**2. Brief history of kinetic vitrification of sperm and cryobiology in general related to the goal of this Chapter**

#### **2.1 Early attempts of** *kinetic* **vitrification of sperm and other cells**

In the dawn of cryopreservation, vitrification of small samples by ultra-fast cooling (tens of thousands OC/min) without additional thickening and ice-blocking agents (VFAs), which is

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 7

Particularly, equilibrium CP of embryos would require much slower pace of cooling (0.3-1

Following cryopreservation of animal spermatozoa, the successful slow freezing of human sperm with glycerol followed, and the first birth was reported by Sherman and colleagues 1964 [Perloff *et al.*, 1964]. It was then followed by the use of frozen spermatozoa for practically all assisted reproduction techniques (ART) mentioned above. Yet, since his first publications, Sherman had questioned the efficiency of glycerol as the ideal CPA for human spermatozoa [Sherman, 1963, 1964]. The addition and especially removal of permeable osmotically active cryoprotective agents (cryoprotectants) during freezing and warming can induce a lethal mechanical stress *per se*. Further problems include the chemical toxicity of cryoprotectants and the possible negative influence on the genetic apparatus of the

On the other hand, Greg Fahy and colleagues [Fahy *et al.*, 1984] reported the vitrification of a whole organ--a rabbit kidney--using high pressure and *extremely high concentrations* of permeable vitrificants. We will call that approach, which for all intents and purposes will be clarified later, *equilibrium* vitrification. The needs of more quick and robust methods of cryopreservation of mammalian embryos had been clear, since Mazur and colleagues and Wilmut had obtained the "frozen mice" by SF in 1972 [Whittingham *et al.*, 1972; Wilmut, 1972].Plus, Fahy's initial report led to the collaboration between him and W. Rall (former Mazur's student, who specialized in freezing embryos) and the first successful vitrification of mouse embryos was reported a year after Fahy's first report [Rall & Fahy, 1985]. The first human baby from a vitrified embryo was reported in 1990 ?? [Gordts *et al.*, 1990]. Since then, vitrification has become an equally spread assisted reproduction technique (ART) as programmed slow freezing of embryos and, especially, oocytes for *in vitro* fertility (IVF)

For detailed state of the art of vitrification of reproduction cells, see several Chapters of this Book and Book 2, as well an excellent book by Tucker and Lieberman [Tucker & Liebermann, 2007]. Several Chapters in that book will be referred throughout this Chapter as well. Particularly, an interesting history and even possible natural occurrence of E-VF in nature is described in the Chapter 1A of that book by Fahy and Rall ("*Certain Alaskan beetles dehydrate sufficiently to generate concentrations of up to 10 mol/L of endogenous glycerol,26 which is enough to vitrify aqueous solutions under laboratory conditions*") [Fahy & Rall, 2007]. Note, however that this particular Chapter 1A is substantially biased against K-VF in favor of E-VF, which we will address throughout the following sub-chapters, and toward the founder of the method, Father Luyet, including some far from diplomatic language escapades. That

**2.4 Vitrification** *of the majority* **of reproductive cells is moving from** *equilibrium* **to** 

While slow freezing showed its limitations for certain cell types (e.g. oocytes), a new era started when Rall and Fahy vitrified mouse embryos [Rall & Fahy, 1985] using essentially the same high concentrations of vitrificants vitrified by Fahy *et al.* used in its original report [Fahy *et al.*, 1984]. However, such high concentrations (40-60 % v/v) of VFA's such as

OC/min) so the whole cryopreservation process would take several hours.

mammalian spermatozoa [Gilmore *et al.*, 1997].

**2.3 The emerging of** *equilibrium* **vitrification** 

(see [Rezazadeh *et al.*, 2009] for examples and background).

part will be addressed at the end of the Chapter.

*kinetic approach*

referred here as kinetic VF, had been considered as the major method of cryopreservation at that time [Graevsky, 1948a, b; Graevsky & Medvedeva, 1948; Hoagland & Pincus, 1942; Jahnel, 1938; Luyet & Hodapp, 1938; Luyet, 1937; Park *et al.*, 2004; Schaffner, 1942]. Note that some authors contributed the first understanding of the importance of vitrification for biopreservation to a an earlier work of Walter Stiles [Stiles, 1930], as it, for example, is done in [Fahy & Rall, 2007]; we think, the Stiles's notion however was vague and had had a marginal impact. It was Luyet's work, which would make cryopreservation a *science*. From the outset, he recognized that ice damage must be avoided and vitrification could be a method for long-term preservation of cell viability [Luyet, 1937]. In 1938 Luyet and Hodapp achieved survival of frog spermatozoa vitrified by plunging into liquid air [Luyet & Hodapp, 1938], and later several Western European groups reported their experiences with attempts in kinetic vitrification of fowl [Schaffner, 1942], human [Hoagland & Pincus, 1942; Jahnel, 1938; Parkes, 1945], and rabbit spermatozoa [Hoagland & Pincus, 1942] with varying success. While not directly related to the K-VF of sperm, a clear notion of vitrification as the only way of viable stabilization of cells has been expressed by Graevsky in USSR. As he worked with bacteria, it was natural to use a bacterial sample collection loop to freeze the cells in thin pellicles [Graevsky, 1948a, b]. A similar approach was used by Hoagland and Pincus in Germany in 1942 [Hoagland & Pincus, 1942], which seems a very natural approach for very fast K-VF. Yet, in the money-driven 21st century, the term *"Cryoloop"* is a registered as a trademark. Apparently, those early scientists would have infringed the trademark law now!

These early efforts of K-VF of sperm did not receive the recognition they deserved, hindered by the low repeatability and poor survival, as well as difficulties in communication due to various "iron walls" that existed between scientists of the Western Allies, Germany and USSR in the era of WWII followed by the Cold War.

#### **2.2 The rise of slow freezing**

The breakthrough came from an independent discovery of the protective role of a permeable CPA glycerol by two groups in 1948-49 [Polge *et al.*, 1949; Smirnov, 1949].The high permeability of glycerol to the sperm membrane in conjunction to relatively low toxicity seemed to be the crucial factor; both groups unsuccessfully tried either non-permeable sugars such as glucose (Parkes's group) or very permeable but very toxic lower alcohols such as ethanol or methanol (Smirnov). The high membrane permeability of glycerol and, thus, fast penetration inside the cells allowed to preserve the cells using slow (10-40 OC/min) freezing, and very moderate warming rates by direct thawing on air or in a water bath. It then became the mainstream of the cryopreservation methods, and a vast variety of cell species of different biological taxa have been preserved by slow (also called *equilibrium*) freezing. It revolutionized two very important fields: the cattle industry (with preservation of bovine sperm and later bovine embryos) and cryopreservation of blood components. It is worth noting that 12 years before the discovery of Parkes's and Smirnov's groups, Bernstein and Petropavlovski had reported the protective role of glycerol during the freezing of sperm [Bernstein & Petropavlovski, 1937] to -20OC, but that work had gone largely unnoticed.

With the development of Peter Mazur's equations and the 2-factor hypothesis of cryodamage [Mazur, 1963; Mazur *et al.*, 1972] and work of other cryobiologists on slow (equilibrium) freezing in 1960's, it became clear that a particular cell would need its own optimal cryopreservation protocol, which would largely depend on the cell cryobiological and physiological parameters as well as on the type of cryoprotective agents (CPA's) used. Particularly, equilibrium CP of embryos would require much slower pace of cooling (0.3-1 OC/min) so the whole cryopreservation process would take several hours.

Following cryopreservation of animal spermatozoa, the successful slow freezing of human sperm with glycerol followed, and the first birth was reported by Sherman and colleagues 1964 [Perloff *et al.*, 1964]. It was then followed by the use of frozen spermatozoa for practically all assisted reproduction techniques (ART) mentioned above. Yet, since his first publications, Sherman had questioned the efficiency of glycerol as the ideal CPA for human spermatozoa [Sherman, 1963, 1964]. The addition and especially removal of permeable osmotically active cryoprotective agents (cryoprotectants) during freezing and warming can induce a lethal mechanical stress *per se*. Further problems include the chemical toxicity of cryoprotectants and the possible negative influence on the genetic apparatus of the mammalian spermatozoa [Gilmore *et al.*, 1997].
