**What is the reason?**

Because:

40 Current Frontiers in Cryobiology

Zhou C. Q., Mai Q. Y., Li T. &Zhuang G. L. (2004). "Cryopreservation of human embryonic


Cryobiology is a rapidly evolving field which only relatively recently has found broad applications in reproductive medicine. However, as any emerging technology, it has both a great potential and a need for further developments (Petrunkina, 2007).

According to worldwide experience, successful cryopreservation of spermatozoa from different kind of animal including human for long-term storage (cryobanking of genome) or relatively short-term storage (artificial insemination) in conjunction with assisted reproductive technologies allows to promote long-term cryopreservation programmes. The use of programmable or non-programmable "slow" (conventional) freezing (McLaughlin et al., 1990; Yin and Seibel, 1999; Stanic et al., 2000) allows to preserve relatively large volume of diluted ejaculate or prepared spermatozoa from 0.25 to 1.0 mL with good rates of motility after thawing (Sawetawan et al., 1993; larson et al, 1997) and acceptable levels of integrity of acrosomal and cytoplasmic membranes, in other words with sufficiently high quality postthaw characteristics (Hammadeh et al., 1999; Duru et al., 2001; Meseguer et al., 2004; Isachenko et al., 2003, 2008). It does also provide acceptable protection against membrane changes and destabilization induced by cryopreservation (Glander and Schaller, 2000; Schuffner et al, 2001).

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 43

than conventional freezing. In spite of that this method has been investigated extensively and successfully applied to female gametes and embryos of different mammalian species including humans (Rall & Fahy, 1985; Chen et al., 2001; Reed et al., 2002; Cervera & Garcia-Ximénez, 2003; Isachenko et al., 2005b, 2007; Silva & Berland, 2004), however, it cannot be directly extrapolated to male gametes, due to deleterious osmotic effect of high

To date, publications dedicated to this topic are rare (Nawroth, et al., 2002; Koshimoto & Mazur, 2002; Isachenko et al., 2004a, b, 2008). Recent work has reversed this situation in that favorable results have been obtained in human spermatozoa after excluding permeable cryoprotectants from cryopreservation solutions, increasing the cooling rate and using carbohydrates, proteins and other extracellular agents, to increase the viscosity of the surrounding medium of cells and prevent the formation of any intra- and big extracellular crystals (Isachenko et al., 2004a, b). It was shown that permeable-cryoprotectants-free vitrification only with protein (Nawroth et al, 2002; Isachenko et al, 2003, 2004a,b, 2005a) or in combination with sucrose (Isachenko et al., 2008 2011a,b,c,d; Sanchez et al., 2011a, b) as a non-permeable cryoprotectant provides a high recovery rate of motile cells and effectively protects the mitochondrial membrane and the DNA integrity of spermatozoa after warming (Isachenko et al., 2004a, b; 2008). And it is not surprising. According to common point of view the non-permeable cryoprotectants plays the supporting role at permeable cryoprotectants. They binds of extracellular water and at the same time plays anti-toxic role (Kuleshova et al., 1999) decreasing of harmful properties of permeable cryoprotectants. In general, the inclusion of osmotically active, non-permeating compounds into the vitrification solution leads to additional rehydration of cells and, as a result, to decreasing toxic effects of the permeable cryoprotectants on intracellular structures. The non-permeable cryoprotectant sugars possess a unique property: stabilization of a cell membrane (Nakagata

& Takeshima, 1992, 1993; Koshimoto et al., 2000; Koshimoto & Mazur, 2002).

Also, the application of this modified cryopreservation technique to human spermatozoa allowed to avoid the toxic effect caused by adding and removing of permeable cryoprotectants including the negative effects on the cells' genetic material (Pérez-Sánchez et al., 1994). In our earlier works we have shown that cryopreserved without permeable cryoprotectants human spermatozoa preserved their relatively high motility rate with ability to fertilize oocytes in vitro (Nawroth et al., 2002; Isachenko *et al.,* 2003; 2004a, b). No statistical differences in parameters such as viability, recovery rate or percentage of morphologically normal spermatozoa with undamaged DNA were noted between vitrified and conventionally frozen cells (Nawroth et al., 2002). However, it was observed that the number of cryopreserved spermatozoa displaying features of acrosome reaction was statistically different from that in freshly prepared swim-up spermatozoa (Isachenko *et al.,*

In contrast to the programmable (slow) conventional freezing the vitrification renders redundant the need for special cooling programs addition of permeable cryoprotectants. It is much faster, simpler and more cost-efficient while still effectively protecting spermatozoa from cryo-injuries (Nawroth et al, 2002; Isachenko et al, 2003, 2004a, b, 2005, 2008) and does not require expensive equipment or special cooling procedures. Spermatozoa, vitrified by such technology, would be ready for further use without any additional treatment (centrifugation, separation in the gradient, removal of cryoprotectant and others)

concentrations of permeable cryoprotectants.

2004a,b, 2005a, 2008, 2011a,b).

immediately after thawing.

To avoid the lethal intracellular ice formation, the cryoprotective solution as ruler contains buffers, carbohydrates (glucose, lactose, raffinose, sucrose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics including permeable cryoprotectant glycerol or other cryoprotectives (Mazur, 1963; Barbas & Mascarenhas, 2009) in combination with comparably slow rates of freezing (Gao et al., 1997) are widely used for these purpose. The aim of slow cooling rates is to maintain a very delicate balance between ice crystal formation and growing concentration of dissolved substances. Conventional freezing procedure for mammalian spermatozoa traditionally includes the following stages of manipulations:


As a rule, a pre-requisite for that is a dilution of semen suspension with culture medium in order to reduce the toxicity of permeable cryoprotectant (according to manufacturer's instructions for cryoprotectant of choice). This is associated with additional costs and, not at least, with environmental and adaptation challenges for spermatozoa. In fact, sensitivity of spermatozoa to additional mechanical manipulation is increased after freezing-thawing, and the negative effects of cryopreservation on cell viability and functional competence can be aggravated by additional procedures. The problem is that the addition and, in particular, the removal of permeable osmotically-active cryoprotective agents (permeable cryoprotectants) before cooling and after warming can induce lethal stress due to intracellular ice formation, intracellular eutectic formation or so-called 'dilution (toxic) effects' (Fraga et al., 1991, Petrunkina, 2007) including chilling injury, cytoplasm fracture or even effects on the cytoskeleton (Critser et al., 1988; Fraga et al., 1991; Pérez-Sánchez et al., 1994). The further problems include the chemical toxicity of cryoprotectants and their possible repercussions on the genome or genome- related structures of mammalian spermatozoa (Hammadeh et al., 1999; Gilmore *et al.,* 1997). Moreover, spermatozoa which survive the cryopreservation stress are likely to have undergone subtle functional changes associated with biophysical and biochemical factors influenced by cryoprotectants, which will affect their fertilizing ability (Petrunkina, 2007).

Actually, the problem of the cooling and warming processes is the lethality which closely associated with the intermediate zone of temperature (-10 to -60 °C) that cells must traverse twice during cooling and once during warming (Mazur, 1963).

One of relatively recent and much discussed cryobiological emerged technologies within the field of the reproductive cryobiology is the spermatozoa vitrification (cryopreservation by direct plunging into liquid nitrogen). Vitrification is an alternative method that can also be applied to achieve the same purpose and does not use the special extenders. This method is based on the rapid cooling of the cells by immersion into liquid nitrogen, and, thereby, is the key factor reducing the chance of the formation of big ice crystals. In contrast to the programmable ("slow") conventional freezing, vitrification has series of technological advantages useful for the practice: it renders the use of permeable cryoprotectants superfluous and, in addition, is much faster, simpler in application and more cost-effective

To avoid the lethal intracellular ice formation, the cryoprotective solution as ruler contains buffers, carbohydrates (glucose, lactose, raffinose, sucrose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics including permeable cryoprotectant glycerol or other cryoprotectives (Mazur, 1963; Barbas & Mascarenhas, 2009) in combination with comparably slow rates of freezing (Gao et al., 1997) are widely used for these purpose. The aim of slow cooling rates is to maintain a very delicate balance between ice crystal formation and growing concentration of dissolved substances. Conventional freezing procedure for mammalian

spermatozoa traditionally includes the following stages of manipulations:

4. treatment of spermatozoa by the density gradient or the swim-up procedure (McLaughlin et al., 1990; Yin and Seibel, 1999; Stanic et al., 2000). 5. The ultimate target of the last manipulation is the removal of permeable

As a rule, a pre-requisite for that is a dilution of semen suspension with culture medium in order to reduce the toxicity of permeable cryoprotectant (according to manufacturer's instructions for cryoprotectant of choice). This is associated with additional costs and, not at least, with environmental and adaptation challenges for spermatozoa. In fact, sensitivity of spermatozoa to additional mechanical manipulation is increased after freezing-thawing, and the negative effects of cryopreservation on cell viability and functional competence can be aggravated by additional procedures. The problem is that the addition and, in particular, the removal of permeable osmotically-active cryoprotective agents (permeable cryoprotectants) before cooling and after warming can induce lethal stress due to intracellular ice formation, intracellular eutectic formation or so-called 'dilution (toxic) effects' (Fraga et al., 1991, Petrunkina, 2007) including chilling injury, cytoplasm fracture or even effects on the cytoskeleton (Critser et al., 1988; Fraga et al., 1991; Pérez-Sánchez et al., 1994). The further problems include the chemical toxicity of cryoprotectants and their possible repercussions on the genome or genome- related structures of mammalian spermatozoa (Hammadeh et al., 1999; Gilmore *et al.,* 1997). Moreover, spermatozoa which survive the cryopreservation stress are likely to have undergone subtle functional changes associated with biophysical and biochemical factors influenced by cryoprotectants, which will affect their fertilizing ability

Actually, the problem of the cooling and warming processes is the lethality which closely associated with the intermediate zone of temperature (-10 to -60 °C) that cells must traverse

One of relatively recent and much discussed cryobiological emerged technologies within the field of the reproductive cryobiology is the spermatozoa vitrification (cryopreservation by direct plunging into liquid nitrogen). Vitrification is an alternative method that can also be applied to achieve the same purpose and does not use the special extenders. This method is based on the rapid cooling of the cells by immersion into liquid nitrogen, and, thereby, is the key factor reducing the chance of the formation of big ice crystals. In contrast to the programmable ("slow") conventional freezing, vitrification has series of technological advantages useful for the practice: it renders the use of permeable cryoprotectants superfluous and, in addition, is much faster, simpler in application and more cost-effective

twice during cooling and once during warming (Mazur, 1963).

1. slow, step-wise adding of freezing solution to the ejaculate

2. cooling during 20 to 45 min 3. warming in water bath and

cryoprotectants.

(Petrunkina, 2007).

than conventional freezing. In spite of that this method has been investigated extensively and successfully applied to female gametes and embryos of different mammalian species including humans (Rall & Fahy, 1985; Chen et al., 2001; Reed et al., 2002; Cervera & Garcia-Ximénez, 2003; Isachenko et al., 2005b, 2007; Silva & Berland, 2004), however, it cannot be directly extrapolated to male gametes, due to deleterious osmotic effect of high concentrations of permeable cryoprotectants.

To date, publications dedicated to this topic are rare (Nawroth, et al., 2002; Koshimoto & Mazur, 2002; Isachenko et al., 2004a, b, 2008). Recent work has reversed this situation in that favorable results have been obtained in human spermatozoa after excluding permeable cryoprotectants from cryopreservation solutions, increasing the cooling rate and using carbohydrates, proteins and other extracellular agents, to increase the viscosity of the surrounding medium of cells and prevent the formation of any intra- and big extracellular crystals (Isachenko et al., 2004a, b). It was shown that permeable-cryoprotectants-free vitrification only with protein (Nawroth et al, 2002; Isachenko et al, 2003, 2004a,b, 2005a) or in combination with sucrose (Isachenko et al., 2008 2011a,b,c,d; Sanchez et al., 2011a, b) as a non-permeable cryoprotectant provides a high recovery rate of motile cells and effectively protects the mitochondrial membrane and the DNA integrity of spermatozoa after warming (Isachenko et al., 2004a, b; 2008). And it is not surprising. According to common point of view the non-permeable cryoprotectants plays the supporting role at permeable cryoprotectants. They binds of extracellular water and at the same time plays anti-toxic role (Kuleshova et al., 1999) decreasing of harmful properties of permeable cryoprotectants. In general, the inclusion of osmotically active, non-permeating compounds into the vitrification solution leads to additional rehydration of cells and, as a result, to decreasing toxic effects of the permeable cryoprotectants on intracellular structures. The non-permeable cryoprotectant sugars possess a unique property: stabilization of a cell membrane (Nakagata & Takeshima, 1992, 1993; Koshimoto et al., 2000; Koshimoto & Mazur, 2002).

Also, the application of this modified cryopreservation technique to human spermatozoa allowed to avoid the toxic effect caused by adding and removing of permeable cryoprotectants including the negative effects on the cells' genetic material (Pérez-Sánchez et al., 1994). In our earlier works we have shown that cryopreserved without permeable cryoprotectants human spermatozoa preserved their relatively high motility rate with ability to fertilize oocytes in vitro (Nawroth et al., 2002; Isachenko *et al.,* 2003; 2004a, b). No statistical differences in parameters such as viability, recovery rate or percentage of morphologically normal spermatozoa with undamaged DNA were noted between vitrified and conventionally frozen cells (Nawroth et al., 2002). However, it was observed that the number of cryopreserved spermatozoa displaying features of acrosome reaction was statistically different from that in freshly prepared swim-up spermatozoa (Isachenko *et al.,* 2004a,b, 2005a, 2008, 2011a,b).

In contrast to the programmable (slow) conventional freezing the vitrification renders redundant the need for special cooling programs addition of permeable cryoprotectants. It is much faster, simpler and more cost-efficient while still effectively protecting spermatozoa from cryo-injuries (Nawroth et al, 2002; Isachenko et al, 2003, 2004a, b, 2005, 2008) and does not require expensive equipment or special cooling procedures. Spermatozoa, vitrified by such technology, would be ready for further use without any additional treatment (centrifugation, separation in the gradient, removal of cryoprotectant and others) immediately after thawing.

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 45

binding intracellular water, the permeable and non-permeable cryoprotectants help to prevent ice formation, and thereby to reduce the cryo-damage (Andrews, 1976; Franks,

Several protocols of spermatozoa separation are available (e.g. swim-up from the ejaculate, single wash of ejaculate and swim-up from pellet, double wash of ejaculate and swim-up from pellet). However, any methodology needs the use of previous centrifugation. Most of the current technologies for sperm vitrification have an obvious shortcoming in terms of standardization of the portion volume. In particular, as the diameter of the pulled part of straw is not uniform, the volumes of the portions packaged in that way can not be standardized. Here we have reported the vitrification methodology using standard capillaries which can be supplied by industrial manufacturers. The technique was performed as follow (Isachenko V et al., 2011c). All specimens used for this study had fulfilled following quality criteria for spermatozoa concentration, motility and morphology: less than 20 millions spermatozoa/mL, 35 % progressive motile and minimum 3 % morphologically normal spermatozoa. Semen analysis was performed according to published guidelines of the World Health Organization (WHO, 1999). Prior to vitrification, the sedimented spermatozoa were diluted with 0.25 M sucrose (end concentration) in sperm preparation medium at room temperature (Isachenko et al., 2008). The final concentration of spermatozoa was approximately of 0.5 x 106 spermatozoa/mL. Diluted suspensions were maintained at room temperature for 5 min before the cooling procedure. Spermatozoa were prepared and portioned for aseptic vitrification in the following way. Specially for our purposes, 50 μL-plastic capillaries (Fig.1) were manufactured from hydrophobic material as vehicles for cooling sperm cell suspensions (Gynemed GmbH & Co. KG, Lensahn, Germany). The end of the straw was labeled on the top to mark the cutting-off position (Fig.1, arrows). The capillary was filled with 10μL of spermatozoa suspension by aspiration (Fig. 1a). It was absolutely crucial to avoid that the inner surface of the capillary become moist during packaging procedure. Aspirating the volume of sperm cell suspension above the mark and correcting it by lowering the fluid level inside the capillary after aspiration is technologically wrong and would result in excess of portion's volume after thawing. After the aspiration was completed, the capillary was inserted into 0.25 ml straw (Medical Technology GmbH, Bruckberg, Germany). One end of this straw was sealed in advance using heat-sealer (Cryo Bio System, Paris, France). After sealing the second end of the straw (Fig. 1b), the straw was plunged into liquid nitrogen and cooled at a cooling speed of 600°C/min. The speed of cooling was determined using a Testo 950 electrical thermometer (Testo AG, Lenzkirch, Germany) using 0.2 mm electrode located inside of the capillary. Hermetical heat-sealing of 0.25 mL straw can be achieved using flame of alcohol burner and forceps or any commercial equipment (including ultrasound equipment because of large distance between spermatozoa suspension and focus of sonographic appliance). Spermatozoa were stored in liquid nitrogen at least for 24 h before warming. For warming, capillary was removed from isolating 0.25 mL straw. The straw was disinfected with ethanol in the area where the marked end of capillary was (Fig. 1, arrows). The second end of capillary is fixed tightly on the inner surface of the straw, and the part of straw containing spermatozoa is still half submerged in liquid nitrogen (Figs. 1c,d). The upper part of the straw was cut off with sterile scissors as close as possible to the marked end of the capillary, just above the mark without touching the marked end of capillary. The capillary

1977).

Successful pregnancies and births have been reported when using vitrified oocytes and embryos, and vitrification protocols have started to form an important part as well of human as of animal reproductive medicine. Although sperm vitrification techniques have been studied in vitro, first successful pregnancies and live birth after fertilization with vitrified spermatozoa have been reported.

This chapter we would like to present contents the interesting results which we have had in the first time achieved using developed us vitrification technique based on using only of protein and carbohydrates as non-permeable cryoprotectants and applying to some mammalian and fish species.

In our presentation we will not touch the historicalquestions of vitrification of spermatozoa as well, but will concentrate us only on own experience according to vitrification of spermatozoa with using only non-permeable cryoprotectants. This theme was well covered in our previous publications (E. Isachenko et al., 2003, 2008, 2011a; Katkov et al., 2006).

In this chapter we will in detail discuss our new data which we have got in our investigation after vitrification of human, dog and fish spermatozoa.
