Novel approaches to the cryopreservation of human spermatozoa: History and development of the spermatozoa vitrification technology

Novel Approaches to the Cryopreservation of Human Spermatozoa: History and Development of the Spermatozoa Vitrification TechnologyNovel approaches to the cryopreservation of human spermatozoa: History and development of the spermatozoa vitrification technology Evgenia Isachenko, PhD1, Gohar Rahimi, MD1, Peter Mallmann, MD1, Raul Sanchez, MD2, Vladimir Isachenko, PhD1
1Department of Gynaecological Endocrinology and Reproductive Medicine, University of Cologne, Kerpener Str. 50931 Cologne, Germany; 2Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Abstract Cryobiology is very intensively applied in reproductive and veterinary medicine for preservation of gametes, embryos and reproductive tissues. Sub-zero temperatures combined with appropriate cryoprotective agents preserve the physiological and reproductive functions of the cells making long-term storage possible without loss of viability.With the use of cryoprotective agents it has become possible to develop cryopreservation techniques, such as the slow conventional freezing and vitrification that are in use in the present times. In slow controlled-rate conventional freezing extracellular ice crystals are formed whereas in vitrification no ice crystals are formed. Glass formation is compatible with the survival of the cell and the preservation of its intracellular structures provided the type(s) and concentrations of cryoprotectant used are not chemo- or osmotoxic. However, irrespective of the type of cooling method employed the cryosurvival of cells and tissues is influenced by the size and maturity of cells, amounts of intracellular water, quality and quantity of intracellular lipids, type of cells, their function and morphology. The intracellular milieu of cryopreserved cells and tissues remain less understood. The application of nanotechnology may help reveal and help advance our knowledge of the cryobiological principles involved in cryosurvival. At this moment the methods of cryopreservation that merit further investigation are vitrification and lyophilization. Vitrification is cheap if reagents are prepared in-house and the procedure can be performed rapidly. It has been successfully applied for gametes and embryos (of different stages of development), and reproductive cells/tissues, somatic cells and stem cells. However, vitrification is more demanding technically and requires operation and storage at sub-zero temperatures. On the other hand lyophilization deserves further investigation because it is a cheaper form of cryopreservation that may enable cryostorage at less demanding temperatures of 4°C and may even allow transport at ambient temperature. These possibilities are explored in this review. Disclaimer: None of the authors have any conflicts of interest, whether of a financial or other nature J Reprod Stem Cell Biotechnol 2(2):128-145 Correspondence: Dr. Evgenia Isachenko, Department of Gynaecological Endocrinology and Reproductive Medicine, University of Cologne, Kerpener Str. 34, 50931 Cologne, Germany, Email: [email protected], T: +49 221 478 4924 ; F: +49 221 478 86201 Keywords: Cryopreservation, history, ICSI, IVF, spermatozoa, vitrification
Introduction The frozen- storage of human spermatozoa is now a routine technique in assisted reproduction. This technique offers the following advantages over the use of fresh ejaculated spermatozoa: Storage of both homologous or donor spermatozoa for subsequent intrauterine insemination, in vitro fertilization and or intracytoplasmic spermatozoa injection; long- term storage of known quality donor semen; allows the quarantine of donor semen until appropriate testing can be completed; preservation of epididymal or testicular
spermatozoa and/or tissue for subsequent intracytoplasmic spermatozoa injection or for diagnostic purposes or prior to radio- /chemotherapy (Keel and Webster, 1989; Nijs and Ombelet, 2001). Fertility preservation has acquired prominence as the next area of scientific inquiry because radio-/ chemotherapy results in a significant reduction of spermatozoa quality and as consequence infertility ensues (Quinn and Kelly. 2000; Meistrich et al. 2005; Trottmann et al. 2007 ). In such instances human genome banking offer the potential for
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preserving the gametes of young boys and adult men before sterilization, chemo- or radiotherapy and onset of autoimmune diseases (Nijs and Ombelet, 2001; Trottmann et al., 2007; Sanger et al., 1992; Brannigan and Sandlow, 2008; Ginsberg et al., 2008) or before other treatment modalities that may adversely affect fertility and before surgical procedures which may lead to testicular failure or ejaculatory dysfunction (Donnelly et al., 2001). Methods of cryopreservation developed in the 1950s are still in use today (Gao et al., 1997; Mazur, 1963). These methods are based on the use of slow cooling rate of 1° to 170°C /min with the critical temperature being in the region of - 10° to -60°C. The major steps used for cryopreservation of different kinds of cells can be summarized as follows: 1) addition of cryoprotective agents (CPAs) before freezing. This substance enhances post-thaw/warm survival by limiting the crystallization of water; 2) seeding of samples at the freezing or around the freezing point; 3) thawing/warming of the cells and 4) removing the CPAs from the cells after thawing/warming (Karow, 1997). Cells have the capability to endure storage at very low temperatures (lower than –100°C). The challenge to cell survival is the lethality of the cooling and warming processes in the intermediate zone of critical temperatures (-10°C to -60°C) that cells must traverse twice: once during cooling and again during warming (Mazur, 1963). The reason for using slow cooling rates is to maintain a very delicate balance between ice crystal formation and the increase in concentration of dissolved substances. An imbalance may lead to cell damage due to crystallization of intracellular water, osmotic and chilling injury, cytoplasm fracture, or even effects on the cytoskeleton, genome or genome-related structures (Critser et al.,1988; Perez-Sanchez et al.,1994; Mazur et al., 1981; Fraga et al.,1991). In spite of this, the use of programmable or non- programmable slow conventional freezing (McLaughlin et al.,1990; Yin and Seibel, 1999) led to acceptable level of survival of frozen- thawed spermatozoa in ejaculates from 0.25 to 1.0 ml. This is demonstrated by resumption of motility of the spermatozoa after thawing (Zavos et al., 1991; Sawetawan et al., 1993; , Larson et
al. 1997), integrity of acrosomal and cytoplasmic membrane (Hammadeh et al. 1999), functional activity of mitochondria (O’Connell et al., 2002; Meseguer et al. 2004), DNA stability (Isachenko et al., 2004a,b) and prevention of phospholipids translocation inside of spermatozoa membranes (Schuffner et al..2001; Duru et al.2001). Vitrification without cryoprotectant Vitrification is not yet routinely applied to cryopreservation of spermatozoa. More investigations are essential in this area. Vitrification of spermatozoa without CPAs has not been attempted except by the authors. It may be worthwhile investigating spermatozoa vitrification because it does not require a cooling program. Furthermore the use of permeable cryoprotectants may be circumvented if the cooling rate is fast enough; it is rapid, less tedious, economical with the possibility of high recovery of motile spermatozoa after warming (Isachenko et al. 2003a,b; 2008; Nawroth et al., 2002). The method is based on the cooling of cells by direct immersion into liquid nitrogen, thereby avoiding the formation of large intracellular ice crystals (Luyet, 1937). Vitrification was successfully applied in 1985 in mouse embryos (Rall and Fahy, 1985) using high concentrations of permeable CPAs. Much later the technique was refined to successfully preserve human female gametes (Nagy et al., 2009; Cao and Chian, 2009) and embryos (Rezazadeh Valojerdi et al.,2009; Son et al. 2009). Vitrification requires very high concentration of CPAs. However, to apply this protocol for spermatozoa cryopreservation may not be impossible because of the resulting osmotic and cytotoxic effects (Critser et al.,1997; Liebermann et al., 2002). The promising results after successful vitrification of frog (Luyet and Hoddap, 1938) and fowl (Schaffner, 1942) spermatozoa were not confirmed in subsequent investigations (Hoagland and Pincus, 1942; Smith, 1962) and work in this direction was stopped for 40 years. At present, as an extremely rapid method of cryopreservation (Silva and Berland, 2004), called vitrification, have been investigated extensively and applied to embryos (Cervera and Garcia-Ximénez, 2003) and oocytes (Isachenko et al, 2004a; Chen et al., 2001), but very seldom to spermatozoa, with the exception of a few reports (Isachenko et al.,2003a; 2004a; 2004b; 2005; 2008; Nawroth et al., 2002;
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Koshimoto C, Mazur, 2002; Schuster et al., 2003; Desai et al., 2004; Hossain AM, Osuamkpe, 2007). At present preservation fall into two categories. 1. In situ preservation: that is the preservation and protection of individual species in their natural setting and 2. ex situ preservation: preservation of the genomic material of individual species in combination with breeding programmes (Seal, 1998). In human medicine the cryopreservation of male gametes offers to men and boys the possibility to preserve their fertility. It was early postulated that after the discovery of the beneficial effects of permeable and non-permeable cryoprotectants on plant cryostability led to the establishment of cryobanks for low temperature storage of genetic material (Maksimov, 1913). Subsequent investigation of the permeable cryoprotectant glycerol led to the successful cryopreservation of mammalian spermatozoa (Bernschtein and Petropavlovski, 1937; Polge et al., 1949; Smirnov, 1949; . Milovanov, 1962). These empirical methods which were developed in the 1950s are still applied today in many species and have led to the use of permeable cryoprotectants. However, until now the technology available for spermatozoa cryopreservation has not provided complete protection because the motility of cryopreserved spermatozoa normally drops to about 50% of their pre-freezing value, with considerable inter-sample fluctuation (Critser et al., 1988; Esteves et al., 2000). The question of diminished spermatozoa motility after freezing is crucial since this variable is known to be the first affected (Watson, 1955) although the mechanism of spermatozoa impairment and its mechanical and/or physic–chemical etiology remain unclear. The reasons for cell damage leading to loss of motility could be due to mechanical cell injury as a consequence of intracellular or extracellular ice crystal formation, and osmotic damage due to extensive cell shrinkage during conventional freezing procedures. The warming process also has a negative influence on cells through the possible excessive osmotic swelling (Gao et al., 1995; 1997). These two factors which can occur during freezing damage the spermatozoa cell membranes, which is the consequence of changes in lipid phase transition and/or increased lipid peroxidation and active production of reactive oxygen species (Aitken et
al., 1989) and subsequent loss of spermatozoa motility (O’Connell et al., 2002; Alvarez and Storey, 1992). The permeable and non- permeable cryoprotectants used during conventional freezing to prevent the intracellular ice formation, can be also be harmful due to CPAs’ toxicity (Isachenko et al., 2003a). The cell membrane due to osmotic and chemical influences of CPA’s during freezing/thawing (Gao et al., 1995; Katkov et al.,1998), activates an apoptosis-like mechanism (Martin et al., 2004) and can also lead to chromatin damage (Sakkas and Tomlinson, 2000). All these findings suggest that slow cooling, and especially thawing of spermatozoa, quite apart from ice crystal formation, is intrinsically deleterious. Compared to slow conventional freezing, vitrification has advantages that could lend itself as the best alternative to standard conventional slow controlled-rate cryopreservation. Why vitrification? Luyet (34) wrote that ice crystallization is incompatible with living systems and should be avoided whenever possible. The cooling of small living systems at ultra-high speeds of freezing was considered possible, if it could circumvent ice nuclei formation and its growth into deleterious crystals. In such a situation the cooled solution and the cell results in a glass- like (vitreous) state (Luyet, 1937). This was the basis for the origin of the idea of vitrification. Vitrification could not be applied to large organs. Early attempts to vitrify large organs failed as they could not be cooled without ice crystal formation or chemotoxicity (Luyet, 1937) but it can be effectively utilized for cryopreservation of cells. It is known that vitrification is utilized as a natural form of cryoprotection in some arctic plants (Hirsh, 1987). In contrast to slow rate freezing protocols, during vitrification the entire solution remains unchanged in appearance and the water does not precipitate, so no ice crystals are formed (Fahy, 1986a). The physical definition of vitrification is the solidification of a solution (water is rapidly cooled and formed into a glassy, vitrified state from the liquid phase) at low temperature, not by ice crystallization, but by extreme elevation in viscosity during cooling (Fahy et al., 1984). Fahy (Fahy, 1986a) expressed this as follows: “... the viscosity of the sample becomes greater and greater until the
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molecules become immobilized and the sample is no longer a liquid, but rather has the properties of a solid…“. However, vitrification with high cooling rates alone is normally insufficient to achieve vitrification unless performed in presence of high concentrations of cryoprotectant(s). Therefore, it is inevitable that the presence of cryoprotectants will affect adversely all living organisms due to their chemotoxicity. Early attempts at vitrification were thus fraught with difficulties as the presence of CPA’s was biologically problematic and technically difficult (Rall, 1987). There are two ways to achieve the vitrification of water inside the cells: 1. To increase the speed of conduction of temperature, and 2. To increase the concentration of cryoprotectant(s). In addition, by using a low volume of high concentration cryoprotectant (<1 ml), very rapid cooling rates from 15,000 to 30,000°C/min can be achieved (e.g., T from -196°C to 25°C = 221°C/0.5 sec = 5). The strategy of vitrification is to achieve decreased amounts of ice crystals from extracellular water and total prevention of ice crystallization of the intracellular water. These physicochemical principles required to achieve the glass-like solidification of solution or vitrification is well documented and have been described by a number of authors (Isachenko et al., 2003a; Cao et al., 1998). In vitrification the solution must remain free of ice during both cooling and warming. Cooling is performed substantially faster than in equilibrium freezing (in the latter ice is formed during cooling). This can be achieved by the combination of relatively slow to moderate speed of cooling (up to 105°C/min) with the use of high concentrations (3.5–8 mol/L) of permeable CPAs; Or by solidification of the bulk solution by abrupt cooling at a very high speed of cooling to temperatures below the glass transition temperature of the solution. This can be achieved by direct plunging of the specimen into liquid nitrogen LN2 with or without direct exposure of the cryoprotectant and specimen to the LN2. Exposure of the cryoprotectant and specimen to LN2 is not recommended as it carries a risk of contamination of the specimen by dangerous pathogenic agents as well as by contaminant toxic chemicals in the LN2.
The molecular structure of the glassy state is that of a liquid but the simultaneous extreme elevation in viscosity (viscosity is proportional to cooling rate) provides it the vitrified state with mechanical properties similar to that of a solid. In the glassy state, all physic-chemical processes are completely arrested. It has been suggested that a number of chemicals can afford cryoprotection to cells (Karow, 1997). These include alcohols (including glycols), amines (including amides), sugars, inorganic salts, and macromolecules (including proteins and polysaccharides) and dimethylsulfoxide. However, it is well known, the rapidly permeable CPAs (such as ethylene and propylene glycol, glycerol, DMSO) are toxic to cells at specific concentrations (Fahy, 1986; Karran and Legge, 1996; Volk et al., 2006; Ali and Shelton, 1993a- c). The least toxic permeating CPA is ethylene glycol (Ali and Shelton, 1993a-c). It is possible to lower the toxic effect of high concentration of permeable CPAs (critical CPA concentration, Cv) by including some non-permeable CPAs. These compounds include carbohydrates (saccharides), polymers (polyvinilpirrolidone), polyols (polyethylene glycol) and polysaccharide (Ficoll), amines (acetamide, formamide), inorganic salts (sodium citrate, ammonium sulphate) proteins (albumin, antifreeze peptide/glycopeptide), phospholipids (hen egg yolk) or by using the combination of two or more permeable and non-permeable cryoprotectants (Fahy, 1985b; Ali and Shelton, 1993a). As a rule, carbohydrates are used for spermatozoa cryopreservation to compensate for the decrease in osmotic pressure caused by the permeable cryoprotectant glycerol, which works as an additional dissolvent and has the property to decrease the medium’s osmotic pressure (Jakobsen,1956). For example, the combination of sugars with permeable CPAs (Kuleshova et al.,1999) leads to a major influence on the vitrification properties of such cryoprotective mixtures resulting in a lowering of permeable CPA- induced chemotoxicity to embryos and oocytes because the use of non- permeable cryoprotectant(s) significantly decreases the concentration of permeable cryoprotectants needed for efficient cryopreservation (Isachenko et al., 2007). In general, the incorporation of non-permeable compounds into the vitrifying solution, and the incubation of the cells in this solution before vitrification helps to withdraw more water from
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the cells, and lessens the prospect of chemotoxicity due to the reduced concentration of permeable cryoprotectants used. Non- permeable cryoprotectants provide protection against osmotoxicity compensating the drop in osmotic pressure and acts as an osmotic buffer to reduce the osmotic shock that might otherwise occur as a result of the dilution of the cryoprotectant after cryostorage thereby stabilizing the cell membrane (Koshimoto and Mazur, 2002; Nakagata and Takeshima, 1992; Nakagata, 2000; Koshimoto et al., 2000). Cells naturally contain high concentrations of protein, which are helpful in vitrification. Higher concentrations of cryoprotectants are needed for extracellular vitrification than for intracellular vitrification. It was demonstrated that under certain conditions a polymer can reduce the Cv on average by 7% and as much as 24% in combination with an increased hydrostatic pressure ( Fahy et al., 1984). Early studies evaluated the potential beneficial effects of adding macromolecular solutes to the vitrification solution to facilitate vitrification (Kasai et al.,1990). These polymers can protect embryos against cryoinjury by mitigating the mechanical stresses occurring during cryopreservation (Dumoulin et al.,1994). They do this by modifying the vitrification properties of these solutions by significantly reducing the amount of cryoprotectant required to achieve vitrification (Shaw et al.1997). They also influence the viscosity of the vitrification solution and reduce the toxicity of the cryoprotectant through lowered concentrations. Furthermore, the polymers may be able to build a viscous matrix for encapsulation of cells, and also prevent crystallization during cooling and warming (Kasai et al.,1990). Indeed, O’Neill (O'Neill et al., 1997) observed that the addition of PEG resulted in greatly improved viability of oocytes following cryopreservation, and vastly reduced the variability seen with vitrification alone. It is possible to reduce the effect of permeable CPAs by exposing the cells to a graded series of pre-cooled concentrated solutions (Rall and Fahy, 1985). The combination of the methods described with the increasing rate of cooling and warming allows the reduction of both the toxic and osmotic effects of cryoprotectants. The higher cooling rate could be achieved by using the electron-microscope grid (Steponkus
et al.,1990 ) or nylon loop, which allowed vitrification at much lower concentrations of the cryoprotectant used due to the significantly decreased volume (Liebermann et al., 2002). The small amount of such cryopreservation solution (0.1 – 1.0 µl) immersed direct into liquid nitrogen promoted ultra-rapid cooling circumventing ice-crystal formation in spite of lower cryoprotectant concentration. Other vehicles used that employs this principle include: open pulled straws (Vajta et al.,1997), the flexipet-denuding pipette (Liebermann et al.,2002), micro drops (Papis et al., 2000), electron microscope copper grids (Martino et al.,1996), the hemi-straw system (Vanderzwalmen et al., 2003); cryotop (Kuwayama and Kato, 2000), small nylon coils (Kurokawa et al., 1996), or nylon meshes (Matsumoto et al.,2001), the cryoloop (Oberstein et al., 2001), cryoleaf (Chian et al., 2008), and the cryotip (Kuwayama et al.,2005). These vehicles have been used with excellent survival rates in routine human reproductive as well as veterinary medicine for vitrification of oocytes and embryos (Chen et al., 2001, Lane et al., 1999; Zhang et al., 2009; Somfai et al., 2009; Campos-Chillòn et al., 2009; Kyono et al., 2009; Liow et al.,2009) but almost all of these methods expose the specimen to contamination hazards because of direct exposure to LN2. The authors’ have observed (Nawroth et al., 2002; Isachenko et al., 2003a) it was possible to achieve similar results in the absence of the ‘conventional’ CPAs, provided that the cooling/ warming speed is high enough to ensure both intra- and extracellular vitrification. In general, the rate of cooling/warming and the concentration of cryoprotective agents required to achieve vitrification…