the United States from 2004–2013.F S Rep. 2020; 1: 71-77Google Scholar) showed that over 1.2 million of these embryos were potentially still in storage, although they could not account for embryos that were discarded or donated or did not survive the thaw; therefore, the actual number in storage is likely lower. At this time, there is no known "shelf-life" a cryopreserved embryo has before it becomes unusable, and to date, the record for the longest embryo stored before transfer and giving birth is 27 years (24Honderich H. Baby girl born from record-setting 27-year-old embryo.https://www.bbc.com/news/world-us-canada-55164607Date: 2020Date accessed: January 4, 2022Google Scholar). Clinical IVF laboratories have an enormous liability in storing gametes and embryos. This has been vividly pointed out in the recent court case of plaintiffs against Pacific Fertility Center, Prelude (their parent company), and Chart Industries, Inc. (Ball Ground, GA), the manufacturer of the tank. On June 11, 2021, a jury awarded the 5 plaintiffs $15 million when a tank's vacuum seal failed at a fertility clinic. Chart Industries, Inc., the manufacturer of the tanks, was found 90% responsible, and Pacific Fertility Center was found 10% responsible for the egg and embryo losses (25Jury awards $14.975 million in IVF tank failure trial. Girard Sharp 2021.https://www.girardsharp.com/newsroom-news-Jury-IVF-TrialGoogle Scholar). Hundreds of other patients also have claims pending against Chart Industries, Inc., and Pacific Fertility Center, and additional trials are scheduled. It is important to understand how common these failures are. One study (26Letterie G. Fox D. Lawsuit frequency and claims basis over lost, damaged, and destroyed frozen embryos over a 10-year period.F S Rep. 2020; 1: 78-82Google Scholar) examined court dockets from January 1, 2009 to July 1, 2019, and found 133 cases relating to lost, damaged, or destroyed embryos in US courts. Eleven of the 133 cases were caused by embryos destroyed due to storage tank failure. If failure of tanks is the most common cause of these cases, what can IVF clinics do to reduce and eliminate these tragic and expensive losses? One must realize that all tanks can fail, and the best way to minimize risk is by having effective methods to detect failure well before tissue can be damaged. One must be prepared for these failures and be able to rescue them before the temperature rises too high. In addition, one needs to set in place protocols that will detect either protocol mishaps (e.g., failure to fill a tank), errors, or damages of the tanks or alarms, as described in the ASRM Practice Committee Opinion (27Practice Committees of the American Society for Reproductive Medicine, Society for Reproductive Biologists and Technologists, Society for Assisted Reproductive TechnologyCryostorage of reproductive tissues in the in vitro fertilization laboratory: a committee opinion.Fertil Steril. 2020; 114: 486-489Google Scholar). There are currently 4 major types of tanks in use in the reproductive laboratory: first, shipper tanks used for transporting tissue and 3 types of storage tanks—small-capacity liquid nitrogen tanks, large-capacity liquid nitrogen tanks, and large-capacity vapor nitrogen tanks. Shipper tanks are charged by filling the tank with liquid nitrogen and then dumping out the liquid nitrogen, leaving the residue trapped in a matrix surrounding the tank. These tanks may be the most common ones to fail, and when they fail, the tissue is often destroyed because there is no real-time monitoring during the shipment process where the tank could be refilled (28Lomanto B, Rush A, Pomeroy KO. Spontaneous liquid nitrogen tank failure, analysis of evaporation rates and temperatures, and comparison of tank monitoring systems. J Assist Reprod Gen. In press.Google Scholar). In addition, the evaporation rate when the vacuum fails on these tanks allows only a 4–6-hour response time before the tank warms up to dangerous levels. One of the newest types of storage tanks is the vapor tank. Instead, of immersing the specimen in liquid nitrogen, the tissue is stored in the vapor phase of nitrogen. Although this temperature is sufficient to keep the tissue below the critical temperature of the glass transition temperature of approximately −130°C, its mechanics (viz, solenoid, heat exchanger, or fan) must be maintained for it to safely store vitrified specimens. A vapor tank was the type that recently failed in an infertility laboratory in Cleveland, Ohio, because of what may have been a faulty solenoid—the device that injects nitrogen into the tank. The most common tank for reproductive tissue storage is the small-capacity tank. It is usually made of an aluminum or aluminum alloy mix and holds <100 L of liquid nitrogen. The most common tank used for storage in clinical IVF laboratories in the United States is the MVE 47/11. There have been several reports of issues with the welds of the neck of the tank to the inner tank where the liquid nitrogen and specimens are stored. Failure at these necks causes a loss of vacuum, resulting in an inefficient tank that increases the evaporation rate of its liquid nitrogen 100-fold when damaged. Such a damaged tank will go from over 70 days of storage to approximately 1 day (28Lomanto B, Rush A, Pomeroy KO. Spontaneous liquid nitrogen tank failure, analysis of evaporation rates and temperatures, and comparison of tank monitoring systems. J Assist Reprod Gen. In press.Google Scholar). If undetected, the specimens will warm, and the tissue will be destroyed. Large-capacity tanks, with liquid nitrogen capacity in hundreds of liters, provide more storage capacity and appear to not be crippled with the Achilles heel of failing neck welds of the small-capacity tanks. They are usually made of steel, are less efficient in their use of liquid nitrogen, and have a weakness of depending on a mechanical/electrical solenoid for automatically filling the tank. This weakness can be eliminated by disarming this function and relying on manually filling these tanks. Large-capacity tanks that are, thus, modified may be the safest of tanks for tissue storage (29Pomeroy K.O. Reed M.L. LoManto B. Harris S.G. Hazelrigg W.B. Kelk D.A. Cryostorage tank failures: temperature and volume loss over time after induced failure by removal of insulative vacuum.J Assist Reprod Genet. 2019; 36: 2271-2278Google Scholar). Any safeguard system for a tank must be able to perform 3 major functions. First, it must be able to detect a failure to fill the tank; second, it should be able to detect catastrophic failure of a tank; and third, it should be able to continuously monitor the temperature of the tank at the level where the tissues are stored. This latter function will be of use if there are ever legal proceedings against a clinic to show that the tissue was never exposed above −130°C. One also needs a method to detect a deteriorating tank that may be slowly losing its vacuum, such as measuring the liquid nitrogen evaporation rates at adequate periods to detect when a tank is starting to lose its vacuum. Measuring tank levels with a ruler may suffice, but weight-based methods using a scale should be more precise in monitoring the volume of remaining nitrogen and, consequently, the evaporation rates of each tank. If a non–weight-based or nitrogen level probe is not used, the tank levels should be checked 3 times a week (30CAP Reproductive Laboratory Checklist. College of American Pathology 2021. Available at www.cap.org.Google Scholar). Tanks should also be visually inspected for leakage (cold spots or sweating) at least twice a day, preferably early in the morning and when the laboratory is being closed for the day. A thermal camera alarm system can provide 24-hour monitoring for vacuum failure. It will alarm should the external tank temperature drop below a certain threshold. Any alarm system should be tested at least quarterly, and one should record the response time. The Practice Committees of the American Society for Reproductive Medicine and Society of Reproductive Biologists and Technologists (27Practice Committees of the American Society for Reproductive Medicine, Society for Reproductive Biologists and Technologists, Society for Assisted Reproductive TechnologyCryostorage of reproductive tissues in the in vitro fertilization laboratory: a committee opinion.Fertil Steril. 2020; 114: 486-489Google Scholar), Pomeroy and Marcon (31Pomeroy K.O. Marcon M. Reproductive tissue storage: quality control and management/inventory software.Semin Reprod Med. 2018; 36: 280-288Google Scholar), and Schiewe et al. (32Schiewe M.C. Freeman M. Whitney J.B. VerMilyea M.D. Jones A. Aguirre M. et al.Comprehensive assessment of cryogenic storage risk and quality management concerns: best practice guidelines for ART labs.J Assist Reprod Genet. 2019; 36: 5-14Google Scholar) provide a more complete discussion of proper quality control and quality management. In the past, the laboratory has relied on temperature probes to detect when either the tank temperature is increasing or the liquid nitrogen level in the tank is too low. Recent studies have shown that if these probes are not checked individually for the time from alarm signal to the time the tank reaches the critical temperature (−130°C), this response time may be too short. This is in part due to the high efficiency of small-capacity tanks where the temperatures within the tank only start increasing when the tank is almost empty (28Lomanto B, Rush A, Pomeroy KO. Spontaneous liquid nitrogen tank failure, analysis of evaporation rates and temperatures, and comparison of tank monitoring systems. J Assist Reprod Gen. In press.Google Scholar). It has been recommended that each tank contain the following alarm probes for adequate monitoring of tanks: a temperature probe located near the level of the topmost tissue in the tank that provides a constant record of this temperature; nitrogen level probe to monitor the amount of liquid nitrogen in the tank; and probe/alarm system that will provide early warning for a catastrophic failure of a tank (31Pomeroy K.O. Marcon M. Reproductive tissue storage: quality control and management/inventory software.Semin Reprod Med. 2018; 36: 280-288Google Scholar). This probe/alarm system could monitor the external temperature of the tank or be a weight-based system that can detect rapid drops in nitrogen volume. There have been few advancements in vitrification protocols since the early 2000s. A few semiautomated vitrification systems have been developed, but the penetrance into the IVF market is minimal. There have been some discussions of developing new vitrification methods where instead of moving the tissue into different media, the tissue would remain stationary and the media as it changes would be moved over the tissue using a flow-cell like device (33Smith G.D. Motta E.E. Serafini P. Theoretical and experimental basis of oocyte vitrification.Reprod Biomed Online. 2011; 23: 298-306Google Scholar). With this method, one could gradually change the concentrations of the cryoprotectants being added or removed. Other advancements may include the use of ice blockers (9Yong K.W. Laouar L. Elliott J.A.W. Jomha N.M. Review of non-permeating cryoprotectants as supplements for vitrification of mammalian tissues.Cryobiology. 2020; 96: 1-11Google Scholar), additives that protect from reactive oxygen species (34Gupta M.K. Uhm S.J. Lee H.T. Effect of vitrification and beta-mercaptoethanol on reactive oxygen species activity and in vitro development of oocytes vitrified before or after in vitro fertilization.Fertil Steril. 2010; 93: 2602-2607Google Scholar, 35Trapphoff T. Heiligentag M. Simon J. Staubach N. Seidel T. Otte K. et al.Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.Mol Hum Reprod. 2016; 22: 867-881Google Scholar), and methods that protect the tissue from radical changes in osmolality or pH or increase cooling velocities (36Yoon T.K. Lee D.R. Cha S.K. Chung H.M. Lee W.S. Cha K.Y. Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies.Fertil Steril. 2007; 88: 952-956Google Scholar). Despite the advancements in cryopreservation, more work needs to be done to improve outcomes and reduce variability in the vitrification process. This advancement is probably impaired by the ethical problems of research in not only the use of human subjects but the issues of working with human gametes and embryos. Somehow, however, we must work to improve cryopreservation in human fertility. Cryopreserved sample recovery also depends on rapid heat penetration during warming to avoid ice recrystallization. The recent development of ultrarapid warming using lasers has improved survival of large frozen specimens preloaded with wavelength-absorbing materials (India ink or nanoparticles). Immediately upon removal from liquid nitrogen, the sample can be exposed to a laser beam that evenly warms the tissue via the dispersed nanoparticles. This approach has resulted in improved viability of murine oocytes (37Jin B. Kleinhans F.W. Mazur P. Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse.Cryobiology. 2014; 68: 419-430Google Scholar), zebrafish embryos (38Khosla K. Wang Y. Hagedorn M. Qin Z. Bischof J. Gold nanorod induced warming of embryos from the cryogenic state enhances viability.ACS Nano. 2017; 11: 7869-7878Google Scholar), and coral larvae (39Daly J. Zuchowicz N. Nuñez Lendo C.I. Khosla K. Lager C. Henley E.M. et al.Successful cryopreservation of coral larvae using vitrification and laser warming.Sci Rep. 2018; 8: 15714Google Scholar). As mentioned earlier, sample structures and functions are currently stabilized by cold temperatures. This state of "suspended animation" ensures long-term longevity and quality of cellular and molecular components (40Hubel A. Spindler R. Skubitz A.P. Storage of human biospecimens: selection of the optimal storage temperature.Biopreserv Biobank. 2014; 12: 165-175Google Scholar). Before, during, and after cryopreservation, samples experience stresses related to exposure to toxic cryoprotectant(s), cooling/freezing, detrimental ice formation, and thawing/rewarming. Resilience of tissues, cells, organelles, and deoxyribonucleic acid depends on protocols that have to be adapted for each type of tissue and each species (41Comizzoli P. Wildt D.E. Mammalian fertility preservation through cryobiology: value of classical comparative studies and the need for new preservation options.Reprod Fertil Dev. 2013; 26: 91-98Google Scholar, 42Comi