**3. New technology for vitrification of spermatozoa in big volume (Isachenko et al., 2011d)**

Actually, the technique which is not acceptable for different volumes of the same object is incomplete and needs subsequent investigations and development. In this case the next aim of our research was development the acceptable vitrification methodology for big volume of spermatozoa with possibility to use cryopreserved ejaculate for intrauterine insemination. At the beginning of 2011 we have published (E. Isachenko et al., 2011a) the prototype of our big-volume vitrification technology the success of which a healthy baby was born after intrauterine insemination with vitrified spermatozoa (Sánchez et al., 2011a). We would like shortly present the history of this case. A 39-year-old patient and her 35-year-old husband, with a 3-year history of primary infertility, were referred to our center for infertility treatment. Laparoscopy revealed patency of the Fallopian tubes and no evidence of endometriosis or pelvic adhesions. Semen analysis of the husband showed oligo-asthenoterato-zoospermia (WHO, 1999). Despite the poor quality of ejaculate parameters, for financial reasons the patients decided to try intra-uterine insemination (IUI). For IUI the spermatozoa from two ejaculates obtained 3 days apart were vitrified. The volume of the first ejaculate was 1.9 ml, concentration 37.8 x 106 spermatozoa/ml, 8% of progressive "a" and "b" motility, 10% of morphologically normal spermatozoa, and 0.2x106 round cells/ml. The volume of the second ejaculate was 3.9 ml, concentration 11.2 x 106 spermatozoa/ml, 27% progressive motility, 10% of morphologically normal spermatozoa and 1.2x106 round cells/ml. The swim up-processed spermatozoa were diluted and proceeded with vitrification solution according our technique, described above, to achieve a final

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 53

In our lectures we have often mentioned that there is a simplified point of view that vitrification is the solidification without formation of crystals. Extending this description, one could say that vitrification is solidification of vitrifying solution without formation of hexagonal (big, lethal) intracellular structures by extreme elevation in viscosity during cooling. Obviously, thereby vitrification appears beneficial in terms of avoiding cryo-injuries traditionally associated with the formation of intracellular ice. Therefore we developed and for the first time reported (V. Isachenko et al., 2011d) the vitrification methodology where a relatively large volume of spermatozoa suspension can be frozen in one cooling pocket (straw). Vitrification medium described here does include sucrose (Isachenko et al., 2008). As a rule, in routine practice the carbohydrates are the standard part of any cryoprotective solution. They are used for spermatozoa cryopreservation to compensate osmotic effects caused by the permeable cryoprotectants and do play an important role as an additional dissolving, membrane stabilizing and dehydrating agents (Wakayama et al., 1998). Therefore, sucrose can be considered as a natural cryoprotectant, lacking most of toxic properties of permeable cryoprotectants. Human spermatozoa can be successfully frozen in the absence of permeable cryoprotectants, using protein- and sugar-rich extracellular nonpermeable cryoprotectants (Koshimoto et al., 2000; Karlsson and Cravalho, 1994). The ability of sucrose to prevent the artificial induction of membrane damages and acrosome reaction during vitrification/warming (Isachenko et al., 2008) corroborated our previous conclusions that the inclusion of sucrose in combination with human serum albumin in the vitrification

In our study (Isachenko et al., 2011d) we reported for the first time a novel technology of aseptic 'cryoprotectant-free' vitrification of human spermatozoa in large volumes. It

1. to obtain 0.5 mL of spermatozoa suspension, free both from seminal plasma (because of swim up procedure preceding vitrification) and free from additives which are part of

2. to cryopreserve spermatozoa, which are ready for further use immediately after thawing without any additional treatment (centrifugation, separation in the gradient,





*Vitrification.* Prior to vitrification, spermatozoa were processed by swim-up technique with subsequent dilution with cryoprotectant medium according to Isachenko (Isachenko et al.,

without influence on warming resalts (non-published data).

spermatozoa into warmed water bath at 42°C.

medium has a visible cryoprotective effect.

conventional freezing procedures;

Tubal Fluid (Quinn et al., 1985).

The technology includes:

liquid nitrogen.

**Technological procedure, shortly** 

removal of cryoprotectant and others).

allows:

concentration of 1 x 106 spermatozoa/ml. All subsequent manipulations were performed at room temperature strictly in a horizontal position to prevent a loss of suspension (E. Isachenko et al., 2011a). Aliquots (100 μl) of the diluted sperm suspension were aspirated into one half of 0.25 ml plastic straws (MTG, Bruckberg, Germany); these were then placed in 0.5 ml plastic straws (MTG) and hermetically sealed from both sides to protect the suspension from direct contact with liquid nitrogen. The closed straw-systems, strictly maintained horizontal, were then immersed into liquid nitrogen and stored until use. From two ejaculates three straws were cryopreserved, each with 100 µl of spermatozoa suspension in concentration of 1x106 spermatozoa/ml. Special for this case we have decided to investigate the presence of reactive oxygen species (ROS) in ejaculated and prepared spermatozoa before and after vitrification. The reason was the following. It is known that poor ejaculate quality is closely associated with elevated concentrations of leucocytes (normal values <1 million/ml). The presence of leucocytes can lead to oxidative stress (Henkel and Schill, 2003; Henkel et al., 2005, 2010). Therefore, we determined the concentration of leucocytes in ejaculates due to leucocytes quantifying by an indirect immunofluorescence (IIF) method (Villlegas et al., 2002) and presence of the following antibodies were checked: anti CD45 for all leukocytes (M 855-DAKO, Hamburg, Germany, in concentration of 1/50 in PBS with 5% BSA), anti CD15 for granulocytes (M 733-DAKO, Hamburg, Germany, in concentration of 1/100 in PBS with 5% BSA) or anti CD68 for macrophages (M 718-DAKO, Hamburg, Germany , in concentration of 1/600 in PBS with 5% BSA). However, in spite of the presence of a large numbers of round cells, the IIF was negative for all tested monoclonal antibodies, indicating high levels of spermatogenic cells. The presence of ROS in ejaculates was tested using a chemiluminescence assay (Aitken and Clarkson, 1987). Only the mild increasing of ROS to 76.960 RLU x 107/live sperm was noted (normal value: 35.000 RLU x 107/live sperm (Henkel et al, 1997). However, it is known that ROS in semen samples of oligozoospermic patients usually is slightly increased (Kumar et al., 2009). On the day of ovulation all three cryopreserved samples of spermatozoa suspension were thawed as described in E. Isachenko et al. (E. Isachenko et al., 2011a), the sperm pellet was resuspended in 500 µl of sperm preparation medium pre-warmed to 37°C and used immediately for intrauterine insemination. The suspension of spermatozoa before insemination (30 min post-warming) had a concentration of 2.7x106 spermatozoa/ml with 60% of progressive motility. Fifteen days after IUI, biochemical pregnancy was confirmed by ß-hCG level of 125 IU/L and on 29 December 2010 a healthy male baby was born.

Our finding has confirmed that the aseptic vitrification technique (without use of permeable cryoprotectants) is not only instrumental in effectively preserving spermatozoal function (Isachenko et al., 2011a, b, c, d, Sánchez et al., 2011), but could also have a massive potential for storage of motile spermatozoa for intrauterine insemination, for example, in cases of oligo-astheno-zoospemic patients.

However, the described methodology for vitrification of big-volume spermatozoa suspension is complicated, because exist often dangerous that sperm suspension will flows out the specimen straw and stick together to the inner wall of packaging straw during vitrification procedure. In this case it will be difficult to remove the specimen straw from the packaging one before warming. According to our opinion the technique must be as simple as possible and at the same time with absolute repeatability and the results have to be compatible with slow conventional freezing.

In our lectures we have often mentioned that there is a simplified point of view that vitrification is the solidification without formation of crystals. Extending this description, one could say that vitrification is solidification of vitrifying solution without formation of hexagonal (big, lethal) intracellular structures by extreme elevation in viscosity during cooling. Obviously, thereby vitrification appears beneficial in terms of avoiding cryo-injuries traditionally associated with the formation of intracellular ice. Therefore we developed and for the first time reported (V. Isachenko et al., 2011d) the vitrification methodology where a relatively large volume of spermatozoa suspension can be frozen in one cooling pocket (straw). Vitrification medium described here does include sucrose (Isachenko et al., 2008). As a rule, in routine practice the carbohydrates are the standard part of any cryoprotective solution. They are used for spermatozoa cryopreservation to compensate osmotic effects caused by the permeable cryoprotectants and do play an important role as an additional dissolving, membrane stabilizing and dehydrating agents (Wakayama et al., 1998). Therefore, sucrose can be considered as a natural cryoprotectant, lacking most of toxic properties of permeable cryoprotectants. Human spermatozoa can be successfully frozen in the absence of permeable cryoprotectants, using protein- and sugar-rich extracellular nonpermeable cryoprotectants (Koshimoto et al., 2000; Karlsson and Cravalho, 1994). The ability of sucrose to prevent the artificial induction of membrane damages and acrosome reaction during vitrification/warming (Isachenko et al., 2008) corroborated our previous conclusions that the inclusion of sucrose in combination with human serum albumin in the vitrification medium has a visible cryoprotective effect.

In our study (Isachenko et al., 2011d) we reported for the first time a novel technology of aseptic 'cryoprotectant-free' vitrification of human spermatozoa in large volumes. It allows:


The technology includes:

52 Current Frontiers in Cryobiology

concentration of 1 x 106 spermatozoa/ml. All subsequent manipulations were performed at room temperature strictly in a horizontal position to prevent a loss of suspension (E. Isachenko et al., 2011a). Aliquots (100 μl) of the diluted sperm suspension were aspirated into one half of 0.25 ml plastic straws (MTG, Bruckberg, Germany); these were then placed in 0.5 ml plastic straws (MTG) and hermetically sealed from both sides to protect the suspension from direct contact with liquid nitrogen. The closed straw-systems, strictly maintained horizontal, were then immersed into liquid nitrogen and stored until use. From two ejaculates three straws were cryopreserved, each with 100 µl of spermatozoa suspension in concentration of 1x106 spermatozoa/ml. Special for this case we have decided to investigate the presence of reactive oxygen species (ROS) in ejaculated and prepared spermatozoa before and after vitrification. The reason was the following. It is known that poor ejaculate quality is closely associated with elevated concentrations of leucocytes (normal values <1 million/ml). The presence of leucocytes can lead to oxidative stress (Henkel and Schill, 2003; Henkel et al., 2005, 2010). Therefore, we determined the concentration of leucocytes in ejaculates due to leucocytes quantifying by an indirect immunofluorescence (IIF) method (Villlegas et al., 2002) and presence of the following antibodies were checked: anti CD45 for all leukocytes (M 855-DAKO, Hamburg, Germany, in concentration of 1/50 in PBS with 5% BSA), anti CD15 for granulocytes (M 733-DAKO, Hamburg, Germany, in concentration of 1/100 in PBS with 5% BSA) or anti CD68 for macrophages (M 718-DAKO, Hamburg, Germany , in concentration of 1/600 in PBS with 5% BSA). However, in spite of the presence of a large numbers of round cells, the IIF was negative for all tested monoclonal antibodies, indicating high levels of spermatogenic cells. The presence of ROS in ejaculates was tested using a chemiluminescence assay (Aitken and Clarkson, 1987). Only the mild increasing of ROS to 76.960 RLU x 107/live sperm was noted (normal value: 35.000 RLU x 107/live sperm (Henkel et al, 1997). However, it is known that ROS in semen samples of oligozoospermic patients usually is slightly increased (Kumar et al., 2009). On the day of ovulation all three cryopreserved samples of spermatozoa suspension were thawed as described in E. Isachenko et al. (E. Isachenko et al., 2011a), the sperm pellet was resuspended in 500 µl of sperm preparation medium pre-warmed to 37°C and used immediately for intrauterine insemination. The suspension of spermatozoa before insemination (30 min post-warming) had a concentration of 2.7x106 spermatozoa/ml with 60% of progressive motility. Fifteen days after IUI, biochemical pregnancy was confirmed by

ß-hCG level of 125 IU/L and on 29 December 2010 a healthy male baby was born.

oligo-astheno-zoospemic patients.

compatible with slow conventional freezing.

Our finding has confirmed that the aseptic vitrification technique (without use of permeable cryoprotectants) is not only instrumental in effectively preserving spermatozoal function (Isachenko et al., 2011a, b, c, d, Sánchez et al., 2011), but could also have a massive potential for storage of motile spermatozoa for intrauterine insemination, for example, in cases of

However, the described methodology for vitrification of big-volume spermatozoa suspension is complicated, because exist often dangerous that sperm suspension will flows out the specimen straw and stick together to the inner wall of packaging straw during vitrification procedure. In this case it will be difficult to remove the specimen straw from the packaging one before warming. According to our opinion the technique must be as simple as possible and at the same time with absolute repeatability and the results have to be

