Cryobiology is a rapidly evolving field that only. Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants: New Capillary Technology

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1 Journal of Andrology, Vol. 33, No. 3, May/June 2012 Copyright E American Society of Andrology Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants: New Capillary Technology V. ISACHENKO,*{ R. MAETTNER,{ A. M. PETRUNKINA, 5 K. STERZIK,{ P. MALLMANN,* G. RAHIMI,* R. SANCHEZ," J. RISOPATRON," I. DAMJANOSKI,# AND E. ISACHENKO*{ From the *Department of Obstetrics and Gynecology, Cologne University, Cologne, Germany; the ÀSection of Gynecological Endocrinology and Reproductive Medicine, University of Ulm, Ulm, Germany; the `Private Maternal Hospital, Endokrinologikum Ulm, Ulm, Germany; the Unit of Reproductive Medicine of Clinics, University of Veterinary Medicine of Hannover, Foundation, Clinic for Horses, Hannover, Germany; the 5Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; the "BIOREN-CEBIOR, Department of Preclinical Sciences, Department of Basic Sciences, La Frontera University, Temuco, Chile; and the #Clinic for Urology, University of Ulm, Ulm, Germany. ABSTRACT: The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 ml of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 ml of spermatozoa vitrified in 50-mL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P,.05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P,.05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P..1) (2% in fresh spermatozoa). 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 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. Key words: Cryoprotectant-free, motility, membrane, acrosome. J Androl 2012;33: Cryobiology is a rapidly evolving field that 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). The use of programmable or nonprogrammable slow (conventional) freezing (McLaughlin et al, 1990; Yin and Seibel, 1999; Stanic et al, 2000) allows preservation of relatively large volumes of diluted ejaculate or sperm preparations, from 0.25 to 1.0 ml, with acceptable rates of motility of spermatozoa after thawing and adequate integrity of acrosomal and cytoplasmic membranes Correspondence to: V. Isachenko, Department of Obstetrics and Gynecology, Cologne University, Kerpener Str. 34, Cologne, Germany ( v.isachenko@yahoo.com). Received for publication April 1, 2011; accepted for publication June 15, Cambridge Institute for Medical Research is in receipt of Wellcome Trust Strategic Award. DOI: /jandrol (Sawetawan et al, 1993; Larson et al, 1997; Hammadeh et al, 1999). It is also instrumental in minimizing cryopreservation-induced cell deterioration, resulting in membrane destabilization processes, such as membrane phosphatidylserine translocation (Glander and Schaller, 2000; Duru et al, 2001; Schuffner et al, 2001). One of relatively recently emerged technologies within the field of reproductive cryobiology is spermatozoa vitrification (cryopreservation by directly plunging into liquid nitrogen). This method is based on rapid cooling of cells by immersion into liquid nitrogen and, thereby, is the key to reducing the chance of forming big ice crystals. In contrast to programmable ( slow ) conventional freezing, vitrification has a series of useful technological advantages: it renders the use of permeable cryoprotectants superfluous and, additionally, is much faster, simpler in application, and more cost effective than conventional freezing. At the same time, vitrification can effectively protect spermatozoa from cryo-injuries associated with cooling and warming (Nawroth et al, 2002; Isachenko 462

2 Isachenko et al N Vitrification of Human Spermatozoa 463 et al, 2003, 2004a,b, 2005, 2008). In particular, the mutagenic effects of permeable cryoprotectants (Fraga et al, 1991) can be avoided. At present, most reported applications of this technology describe vitrification of only very small volumes of spermatozoa suspensions: from 1 to 10 ml with isolation from liquid nitrogen (Isachenko et al, 2005) and from 20 to 30 ml with direct plunging or dropping of the suspension with spermatozoa into liquid nitrogen (Nawroth et al, 2002; Isachenko et al, 2005, 2008). Vitrification of up to 10 ml of a spermatozoon suspension, as it is being used in daily practice (Isachenko et al, 2005), presumes that open pulled straws (Vajta et al, 1998) are used. Current industrial technology does not yet enable the manufacturing of such open pulled straws with a uniform (standard) diameter of the pulled part of the straw. This shortcoming is reflected in nonuniformity of the vitrification regime. On the other hand, industrial suppliers offer plastic capillaries of regular cylinder shape with stable (fixed) diameter for medical applications. The vitrification process can be standardized using these standard capillaries, which would unfold a great potential for assisted reproduction using vitrified spermatozoa. The aim of this study was to develop and test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries for intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Materials and Methods This study was approved by the Review Boards of Cologne and Ulm universities. Except where otherwise stated, all chemicals were obtained from Sigma (Sigma Chemical Co, St Louis, Missouri). Spermatozoa Preparation After informed consent, 68 ejaculates were obtained from 68 patients with oligo-astheno-terato-zoospermia in anamneses who were either patients of an infertility clinic or had donated sperm for research. All specimens used for this study had fulfilled the following quality criteria for spermatozoa concentration, motility, and morphology: less than 20 million spermatozoa/ ml, 35% progressive motile spermatozoa, and a minimum of 3% morphologically normal spermatozoa. Semen analysis was performed according to published guidelines of the World Health Organization (WHO, 1999). All investigations were carried out on swim-up prepared spermatozoa. Human tubal fluid (HTF; Quinn et al, 1985) with 1% human serum albumin (HSA) was the basic medium for spermatozoa preparation. After swim-up, each sample was centrifuged, resuspended with the same medium to achieve a concentration of spermatozoa/ml, and finally aliquoted into 3 equal subsamples. Each of these aliquots was assigned to 1 of 3 groups: Group 1 included fresh, non-cryopreserved swim-up spermatozoa preparations and served as control. Spermatozoa of Group 2 were conventionally frozen, and spermatozoa of Group 3 were vitrified. Vitrification and Warming For preparation of vitrification solution, the basic medium (HTF + 1% HSA) was diluted 1:1 with 0.5 M sucrose. Spermatozoa were centrifuged and resuspended with this vitrification solution (Isachenko et al, 2008). The sucrose stock solution was prepared with bi-distillate water, passed through a 0.22-mm filter, and then stored frozen until use. Diluted suspensions were maintained at room temperature for 5 minutes before the cooling procedure. Spermatozoa were prepared and portioned for aseptic vitrification in the following way. Specifically, for our purposes, 50-mL plastic capillaries were manufactured from hydrophobic material as vehicles for cooling sperm cell suspensions (Gynemed GmbH & Co KG, Lensahn, Germany; Figure 1). The end of the straw was labeled on the top to mark the cut-off position (Figure 1, arrows). The capillary was filled with 10 ml of spermatozoa suspension by aspiration (Figure 1a). It was absolutely crucial that the inner surface of the capillary not become moist during the 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 an excess volume after thawing. After aspiration was completed, the capillary was inserted into a 0.25-mL straw (Medical Technology GmbH, Bruckberg, Germany). One end of this straw was sealed in advance using a heat-sealer (Cryo Bio System, Paris, France). After sealing the second end of the straw (Figure 1b), the straw was plunged into liquid nitrogen and cooled at 600uC/min. The speed of cooling was determined using a Testo 950 electrical thermometer (Testo AG, Lenzkirch, Germany) using a 0.2-mm electrode located inside the capillary. Hermetical heat sealing of the 0.25-mL straw can be achieved with the flame of an alcohol burner and forceps or any commercial equipment (including ultrasound equipment because of the large distance between the spermatozoa suspension and the focus of the sonographic appliance). Spermatozoa were stored in liquid nitrogen for at least 24 hours before warming. For warming, the capillary was removed from the isolating 0.25-mL straw. The straw was disinfected with ethanol at the marked end of the capillary (Figure 1, arrows). The second end of the capillary was fixed tightly on the inner surface of the straw, and the part of the straw containing spermatozoa was still half submerged in liquid nitrogen (Figure 1c and 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 the capillary. The capillary was expelled with a conical bolt (Figure 1d). For this purpose, the conical bolt (or, instead, the conical bolt forceps can be used) is inserted into the inner part of the capillary and pulled off the straw. The final warming of spermatozoa is achieved by immersing the capillary without the conical apex (the capillary must be

3 464 Journal of Andrology N May ÙJune 2012 using an aspirator (Medical Technology GmbH). These capillaries were placed into 0.25-mL standard insemination straws (Medical Technology GmbH). Then, straws were put horizontally into liquid nitrogen vapor (280uC, 10 cm over the liquid nitrogen surface), kept for 30 minutes, and finally placed into liquid nitrogen, where they were stored a minimum of 24 hours until evaluation. Thawing of spermatozoa was performed in accordance with the technique described above for vitrified spermatozoa. After thawing and expelling 10 ml of spermatozoa from capillaries, 10 ml of basic (HTF-HSA) medium was added to the thawed sample and centrifuged for 5 minutes at g. The supernatant was removed and the pellet resuspended with the same basic medium to obtain a final concentration of spermatozoa/ml. Then, the evaluation of spermatozoa quality was performed. Motility Motility of spermatozoa was assessed 1 hour after liquefaction, swim-up procedure, cryopreservation, and warming of samples and after 24 and 48 hours in vitro in culture for evaluation of long-term spermatozoa survival. The Makler chamber was used for all motility studies. Motility was estimated under light microscope (Zeiss Axiolab epifluorescence microscope) under 6400 magnification. Only spermatozoa with progressive motility (categories a [rapid, regular forward progression] and b [moderate, slow, or sluggish forward progression]) according to WHO (1999) were assessed. Figure 1. Schematic illustration of spermatozoa vitrification using 50- ml capillaries. (Arrows) Marked end of 50-mL capillary; (a) aspiration of spermatozoa suspension in straw, (b) 50-mL capillary sealed in a 0.25-mL straw, (c) cutting of 0.25-mL straw, (d) expelling of 50-mL capillary from 0.25-mL straw, (e) warming of spermatozoa. Color figure available online at open from both sides) with vitrified spermatozoa into a 1.8-mL centrifuge tube with 0.7 ml of vitrification medium prewarmed to 37uC for approximately 20 seconds (Figure 1e). It is important to note that the volume of vitrified suspension after warming is not increased (Figure 1e). Finally, the suspension of spermatozoa was expelled from the capillary for immediate evaluation of spermatozoa quality and use in IVF or ICSI. Conventional Freezing and Thawing After the swim-up procedure, the sperm suspension was diluted 1:2 with freezing medium (12% [v/v] glycerol and 20% [v/v] egg yolk; Freezing Medium TYB, Irvine Scientific, Santa Ana, California) to achieve a concentration of spermatozoa/ml. Subsequently, diluted sperm preparations were equilibrated at room temperature for 10 minutes. Thereafter, 10 ml of spermatozoa suspension was portioned by aspiration into 50-mL plastic capillaries (Gynemed GmbH) Plasma Membrane Integrity Integrity of the plasma membrane was assessed with the LIVE/ DEAD sperm viability kit (Molecular Probes L-7011, Eugene, Oregon). This kit includes SYBR-14 dye and propidium iodide (PI), which stain nucleic acid. With this kit, one can differentiate intact and damaged spermatozoa. Because of its properties, SYBR-14 accumulates inside of living cells and emits green fluorescence; therefore it can be used for identification of live spermatozoa and exclusion of non DNA-containing particles and debris (Garner et al, 1994; Petrunkina and Harrison, 2010). The dead cells membrane will be permeable for PI, which would intercalate DNA and emit red fluorescence. For estimation of cytoplasmic membrane integrity, the 50-mL suspension with spermatozoa/ml were add to 200 ml of phosphate-buffered saline (PBS) with 1.25 ml of SYBR-14 (1:100, Molecular Probes, Grand Island, New York) and exposed for 5 minutes at 37uC. Then, 1 ml of 2.4 mm of PI was added, and this solution with spermatozoa was incubated additionally for 7 minutes at 37uC. Spermatozoa were visualized microscopically using a Zeiss Axiolab epifluorescence microscope. Acrosomal Integrity and Capacitation-like Changes Changes in the membrane status and permeability associated with capacitation are traditionally evaluated by using the double fluorescence chlortetracycline (CTC) Hoechst staining technique (Kay et al, 1994). In short, 100 ml of suspension with spermatozoa/ml were incubated with

4 Isachenko et al N Vitrification of Human Spermatozoa 465 Figure 2. Motility of spermatozoa after conventional freezing and vitrification. All rates in respective groups are significantly different (P,.05). 1 mlof10mg/100ml Hoechst water solution for 5 minutes at room temperature in the dark. After this, the spermatozoa suspension was centrifuged at 340 6g for 5 minutes. The pellet was resuspended in 1 ml of PBS and centrifuged again at g for 5 minutes. After discarding the supernatant, the pellet was gently agitated, and a 5-mL suspension was placed in the center of aslide.next,5ml of 1 mm CTC in 20 mm Tris (ph 7.8) solution was added to the droplet on the slide and incubated for 30 seconds. The cells were fixed with a 1:1 ratio of 25% glutaraldehyde in 1 M Tris buffer. To delay photobleaching during the fluorescence procedure, 0.22 mm 1,4-diazabicyclo[2.2.2]octane was used. The probes were stored in the dark at 4uC until reading (a maximum of 48 hours). At least 200 spermatozoa were observed on each plate, and 3 patterns were identified: a uniform fluorescence on the head of the spermatozoa (classified as noncapacitated ), a band of fluorescence diminished in the postacrosomal region and a relatively shining fluorescence in the acrosomal region (spermatozoa identified as undergoing capacitation-like changes); and a fluorescence of the complete head of the spermatozoa, except for a tenuous band of fluorescence in the equatorial segment (identified as acrosome-reacted spermatozoa). The slides were viewed using a Zeiss Axiolab epifluorescence microscope that was equipped with an excitation/emission filter of 485/520 nm under 6400 magnification. The nonviable spermatozoa were observed with filter set 09 ( nm). The dead spermatozoa displayed a pattern of blue fluorescence in the whole head. Statistical Analysis An ANOVA (Kruskal Wallis test) with a significance of 0.05, for nonparametric statistical analysis, and the Dunn multiple comparison to establish differences between groups were used. Data presented are x + SEM unless otherwise specified. All data refer to 68 observations for each group. Results Motility Motility of small volumes of vitrified spermatozoa (10 ml) in the absence of permeable cryoprotectants displayed statistically higher levels of motility compared Figure 3. Plasma membrane integrity of spermatozoa after conventional freezing and vitrification. All rates in respective groups are significantly different (P,.05). with slow conventional freezing ( % vs %, respectively; P,.05) (in fresh, %). Motility of vitrified spermatozoa in culture after 24 hours in vitro was also statistically different from that of frozen spermatozoa ( % vs %, respectively; P,.05) (in fresh, %) (Figure 2). Motility rates of vitrified spermatozoa in culture after 48 hours in vitro was also statistically different from those of frozen spermatozoa ( % vs %, respectively; P..1) (in fresh, %) (Figure 2). Plasma Membrane Integrity The effect of different modes of cryopreservation on plasma membrane integrity is shown in Figure 3. It was observed that higher rates of membrane integrity were achieved in vitrified spermatozoa compared with slow conventional freezing ( % vs %, respectively; P,.05). However, compared with nontreated control (fresh spermatozoa), spermatozoa viability was significantly diminished in frozen-thawed spermatozoa in both cases (in fresh spermatozoa, %; P,.05). Acrosomal Integrity and Capacitation-like Changes The effect of 2 procedures used for cryopreservation on acrosomal membrane status by CTC staining is shown in Figure 4. There was a statistically significant difference between percentages of spermatozoa with intact acrosome after vitrification compared with conventional freezing ( % vs %, respectively; P,.05) (in fresh, %; P,.05). The presence of capacitation-like changes after different modes of cryopreservation is shown in Figure 5. It was noted that the aseptic vitrification technique provides significantly stronger prevention against cryo-capacitation compared with conventional freezing ( % vs %, respectively; P,.01) (in both cases, the

5 466 Journal of Andrology N May ÙJune 2012 Figure 4. Acrosomal integrity of spermatozoa after conventional freezing and vitrification. All rates in respective groups are significantly different (P,.05). Figure 5. Capacitation-like changes of spermatozoa after conventional freezing and vitrification. Rates in groups after freezing and vitrification are similar (P..5). percentage of cells with capacitation-like changes was higher than in fresh spermatozoa: %; P,.05). Discussion Varied methods of vitrifying spermatozoa have been described previously: cryo-loops, the droplets method, and the open pulled straw method (Nawroth et al, 2002; Isachenko et al, 2004a,b, 2005, 2008). Depending on the vitrification method selected and the quality of the original ejaculate, one can achieve motility levels of up to 60% and 20% after thawing in normospermic and oligo-astheno-terato-zoospermic patients, respectively. Vitrified spermatozoa can be processed for further use immediately after warming. No additional treatment (eg, centrifugation, gradient separation, removal of cryoprotectants, etc) is required. For practical purposes, this simplicity represents one of the most attractive advantages of our technology. It is worth mentioning that the protocol for vitrification does include swim-up treatment; therefore, after swim-up, vitrified and warmed spermatozoa are also free of seminal plasma with potential pathogens. Slow freezing of human spermatozoa traditionally includes the following stages of manipulations: 1) slow, stepwise addition of freezing solution to the ejaculate; 2) cooling for 20 to 45 minutes; 3) warming in a waterbath; and 4) preparation of spermatozoa by density gradient or swim-up for removal of permeable cryoprotectants. The removal of permeable cryoprotectants is the ultimate target of the last manipulation. As a rule, a prerequisite for that step is a dilution of semen suspension with culture medium to reduce toxicity of the permeable cryoprotectant (according to manufacturer s instructions for the cryoprotectant of choice). This step is associated with additional costs and, not the least, with environmental and adaptational challenges for spermatozoa. In fact, the sensitivity of spermatozoa to additional mechanical manipulation increases after freezing-thawing, and the negative effects of cryopreservation on cell viability and functional competence can be aggravated by additional procedures (Petrunkina, 2007). Several protocols of spermatozoa separation are available (eg, swim-up from the ejaculate, single wash of ejaculate and swim-up from the pellet, and double wash of ejaculate and swim-up from the pellet). However, any methodology needs previous centrifugation. Most of the current technologies for sperm vitrification have an obvious shortcoming in terms of standardization of the portion volume. In particular, because the diameter of the pulled part of the straw is not uniform, the volumes of the portions packaged in that way cannot be standardized. Here, we have reported a vitrification methodology that uses standard capillaries supplied by industrial manufacturers. Using the technique described here, exact quantifiable volumes of spermatozoa samples were obtained: 10-mL suspension of spermatozoa were vitrified, 10 ml was thawed, and the same 10 ml was 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 instrumental in clinical practice. The results of the present study suggest that cryopreservation by vitrification helps 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 function parameters (Petrunkina, 2007). In the present study, we have compared spermatozoa quality after vitrification by our method with spermato-

6 Isachenko et al N Vitrification of Human Spermatozoa 467 zoa quality after conventional freezing with the addition of permeable cryoprotectant. The outcomes indicated that motility of spermatozoa and membrane integrity were better preserved after vitrification in capillaries than after conventional freezing. 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 spermatozoon intracellular signaling pathways can be affected during cryopreservation and cooling, 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 cryopreservation 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 less damage to acrosomes. However, the levels of membrane changes related to cryo-capacitation assessed by CTC in vitrified spermatozoa were comparable to those after conventional freezing. Nevertheless, exposure to low temperatures can affect those crucial signaling mechanisms that cannot be monitored by CTC. Thus, further studies with additional, advanced techniques are needed to investigate the changes induced by vitrification in its complexity (eg, targeting specific pathways and membrane processes such as changes in lipid architecture, protein kinase/phosphatase-regulated pathways, or a combination of processes). Given that the outcome of basic spermatozoon quality was comparable (or even better) than that after conventional freezing, other advantages of the vitrification process must be taken into account. During conventional procedures, the success in applying permeable cryoprotectants for cryopreservation of various cells and tissues is inseparably linked to such cryoprotectant properties as their ability to permeate rapidly through the 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 the time of cryoprotectant removal 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 our university s maternity hospital ( IVF Centers in Temuco, Chile (about 200 IUI cycles/y), and in Ulm, Germany ( about 1000 IVF cycles/y). The first successful pregnancies and births of healthy babies have been recently achieved with spermatozoa vitrified without permeable cryoprotectants (Isachenko et al, 2012). 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 of seminal plasma, owing to swim-up preceding vitrification and are free of 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. References Cormier N, Bailey JL. A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa. Biol Reprod. 2003;69: Duru NK, Morshedi M, Schuffner A, Oehninger S. Cryopreservationthawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine. Fertil Steril. 2001;75: Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ, Jacob RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Nat Acad Sci U S A. 1991;88: Gao DY, Liu J, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P, Critser ES, Critser JK. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod. 1995;10: Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J Androl. 1994;15: Gilmore JA, Liu J, Gao DY, Critser JK. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum Reprod. 1997;12: Glander HJ, Schaller J. Hidden effects of cryopreservation on quality of human spermatozoa. Cell Tissue Bank. 2000;1: Green CE, Watson PF. Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction. 2001;122: Hammadeh ME, Askari AS, Georg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl. 1999;22: Isachenko E, Isachenko V, Katkov II, Nawroth F. Vitrification of human spermatozoa without cryoprotectants: review of problem and practical success. Reprod Biomed Online. 2003;6: Isachenko E, Isachenko V, Katkov II, Rahimi G, Schondorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19: Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat AG, Risopatrón MJ, Sánchez R. Acrosomal status

7 468 Journal of Andrology N May ÙJune 2012 and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136: Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, van der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71: Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko A, Nawroth F, Dessole S, Katkov I, van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10: Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012; 24: Kay VJ, Coutts JR, Robertson L. Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction. Hum Reprod. 1994;9: Larson JM, McKinney KA, Mixon BA, Burry KA, Wolf DP. An intrauterine insemination-ready cryopreservation method compared with sperm recovery after conventional freezing and postthaw processing. Fertil Steril. 1997;68: McLaughlin EA, Ford WC, Hul MG. A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer. Hum Reprod. 1990;5: Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P. Vitrification of human spermatozoa without cryoprotectants. CryoLetters. 2002;23: Petrunkina A. Fundamental aspects of gamete cryobiology. J Reprod Med Endokrinol. 2007;4: Petrunkina AM, Harrison RAP. Systematic misestimation of cell subpopulations by flow cytometry: a mathematical analysis. Theriogenology. 2010;73: Petrunkina AM, Volker G, Weitze KF, Beyerbach M, Töpfen-Petersen E, Waberski D. Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions. Theriogenology. 2005;63: Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci. 1985;442: Sawetawan C, Bruns ES, Prins GS. Improvement of post-thaw sperm motility in poor quality human semen. Fertil Steril. 1993;60: Schuffner A, Morshedi M, Oehninger S. Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation. Hum Reprod. 2001;16: Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B, Suchanek E. Comparison of protective media and freezing techniques for cryopreservation of human semen. Eur J Obstet Gyn RB. 2000;91: Vadnais ML, Roberts KP. Seminal plasma proteins inhibit in vitroand cooling-induced capacitation in boar spermatozoa. Reprod Fertil Dev. 2010;22: Vajta G, Kuwayama M, Holm P, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev. 1998;51: World Health Organization (WHO). WHO Laboratory Manual for the Examination of Human Semen Cervical Mucus Interaction. 4th ed. Cambridge, United Kingdom: Cambridge University Press; Yin HZ, Seibel MM. Human sperm cryobanking. Use of modified liquid nitrogen vapor. J Reprod Med. 1999;44:87 90.