**2. New capillary technology (Isachenko et al., 2011c) for vitrification of small volume of human spermatozoa and practical application**

Varied methods to vitrify spermatozoa have been described previously: cryo-loops, droplets- and open pulled straw method (Nawroth et al, 2002; Isachenko et al., 2004a,b, 2005, 2008). According to these results it is possible to achieve up to 60%- and 20% -motility levels after thawing in normospermic and oligo-astheno-terato-zoospermic patients, respectively, depending on vitrification method selected and the quality of the original ejaculate. Independent from the vitrification technique the vitrified spermatozoa can be processed for further use immediately after warming without additional treatment such as centrifugation, gradient separation, removal of cryoprotectants etc is required. This simplicity for practical purposes represents one of the most attractive advantages of our technology. It is worth to mention that the protocol for vitrification does include swim up treatment, therefore, after swim up, vitrification and warming spermatozoa are also free from seminal plasma with potential pathogens. "Slow" freezing of human spermatozoa traditionally proposes the removing of permeable cryoprotectant after thawing.

This «*removal of permeable cryoprotectants*» is the ultimate target of the last manipulation. 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 (Petrunkina, 2007). Cryopreservation induces extensive damage to cells during both freezing and thawing. According to present knowledge, the effective induction of anabiosis in cells at very low temperatures (in liquid nitrogen at -196°C, for example) can be achieved by optimizing the multi-factorial freezing process (Lozina-Lozinski, 1982), commonly with the use of permeable cryoprotectants (Levin, 1982). Acting by depressing the freezing point and by

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

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

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

**2. New capillary technology (Isachenko et al., 2011c) for vitrification of small**

Varied methods to vitrify spermatozoa have been described previously: cryo-loops, droplets- and open pulled straw method (Nawroth et al, 2002; Isachenko et al., 2004a,b, 2005, 2008). According to these results it is possible to achieve up to 60%- and 20% -motility levels after thawing in normospermic and oligo-astheno-terato-zoospermic patients, respectively, depending on vitrification method selected and the quality of the original ejaculate. Independent from the vitrification technique the vitrified spermatozoa can be processed for further use immediately after warming without additional treatment such as centrifugation, gradient separation, removal of cryoprotectants etc is required. This simplicity for practical purposes represents one of the most attractive advantages of our technology. It is worth to mention that the protocol for vitrification does include swim up treatment, therefore, after swim up, vitrification and warming spermatozoa are also free from seminal plasma with potential pathogens. "Slow" freezing of human spermatozoa

traditionally proposes the removing of permeable cryoprotectant after thawing.

This «*removal of permeable cryoprotectants*» is the ultimate target of the last manipulation. 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 (Petrunkina, 2007). Cryopreservation induces extensive damage to cells during both freezing and thawing. According to present knowledge, the effective induction of anabiosis in cells at very low temperatures (in liquid nitrogen at -196°C, for example) can be achieved by optimizing the multi-factorial freezing process (Lozina-Lozinski, 1982), commonly with the use of permeable cryoprotectants (Levin, 1982). Acting by depressing the freezing point and by

vitrified spermatozoa have been reported.

after vitrification of human, dog and fish spermatozoa.

**volume of human spermatozoa and practical application**

mammalian and fish species.

binding intracellular water, the permeable and non-permeable cryoprotectants help to prevent ice formation, and thereby to reduce the cryo-damage (Andrews, 1976; Franks, 1977).

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

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 47

by CTC. Thus, further studies with additional, advanced techniques are needed to investigate the changes induced by vitrification in its complexity (e.g. targeting specific pathways and membrane processes such as changes in lipid architecture and/or protein kinases/phosphatases regulated pathways). Given the fact that the outcome of basic spermatozoa quality was comparable (or even better) that after conventional freezing, other advantages of the vitrification process must be taken into account. During conventional procedure, the success of applying permeable cryoprotectants for cryopreservation of varied cells and tissues is inseparably linked to such cryoprotectant properties as their ability to permeate rapidly through cellular membrane and their toxicity (Gilmore et al, 1997). These properties are directly connected to osmotic damages of cells during saturation with permeable cryoprotectants before freezing and then at time of cryoprotectants removing after thawing (Gao et al, 1995, Petrunkina, 2007). It is known that human spermatozoa contain large quantities of proteins, sugars, and other components that may act as natural cryoprotectants. Our technology does not presuppose the use of permeable cryoprotectant. In practical terms, permeable cryoprotectant-free vitrification technology for the cryopreservation of spermatozoa (in straws) instead traditional slow freezing with permeable cryoprotectants is already used in following centers: our university's maternity hospital (www.uniklinik-ulm.de): IVF Centers in Temuco, Chile (about 200 IUI cycles/year) and in Ulm, Germany (www.kinderwunschulm.de) (about 1,000 IVF cycles/year). First successful pregnancies and birth of healthy babies has been recently achieved with spermatozoa vitrified without permeable cryoprotectants (Isachenko et al., 2011b). In summary, the newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim up procedure preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in

As successful application of this vitrification technology for routine practice is born of two healthy babies (Isachenko et al., 2011b). We would like shortly present here the history of this case. A couple, both 39 years old, underwent assisted reproduction due to severe endometriosis and oligo-astheno-terato-zoospermia (13 x 106 motile spermatozoa/ml; with 42% of progressive motility and 8% morphologically normal spermatozoa [WHO, 1999]). Cryopreserved spermatozoa were used because the partner was absent during the oocyte retrieval procedure. The swim up-processed spermatozoa were diluted and proceeded with vitrification solution according our technique, described above, to achieve a final concentration of 2,5 x 106 spermatozoa/ml and 10µl aliquots were vitrified with using of Cut-Standard-Straws (CSS, Isachenko *et al.,* 2007) which was chosen as the prototype of our capillary technology. The spermatozoa were kept frozen in liquid nitrogen (at -196ºC) for 7 months. Only the warming technique was different from newly developed one and supposed the concentrations of spermatozoa by centrifugation after warming. The changes in following physiological and morphological parameters of thirty minutes after warming of vitrified spermatozoa and the freshly prepared swim-up were investigated for progressive motility, capacitation-like membrane changes due to determining of phosphatidylserine translocation (PST). The capacitation-like membrane changes was investigated due to determining of phosphatidylserine translocation (PST) in the sperm with appling the anexin

V-FITC staining technique (APOPTESTTM-FITC, Nexins Research, the Netherlands).

ICSI / IVF.

was expelled with a conical bolt (Fig. 1d). For this purpose, conical bolt (instead the conical bolt forceps can be used in place) is inserted into inner part of the capillary and pulled off the straw. The final warming up of spermatozoa is achieved by immersing of capillary without conical apex (capillary must be open from both sides) with vitrified spermatozoa into 1.8 mL centrifuged tube with 0.7 mL pre-warmed to 37°C vitrification medium for approximately 20 sec. (Fig. 1e). It is important to note that the volume of vitrified suspension after warming is not decreased (Fig 1e). Finally, the suspension of spermatozoa was expelled from the capillary for immediate evaluation of spermatozoa quality. Using this technique the exactly quantifiable volumes of spermatozoa samples were obtained: 10 μL suspension of spermatozoa were vitrified, 10 μL were thawed and the same 10 μL added to the respective volume of medium for ICSI or IVF. Thus, one of the most important features of this novel method of vitrification in capillaries is its potential for standardization which can be used for the routine clinical practice. The results of the present study let suggest that cryopreservation by vitrification helps to preserve essential determinants of spermatozoa function, such as motility and plasma membrane integrity. It is well known that spermatozoa cryopreservation is associated with a large decline in spermatozoa viability and other sperm functional parameters (Petrunkina, 2007). In the present study we have compared spermatozoa quality after vitrification by our method with spermatozoa quality after conventional freezing with addition of permeable cryoprotectant. The outcomes indicated that vitrification in capillaries compare to conventional freezing preserved better the motility of spermatozoa (after warming/thawing: 28.0 +6.0 % *vs* 18.0 + 9.2 %, respectively, P<0.05 and in fresh control 35.0 + 9.5%; after 24 h *in vitro* culture: 12.0 +2.8 % *vs* 5.0 + 3.1 %, respectively, P<0.05 and in fresh control 20.0 + 3.9%; after 48 h in vitro culture: 6.0 +1.0 % *vs* 0.5 + 0.02 %, respectively P>0.1 and in fresh control 10.0 + 1.9% [Fig. 2]) and their plasma membrane integrity (56.0 ± 5.1 % *vs* 22.0 ± 3.5 %, respectively, P<0.05 and in fresh control 96.0 ± 0.6 %, P<0.05 [Figure 3]) which was assessed with LIVE / DEAD sperm viability kit (LIVE/DEAD Sperm Viability Kit, Molecular Probes cat no. L-7011, Eugene, OR, USA). Pilot results have been obtained with respect to evaluating capacitation-like changes associated with cryopreservation, so called "cryo-capacitation" (Cormier and Bailey, 2003). A body of evidence suggests that some spermatozoa' intracellular signaling pathways can be affected during cryopreservation, and after warming spermatozoa display features commonly observed in capacitating or capacitated spermatozoa (Green and Watson, 2001; Petrunkina et al., 2005; Vadnais and Roberts, 2010). It is important, however, to emphasize that the changes induced by cryo-preservation are similar to those of capacitation only at the functional level, and they seem to differ at the molecular level, and with respect to pathways and signaling mechanisms involved (Cormier and Bailey, 2003). Our observations imply that permeable cryoprotectant-free aseptic vitrification is associated with lesser damage to acrosomes compare to conventional freezing (55.0 ± 5.8 % *vs* 21.0 ± 3.8 %, respectively, P<0.05 and in fresh control 84.0 ± 3.1%, P<0.05 [Fig. 4]). However, the levels of membrane changes related to "cryo-capacitation" assessed by CTC in vitrified spermatozoa were comparable with those after conventional freezing (8.0 ± 1.1% *vs* 9.0 ± 2.2%, respectively, P <0.01 and in fresh control 2.0 ± 0.3%, P<0.05, [Figure 5]). Changes in the acrosomal membrane status and permeability associated with the capacitation we have evaluated by using the double fluorescence chlortetracycline (CTC)- Hoechst 33258 staining technique (Kay et al, 1994). Nevertheless, the exposure to low temperatures can affect those crucial signaling mechanisms which can not be monitored

was expelled with a conical bolt (Fig. 1d). For this purpose, conical bolt (instead the conical bolt forceps can be used in place) is inserted into inner part of the capillary and pulled off the straw. The final warming up of spermatozoa is achieved by immersing of capillary without conical apex (capillary must be open from both sides) with vitrified spermatozoa into 1.8 mL centrifuged tube with 0.7 mL pre-warmed to 37°C vitrification medium for approximately 20 sec. (Fig. 1e). It is important to note that the volume of vitrified suspension after warming is not decreased (Fig 1e). Finally, the suspension of spermatozoa was expelled from the capillary for immediate evaluation of spermatozoa quality. Using this technique the exactly quantifiable volumes of spermatozoa samples were obtained: 10 μL suspension of spermatozoa were vitrified, 10 μL were thawed and the same 10 μL added to the respective volume of medium for ICSI or IVF. Thus, one of the most important features of this novel method of vitrification in capillaries is its potential for standardization which can be used for the routine clinical practice. The results of the present study let suggest that cryopreservation by vitrification helps to preserve essential determinants of spermatozoa function, such as motility and plasma membrane integrity. It is well known that spermatozoa cryopreservation is associated with a large decline in spermatozoa viability and other sperm functional parameters (Petrunkina, 2007). In the present study we have compared spermatozoa quality after vitrification by our method with spermatozoa quality after conventional freezing with addition of permeable cryoprotectant. The outcomes indicated that vitrification in capillaries compare to conventional freezing preserved better the motility of spermatozoa (after warming/thawing: 28.0 +6.0 % *vs* 18.0 + 9.2 %, respectively, P<0.05 and in fresh control 35.0 + 9.5%; after 24 h *in vitro* culture: 12.0 +2.8 % *vs* 5.0 + 3.1 %, respectively, P<0.05 and in fresh control 20.0 + 3.9%; after 48 h in vitro culture: 6.0 +1.0 % *vs* 0.5 + 0.02 %, respectively P>0.1 and in fresh control 10.0 + 1.9% [Fig. 2]) and their plasma membrane integrity (56.0 ± 5.1 % *vs* 22.0 ± 3.5 %, respectively, P<0.05 and in fresh control 96.0 ± 0.6 %, P<0.05 [Figure 3]) which was assessed with LIVE / DEAD sperm viability kit (LIVE/DEAD Sperm Viability Kit, Molecular Probes cat no. L-7011, Eugene, OR, USA). Pilot results have been obtained with respect to evaluating capacitation-like changes associated with cryopreservation, so called "cryo-capacitation" (Cormier and Bailey, 2003). A body of evidence suggests that some spermatozoa' intracellular signaling pathways can be affected during cryopreservation, and after warming spermatozoa display features commonly observed in capacitating or capacitated spermatozoa (Green and Watson, 2001; Petrunkina et al., 2005; Vadnais and Roberts, 2010). It is important, however, to emphasize that the changes induced by cryo-preservation are similar to those of capacitation only at the functional level, and they seem to differ at the molecular level, and with respect to pathways and signaling mechanisms involved (Cormier and Bailey, 2003). Our observations imply that permeable cryoprotectant-free aseptic vitrification is associated with lesser damage to acrosomes compare to conventional freezing (55.0 ± 5.8 % *vs* 21.0 ± 3.8 %, respectively, P<0.05 and in fresh control 84.0 ± 3.1%, P<0.05 [Fig. 4]). However, the levels of membrane changes related to "cryo-capacitation" assessed by CTC in vitrified spermatozoa were comparable with those after conventional freezing (8.0 ± 1.1% *vs* 9.0 ± 2.2%, respectively, P <0.01 and in fresh control 2.0 ± 0.3%, P<0.05, [Figure 5]). Changes in the acrosomal membrane status and permeability associated with the capacitation we have evaluated by using the double fluorescence chlortetracycline (CTC)- Hoechst 33258 staining technique (Kay et al, 1994). Nevertheless, the exposure to low temperatures can affect those crucial signaling mechanisms which can not be monitored by CTC. Thus, further studies with additional, advanced techniques are needed to investigate the changes induced by vitrification in its complexity (e.g. targeting specific pathways and membrane processes such as changes in lipid architecture and/or protein kinases/phosphatases regulated pathways). Given the fact that the outcome of basic spermatozoa quality was comparable (or even better) that after conventional freezing, other advantages of the vitrification process must be taken into account. During conventional procedure, the success of applying permeable cryoprotectants for cryopreservation of varied cells and tissues is inseparably linked to such cryoprotectant properties as their ability to permeate rapidly through cellular membrane and their toxicity (Gilmore et al, 1997). These properties are directly connected to osmotic damages of cells during saturation with permeable cryoprotectants before freezing and then at time of cryoprotectants removing after thawing (Gao et al, 1995, Petrunkina, 2007). It is known that human spermatozoa contain large quantities of proteins, sugars, and other components that may act as natural cryoprotectants. Our technology does not presuppose the use of permeable cryoprotectant. In practical terms, permeable cryoprotectant-free vitrification technology for the cryopreservation of spermatozoa (in straws) instead traditional slow freezing with permeable cryoprotectants is already used in following centers: our university's maternity hospital (www.uniklinik-ulm.de): IVF Centers in Temuco, Chile (about 200 IUI cycles/year) and in Ulm, Germany (www.kinderwunschulm.de) (about 1,000 IVF cycles/year). First successful pregnancies and birth of healthy babies has been recently achieved with spermatozoa vitrified without permeable cryoprotectants (Isachenko et al., 2011b). In summary, the newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim up procedure preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI / IVF.

As successful application of this vitrification technology for routine practice is born of two healthy babies (Isachenko et al., 2011b). We would like shortly present here the history of this case. A couple, both 39 years old, underwent assisted reproduction due to severe endometriosis and oligo-astheno-terato-zoospermia (13 x 106 motile spermatozoa/ml; with 42% of progressive motility and 8% morphologically normal spermatozoa [WHO, 1999]). Cryopreserved spermatozoa were used because the partner was absent during the oocyte retrieval procedure. The swim up-processed spermatozoa were diluted and proceeded with vitrification solution according our technique, described above, to achieve a final concentration of 2,5 x 106 spermatozoa/ml and 10µl aliquots were vitrified with using of Cut-Standard-Straws (CSS, Isachenko *et al.,* 2007) which was chosen as the prototype of our capillary technology. The spermatozoa were kept frozen in liquid nitrogen (at -196ºC) for 7 months. Only the warming technique was different from newly developed one and supposed the concentrations of spermatozoa by centrifugation after warming. The changes in following physiological and morphological parameters of thirty minutes after warming of vitrified spermatozoa and the freshly prepared swim-up were investigated for progressive motility, capacitation-like membrane changes due to determining of phosphatidylserine translocation (PST). The capacitation-like membrane changes was investigated due to determining of phosphatidylserine translocation (PST) in the sperm with appling the anexin V-FITC staining technique (APOPTESTTM-FITC, Nexins Research, the Netherlands).

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 49

All rates in respective groups are significantly different (P<0.05).

All rates in respective groups are significantly different (P<0.05).

vitrification.

Fig. 2. Motility of human spermatozoa after conventional freezing and vitrification.

Fig. 3. Plasma membrane integrity of human spermatozoa after conventional freezing and

(Arrows) marked end of 50 μL capillary, (a) aspiration of spermatozoa suspension in straw, (b) 50 μL capillary sealed in 0.25 mL straw, (c) cutting of 0.25 mL straw, (d) expelling of 50 μL capillary from 0.25 mL straw, (e) warming of spermatozoa.

Fig. 1. Schematic illustration of human spermatozoa vitrification with 50 μL capillary.

(Arrows) marked end of 50 μL capillary, (a) aspiration of spermatozoa suspension in straw, (b) 50 μL capillary sealed in 0.25 mL straw, (c) cutting of 0.25 mL straw, (d) expelling of 50 μL capillary from

Fig. 1. Schematic illustration of human spermatozoa vitrification with 50 μL capillary.

0.25 mL straw, (e) warming of spermatozoa.

All rates in respective groups are significantly different (P<0.05).

Fig. 2. Motility of human spermatozoa after conventional freezing and vitrification.

All rates in respective groups are significantly different (P<0.05).

Fig. 3. Plasma membrane integrity of human spermatozoa after conventional freezing and vitrification.

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 51

transferred to the uterus cavity under ultrasonographic guidance. Fifteen days after embryo transfer, the maternal ß-hCG level was 360 IU/L and two healthy boys were born at term. These data supports the notion that: i) cells can be frozen effectively without toxic permeable cryoprotectants, and ii) such frozen material could in principle be lyophilized. It is, however, critical to ensure that freeze-drying is not associated with the genetic and developmental abnormalities that have been observed after fertilization with mouse freeze-

Cryopreservation is normally achieved through a tertiary combination of cells, permeable cryoprotectants and low temperature environment. In contrast, our cryopreservation protocol can be considered as a simplified binary combination of cells (in a simplified medium containing sucrose as a natural cryoprotectant) and a cold environment. The birth of two healthy babies using this *in vitro* fertilization technique is not only the first report on successful fertilization using vitrified spermatozoa (which has obvious practical advantages for assisted reproduction techniques). The above protocol also demonstrates that highly organized cells (human spermatozoa) may be effectively frozen-dried (lyophilized) with the recovery of their most important physiological function after thawing – propagation of genetic hereditary information and subsequent birth of new individuals. Of course, it would need to be proved on a large number of ejaculates that the damage produced by vitrification does not exceed the damage produced by conventional freezing and that there are no deleterious effects on the genetic integrity of sperm after vitrification (Ward *et al.,* 2003).

These aspects, however important, are outside the scope of this case report.

**3. New technology for vitrification of spermatozoa in big volume (Isachenko**

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

dried sperm (Ward *et al.,* 2003).

**et al., 2011d)**

All rates in respective groups are significantly different (P<0.05).

Fig. 4. Acrosomal integrity of human spermatozoa after conventional freezing and vitrification.

Rates in groups after freezing and vitrification are similar (P>0.5).

Fig. 5. Capacitation-like changes of human spermatozoa after conventional freezing and vitrification.

The mitochondrial membrane potential integrity was evaluates due to measurement of the changes in the (M ) using a unique fluorescent cationic dye, 5,5', 6,6'-tetachloro-1–1', 3,3' tetraethyl-benzamidazolocarbocyanin iodide. The results were as following: progressive motility 60% *vs* 90%, correspondingly, 10% were identified as displaying a 'capacitation' CTC pattern and 5% as displaying an 'acrosome reaction' pattern, as compared to 8% and 5% in freshly prepared swim-up sperm respectively; 63% of spermatozoa were classified as having high mitochondrial membrane potential (*vs* 96% in freshly prepared spermatozoa).

From ten ICSI-ed with vitrified spermatozoa oocytes 6 oocytes showed signs of normal fertilization and two PN-oocytes were culture subsequent 24 hours. At day of embryo transfer two 4-blastomere embryos of Grades "a" (4a) and "b" (4b) (Steer et al., 1992) were

All rates in respective groups are significantly different (P<0.05).

Rates in groups after freezing and vitrification are similar (P>0.5).

vitrification.

vitrification.

Fig. 4. Acrosomal integrity of human spermatozoa after conventional freezing and

Fig. 5. Capacitation-like changes of human spermatozoa after conventional freezing and

The mitochondrial membrane potential integrity was evaluates due to measurement of the changes in the (M ) using a unique fluorescent cationic dye, 5,5', 6,6'-tetachloro-1–1', 3,3' tetraethyl-benzamidazolocarbocyanin iodide. The results were as following: progressive motility 60% *vs* 90%, correspondingly, 10% were identified as displaying a 'capacitation' CTC pattern and 5% as displaying an 'acrosome reaction' pattern, as compared to 8% and 5% in freshly prepared swim-up sperm respectively; 63% of spermatozoa were classified as having high mitochondrial membrane potential (*vs* 96% in freshly prepared spermatozoa). From ten ICSI-ed with vitrified spermatozoa oocytes 6 oocytes showed signs of normal fertilization and two PN-oocytes were culture subsequent 24 hours. At day of embryo transfer two 4-blastomere embryos of Grades "a" (4a) and "b" (4b) (Steer et al., 1992) were transferred to the uterus cavity under ultrasonographic guidance. Fifteen days after embryo transfer, the maternal ß-hCG level was 360 IU/L and two healthy boys were born at term.

These data supports the notion that: i) cells can be frozen effectively without toxic permeable cryoprotectants, and ii) such frozen material could in principle be lyophilized. It is, however, critical to ensure that freeze-drying is not associated with the genetic and developmental abnormalities that have been observed after fertilization with mouse freezedried sperm (Ward *et al.,* 2003).

Cryopreservation is normally achieved through a tertiary combination of cells, permeable cryoprotectants and low temperature environment. In contrast, our cryopreservation protocol can be considered as a simplified binary combination of cells (in a simplified medium containing sucrose as a natural cryoprotectant) and a cold environment. The birth of two healthy babies using this *in vitro* fertilization technique is not only the first report on successful fertilization using vitrified spermatozoa (which has obvious practical advantages for assisted reproduction techniques). The above protocol also demonstrates that highly organized cells (human spermatozoa) may be effectively frozen-dried (lyophilized) with the recovery of their most important physiological function after thawing – propagation of genetic hereditary information and subsequent birth of new individuals. Of course, it would need to be proved on a large number of ejaculates that the damage produced by vitrification does not exceed the damage produced by conventional freezing and that there are no deleterious effects on the genetic integrity of sperm after vitrification (Ward *et al.,* 2003). These aspects, however important, are outside the scope of this case report.