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BOR - Papers in Press, published online ahead of print June 2, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028811
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BIOLOGY OF REPRODUCTION 71, 1167–1173 (2004)
DOI: 10.1095/biolreprod.104.028811
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Cryoprotectant-Free Cryopreservation of Human Spermatozoa by Vitrification and Freezing in Vapor: Effect on Motility, DNA Integrity, and Fertilization Ability

Vladimir Isachenko1,3, Eugenia Isachenko3,4, Igor I. Katkov5, Markus Montag3, Salvatore Dessole6, Frank Nawroth2,4, and Hans van der Ven3

Department of Gynecological Endocrinology and Reproductive Medicine,3 University of Bonn, 53105 Bonn, Germany Department of Obstetrics and Gynecology,4 University of Cologne, D-50924 Cologne, Germany Cancer Center,5 University of California at San Diego, La Jolla, California 92093 Department of Obstetrics and Gynecology,6 University of Sassari, 07100 Sassari, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 105 °C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (–160°C) with speed in the range 150–250°C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

assisted reproductive technology, cooling rate, cryopreservation without cryoprotectants, DNA integrity, human spermatozoa, in vitro fertilization, viability, vitrification


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many years ago, spermatozoa of many mammalian species were cryopreserved successfully.

Bernstein and Petropavlovski in 1937 [1] used 0.5–3 M glycerol for freezing of bull, ram, stallion, boar, and rabbit spermatozoa to a temperature of –21°C. Authors reported that they have obtained the best results at concentrations of glycerol 0.5–2 M. Observations of Jahnel in 1938 and Parkes in 1945 on freezing of human spermatozoa were published [2, 3]. The work of Polge and colleagues is now considered as a milestone of modern cryobiology [4].

Hoagland and Pincus, in 1942, described the freezing of human and rabbit spermatozoa using a bacteriological loop to cool small specimens at rapid cooling [5]. They obtained up to 40% of viable human spermatozoa after cooling of a sperm emulsion or a sperm film in liquid nitrogen followed by quick warming of these microvolumes.

Smirnov, in 1949 [6], reported successful freezing of rabbit sperm in liquid nitrogen and liquid oxygen using a 0.05-ml aluminum container.

It is well established that radiotherapy, chemotherapy, or even invasive surgery may lead to testicular failure or ejaculatory dysfunction [7–9]. Thus, by cryopreserving sperm as part of an assisted reproduction program, we can offer couples the option of having children in the future. Moreover, in situations of impaired male fertility, sperm storage will provide the necessary time for a reasonable amount of sperm to be obtained for successful artificial insemination or in vitro fertilization. Nevertheless, because of the damage associated with freezing, the motility of cryopreserved spermatozoa after thawing is statistically reduced with respect to prefreezing motility, and this factor also shows wide interindividual variability [10, 11]. Sperm quality may also be affected by the subsequent slow-thawing process. This process induces further cell damage [12]. Further, the addition and removal of osmotically active cryoprotective agents (CPAs) during freezing and warming can induce lethal mechanical stress per se. Even further problems include the chemical toxicity of CPAs and possible negative influence on the genetic apparatus of mammalian spermatozoa [13, 14].

We recently developed a new technique of ice- and CPA-free cryopreservation (vitrification) by direct plunging of a sperm suspension into liquid nitrogen. After storage, warming is achieved by direct melting of the frozen suspension. This freezing/warming method is performed at cooling and warming rates of up to hundreds of thousands of °C/min [15, 16]. This simple, straightforward approach preserves the ability of the spermatozoa to move and fertilize the oocyte. Its improved results over the conventional method of slow freezing (ice-equilibrium) may be attributed to avoiding the use of the classic permeable cryoprotectants, thus preventing the lethal effects of osmotic shock. Vitrification is also able to avoid lethal intracellular ice formation and the detrimental effects of high salt concentrations during freezing and rewarming (thawing). In addition, the entire process only takes a few seconds.

The conventional method of vitrification used to preserve large cells (embryos, oocytes), tissues, or organs requires high CPA concentrations with the consequent lethal osmotic and toxic effects. As a result, it has not been possible to successfully cryopreserve the osmotically sensitive mammalian spermatozoon by conventional vitrification, which implies high CPA concentrations and a relatively high (≤5 x 103 °C/min) cooling rate.

When we compared the outcome of vitrification in the presence or absence of low CPA concentrations, variables such as postwarm sperm morphology, viability, motility, and percentage of acrosome-reacted cells remained the same [15].

The protocol for the cryopreservation of spermatozoa by vitrification we proposed at that time for application in assisted reproduction was based on the two main requirements of very fast cooling before storage in liquid nitrogen and very rapid warming after this storage [15]. We speculated that this rapid cooling would prevent the formation of sizeable intracellular ice crystals that are potentially lethal. Similarly, the very fast warming rate would serve to prevent the recrystallization that may otherwise occur in the supercooled vitrified glass state [16]. It was emphasized that devitrification (especially intracellular growth of crystals) and mechanical damage (cracking) of the vitrified matrix during rewarming could be highly detrimental to the cell suspension [17]. This prompted a change in our working hypothesis to consider that, while warming should be undertaken as rapidly as possible (we refer to the direct immersion of the sperm suspension in a warm medium as instant warming due to the thinness of the sperm suspension film), the cells might be less sensitive to the rate of cooling during vitrification. The essential question to address is thus: What effect will CPA-free cryopreservation by slow versus rapid cooling have on sperm viability? The answer to this question has implications both for future assisted reproduction technology and for our current understanding of the fundamental issues of cryobiology.

The goal of this study was to compare the quality of spermatozoa (in terms of motility, DNA integrity, and fertilization ability) cryopreserved using a very fast cooling rate (that leads to practically instant vitrification) by plunging into liquid nitrogen, with cryopreservation at a slower rate (but still fast in the conventional sense) by freezing in liquid nitrogen vapor before immersion into liquid nitrogen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The study, performed in Italy and Germany, was approved by the University Review Board (Italy) and University Ethics Committees (Germany).

Sampling and Spermatozoa Evaluation

Ejaculates were obtained from 38 healthy men by masturbation after at least 48 h of sexual abstinence. The ejaculates were required to contain 20 million or more spermatozoa/ml and to show at least 50% progressive sperm motility and 15–30% morphologically normal spermatozoa. Informed consent was obtained from each donor. Semen analysis was performed according to published guidelines of the World Health Organization [18].

Swim-Up

Each ejaculate was swim-up prepared and divided into three equal parts for instant vitrification, slow vapor freezing, or no treatment (control, fresh spermatozoa). Swim-up preparation using standard preservation medium (SPM; Scandinavian IVF Science, Gothenburg, Sweden) was performed according to World Health Organization instructions [18]. In brief, each ejaculate was washed twice by centrifugation at 380 x g for 10 min in a double volume of SPM. After washing, 0.8 ml of SPM was pipetted over the pellet. The samples were then incubated for 30 min for swim-up and obtaining of concentration >1.5 x 107 sperm/ml. The controls for all the experimental groups were swim-up-prepared fresh spermatozoa.

Instant Vitrification and Warming

The prepared spermatozoa were loaded onto copper loops of 5-mm diameter by dipping the loops in a sperm suspension to obtain a thin film (supported by surface tension) of 20 ± 2 µl. The volume of sperm suspension included on the film was determined by the following way. After plunging of the loop rind into the suspension of spermatozoa and formation of the film, we shook down a suspension on the flat of a plastic Petri dish. Then, using a micromeasurer (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany), we had a possibility of determining the volume of the film, which had formed a droplet on the flat of the Petri dish. A potential negative effect of copper on spermatozoa was ignored due to their short contact with this material. The loaded loops were then plunged into liquid nitrogen. After storage for a minimum of 24 h, the samples were thawed by plunging the loops into a 15-ml tube containing 10 ml SPM at 37°C under intense agitation. After warming 5 loops in one tube, the tube was placed in a CO2 incubator for 5–10 min. Next, the spermatozoa were concentrated by centrifugation at 380 x g for 10 min, and the resultant pellet was resuspended in 100 µl of SPM and processed for further evaluation.

Freezing in Liquid Nitrogen Vapor

Spermatozoa were vitrified and thawed according to the procedure described above with the following modifications. Before plunging into liquid nitrogen, the loops were cooled for 3 min in liquid nitrogen vapor at –160°C. This was achieved by placing the loop in a Styrofoam box (5 x 5 x 10 cm) containing a 0.5- to 0.8-cm depth of liquid nitrogen ~1 cm above the liquid nitrogen level. The temperature of the vapor was determined using a Testo 950 electrical thermometer (Testo AG, Lenzkirch, Germany).

Estimation of Cooling Rates

The speed of cooling during direct plunging into liquid nitrogen was calculated by introducing variables such as the geometry of the loop, amount of attached material, and physical characteristics of the sperm suspension.

The cooling speed of a sperm suspension film on a loop frozen in liquid nitrogen vapor was determined according to a method designed by us (Fig. 1). A loaded loop was placed in the same position in the same Styrofoam box containing liquid nitrogen, in which the experimental spermatozoa were frozen. The film was then periodically (at ~1-sec intervals) pierced by a thin (27-gauge) needle at different locations (center, near the copper ring, and at the periphery). When the film is liquid, it is possible to punch through it many times without disruption of the film, and after the needle is removed, the film remains intact. Upon freezing, the film solidifies (starting from the copper ring area toward the center) and piercing without disruption of the film becomes impossible: the ring begins to move. The time elapsed (visually indicated) from placing the loop in the box at room temperature (+23°C) to the beginning of solidification of the suspension (–4°C) allowed us to calculate the speed of cooling of the spermatozoa. This experimental freezing-transfixing procedure was repeated 50 times.



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  		FIG. 1. Scheme of the mode of estimation of spermatozoa cooling rate in vapor of liquid nitrogen. 1) Copper loop with film of spermatozoa suspension, (2) foam box, (3) liquid nitrogen, (4) foot for loop, (5) needle

Motility of Spermatozoa

The motility of control (swim-up prepared, unfrozen) spermatozoa was assessed immediately after swim-up treatment. The motility of cryopreserved spermatozoa was determined immediately after instant thawing by melting in warm SPM and concentrating the sample by centrifugation as described above. A Makler Counting Chamber (Sefi-Medical Instruments Ltd., Haifa, Israel) was used for motility scoring. This index was estimated under the light microscope at a magnification of 400x. Only spermatozoa showing progressive motility were assessed. The recovery of motile spermatozoa was defined as the percentage of postthaw motility x 100% divided by the percentage of motility before cryopreservation.

DNA Integrity of Spermatozoa

The Comet assay was performed using the CometAssay Reagent Kit for Single Cell Gel Electrophoresis Assay (R&D Systems GmbH, Wiesbaden-Norgenstadt, Germany) according to the manufacturer's instructions. Briefly, spermatozoa samples were washed twice with SPM and the pellet was resuspended in Dulbecco phosphate buffered saline (PBS, Ca2+ and Mg2+ free; Bio-Wittaker, Verviers, Belgium). Samples were then put on ice to avoid endogenous damage occurring during sample preparation. During preparation, cell samples were handled under yellow light to prevent DNA damage from ultraviolet light. As controls (comet tails-positive), cells were treated with 25 µM KMnO4 for 20 min at 4°C. Subsequent treatment of DNA-damaged and undamaged cells was performed as follows. Freshly prepared lysis solution supplemented with 1% dimethylsulfoxide was chilled at 4°C for at least 20 min before use. The lysis solution contains 2.5 M sodium chloride, 100 mM EDTA, pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100. After mixing the spermatozoa suspension (containing approximately 1 x 105 cells/ml) with 1% molten low-melting point agarose at 40°C at a ratio of 1:10 (v/v), 75 µl of suspension was immediately pipetted onto the Trevigen CometSlide (Trevigen, Inc., Gaithersburg, MD) and was gently spread over the slide and put flat in the dark at 4°C for 10 min. The slides were then immersed into a prechilled lysis solution for 60 min for dissolution of the cell membranes. After cell lysis, DNA decondensation was achieved by incubating the slides with 10 mM dithiothreitol (DTT; Sigma-Aldrich, Steinheim, Germany) for 30 min at 4°C and then with 4 mM 3.5-diodosalicylic acid lithium salt (LIS; Sigma-Aldrich) for 90 min at 20°C. After tapping off excess solution from the slides, they were immersed into freshly prepared alkaline solution (300 mM NaOH, 1 mM EDTA, pH > 13) in the dark for 20 min at room temperature. A horizontal gel electrophoresis apparatus was filled with the same alkaline solution at 4°C. Slides were placed flat onto a gel tray and aligned equidistantly from the electrodes. Electrophoresis was performed at 1 V/cm adjusted to 30 0 mA by either raising or lowering the buffer level in the apparatus for 10 min. After electrophoresis, the excess solution was gently tapped from the slides, which were dipped in 70% ethanol for 5 min with subsequent air drying at room temperature before being stored in an airtight desiccator. The slides were viewed using a Zeiss i.m. epifluorescence microscope equipped with an excitation/emission filter of 485 nm/520 nm under 400x magnification. Fluorescent staining was performed using the SYBR Green Stain (component 4250-50-01; working concentration 1:200; R&D Systems GmbH, Wiesbaden-Norgenstadt, Germany). In healthy cells, fluorescence was confined to the nucleoid: undamaged DNA is supercoiled and does not migrate very far from the nucleus (Fig. 2). In cells with accrued damage to their DNA, the alkali treatment unwinds the DNA-releasing fragments that migrate from the nucleoid (Fig. 2). A total of 200 cells were analyzed on each slide.



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  		FIG. 2. Spermatozoa processed using Comet assay. Cells with undamaged DNA (arrows), cells with damaged DNA exhibit comet tail (arrowheads). A) Spermatozoa treated by KMnO4 (control). B and C) Cryopreserved spermatozoa. Scale bar = 20 µm

Fertilization Ability of Spermatozoa

The in vitro fertilizing capacity of swim-up fresh (control), vitrified (rapid-cooled), and frozen (slow-cooled) spermatozoa was assessed using oocytes from eight patients (35 ± 4.2 yr old) after obtaining informed consent (University of Sassari). These patients suffered from unexplained infertility and were stimulated for in vitro fertilization (IVF) with triptorelin (Decapeptyl; Ipsen SPA, Milan, Italy) and recombinant follicle-stimulating hormone (Gonal F; Serono Pharma, Rome, Italy) according to the long protocol. For oocyte retrieval, IVF and subsequent embryo culture, we used Universal IVF Medium (I and II) (Medicult, Redhill Surrey, UK) in a routine protocol. Eighty oocytes were obtained after follicular puncture and divided into three groups (Table 1): 39 oocytes were fertilized using control spermatozoa, 19 using vitrified (rapidly cooled) spermatozoa, and a group of an additional 19 oocytes using frozen (slowly cooled) spermatozoa. Oocytes were cultured in 5% CO2 at 39°C in Universal IVF Medium (MediCult, Jyllinge, Denmark). Pronucleus formation evaluation and scoring of embryos were performed routinely.


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  		TABLE 1. Fertilization properties of spermatozoa after cryroprotectant-free cryoperservaton with rapid and slow cooling: results of in vitro fertilization and culture.*

Statistical Analysis

Treatment effects on the parameters assessed were evaluated by ANOVA. The data are given as mean values ± SD. The level of statistical significance was set at a P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estimation of Cooling Rates

The time elapsed from 23°C to the beginning of the pellicle solidification (beginning of ring to move at –4°C) was in accordance with the place of piercing of the film by needle: from 6 sec (piercing near the copper ring) to 10 sec (piercing at the center of the ring). Thus, the rate of cooling of spermatozoa in vapors of liquid nitrogen was in the range 162–270°C/min.

Motility of Spermatozoa

Quality of spermatozoa before instant vitrification (direct plunging into liquid nitrogen) and freezing in vapor at –160°C is shown in Figure 3. Both regimes of cryopreservation gave about 40% reduction of motility of spermatozoa (P < 0.05) in comparison with swim-up-treated control. No statistically significant difference was found in this parameter between the two methods of cryopreservation.



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  		FIG. 3. Motility and DNA integrity of spermatozoa after cryoprotectant-free cryopreservation with rapid (vitrification) and slow (freezing) coolings. No differences between respective rates of vitrified and frozen spermatozoa. Pa–b < 0.05

DNA Integrity of Spermatozoa

In contrast with motility, DNA integrity in both groups of cryopreserved spermatozoa was found to be unaffected by the vitrification mode (Fig. 2; P > 0.05).

Fertilization Ability of Spermatozoa

Instead of motility, three parameters of viability of spermatozoa after vitrification with rapid and slow cooling compared with control (fresh samples) were taken into account: fertilization rate (formation of pronuclei), early cleavage of zygotes (formation of 4–6 blastomeres), and late development of embryos (formation of blastocoel). The results of IVF prove the approximately equal fertilization potential of human spermatozoa, which were CPA-free vitrified and frozen in vapor of liquid nitrogen (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cell Damage During Conventional Freezing and Thawing

The cryopreservation of sperm is an important routine technique used in the management of human male infertility. The question of diminished spermatozoon motility after cryopreservation is crucial because this variable is known to be the first affected [19, 20], although the mechanism of sperm impairment and its mechanical and/or physical-chemical etiology remain unclear.

Mechanical cell injury by conventional (ice-equilibrium) freezing is a consequence of intracellular or extracellular ice crystal formation and osmotic damage due to extensive cell shrinkage. Subsequent rewarming and thawing of the cells can further deteriorate their viability through possible excessive osmotic swelling [20–22]. As a result, average velocity in terms of the percentage of motile spermatozoa drops significantly after cryopreservation with respect to that of fresh sperm [20, 23–26]. Conventional slow freezing may also cause extensive chemical and physical damage to sperm cell membranes due to changes in lipid-phase transition and/or increased lipid peroxidation. It is well established that the production of reactive oxygen species leads to an increase in lipid peroxidation after cryopreservation [27] and that this event is associated with a loss of sperm motility [28, 29]. As previously suggested [28, 30, 31], the injury to human spermatozoa induced by conventional cryopreservation occurs mainly during thawing and has been related to diminished antioxidant defense activity during cooling and/or structural damage to the cytoskeleton and/or antioxidant enzymes during cryopreservation [28, 30]. All these findings suggest that slow cooling, and especially thawing of spermatozoa, aside from ice crystal formation, is intrinsically deleterious.

To prevent excessive cell shrinkage during slow cooling, permeable CPAs are used. However, the effectiveness (prevention of intracellular ice formation) of permeable and nonpermeable cryoprotectants during conventional freezing can only be achieved with a low cooling rate [22], which, as we indicated before, can be damaging in itself. In addition to this, the introduction (during freezing) and removal (after thawing) of CPAs can produce damage per se even at room temperature in the absence of freezing/thawing. The main mechanisms of CPA toxicity have been discussed by us elsewhere [16] and include osmotic damage as well as chemical cell and membrane toxicity [10, 19, 31–33]. All these negative effects of conventional slow (ice-equilibrium) freezing and thawing on cells can also lead to chromatin damage. The assessment of sperm nucleus integrity due to such possibilities is very important because, as recently noted [34, 35], chromatin abnormalities have repercussions on sperm quality and male fertility status.

It was shown that sperm DNA damage is strongly correlated with mutagenic effects [36]. It has been noted that freezing/thawing the sperm of fertile and infertile men leads to significant chromatin damage as well as significant effects on sperm morphology and membrane integrity [8, 9, 37, 38]. Other studies have demonstrated that any defects in the chromatin structure of spermatozoa from infertile men showing increased DNA instability are sensitive to denaturing stress [39]. This denaturing stress may be induced by several treatments, including freezing. Despite this, oocytes have the ability to repair a small amount of sperm DNA damage, although this seems to be insufficient to support subsequent embryo development [40], and DNA damage can lead to decreased conception rates or conception failure [37, 41]. The percentage of spermatozoa with fragmented DNA has also been negatively correlated with in vitro fertilization rates [42] and intracytoplasmic sperm injection (ICSI) [43].

In contrast with the conventional slow-freezing, ice-forming techniques, the protocols of vitrification currently used for the cryopreservation of oocytes, embryos, and tissues as a rule involve the use of very high concentrations (3.5–8 M) of permeating cryoprotectants and relatively high cooling rates (up to 104 K/min) compared with rates associated with conventional slow freezing. According to the literature, the critical cooling speed for the vitrification of pure water varies drastically, depending on the method used, from 107 to 1013 K/min (see Fig. 9 in [44] for reference). The presence of very high concentrations of CPAs substantially decreases the critical rate of freezing and warming. However, it is known that high concentrations of cryoprotectants have a marked toxic effect [45–47]. It is possible to decrease cryoprotectant toxicity by the stepwise exposure of cells to precooled concentrated solutions [45, 48] and/or by reducing the amount of cryoprotectant and at the same time increasing cooling and warming rates [49]. This lowering of the CPA concentration consequently requires an increased rate of cooling and warming, which can be achieved by decreasing the volume of the cooled suspension and increasing the surface-to-volume ratio of the sample. The sample size can be minimized using different kinds of packages [49–51], microdrops [52], electron microscopic copper grids [53, 54], original cryoloops [55], nylon mesh [56], and a minimal size of the sample and maximal surface-to-volume ratio and rate of cooling can be obtained using metallic cryoloops [51].

In our experiments, when the specimen is kept above the surface of the liquid nitrogen in its vapor at –160°C, given the thermal conductivity of the vapor is substantially lower than that of the liquid nitrogen, the cooling rate can be several orders of magnitude lower than when plunging into liquid nitrogen. The main cooling front will extend from the surface of the copper ring in a radial direction toward the center of the pellicle. The mathematical calculations of this scenario are very complex. However, we were able to estimate the average rate of cooling, by first measuring the time of solidification of the film near the ring and then estimating the time taken for the surface of the film to completely solidify at the center. This gave an initial cooling rate in the range 270°C/min, near the copper ring, to 162°C/ min, at the center of the film. It is clear that, at this rate and in the absence of a viscous vitrification solution in the SBM medium, the extracellular milieu of the cells will not vitrify, but start freezing with the initiation of ice crystal formation. However, human spermatozoa contain large amount of proteins, sugars, and other components that make the intracellular matrix highly viscous and compartmentalized. The quantity of high-molecular-weight macromolecules and polymers diluted in the cytosol can be estimated from the fraction of the osmotically inactive volume, which is about 20–25% for embryos and oocytes and much higher (45–77%) for spermatozoa [22]. As a result, we can speculate that we were able to achieve intracellular vitrification of the human spermatozoa even at such a low range of cooling rate. A further factor to consider is the small size and high degree of compartmentalization of the sperm head, such that, even if small (nonlethal) crystals start to form during this relatively slow cooling, there would be insufficient time for substantial growth during cooling. It is known that a major problem for such metastable systems can be the regrowth of crystals and devitrification during warming. However, our method of instant thawing seemed to prevent cell damage even after relative slow freezing in liquid nitrogen vapor.

The cooling rate of 160–250°C/min is 5–10 times higher than the rate of conventional slow freezing with the use of permeable cryoprotectants. At such a slow freezing speed, water has time to escape from the cells upon freezing. As a result, the cell shrinks and can be osmotically damaged unless nonpermeable CPAs are used to prevent hydration. In our case, however, the freezing rate is much faster, so the cell maintains its volume without the need for a conventional cryoprotectant.

Our results indicate that the relatively small size of the mammalian spermatozoon head and reduced water content compared with the larger embryos and oocytes may be an additional benefit for ensuring intracellular vitrification without substantial large ice crystal formation (detailed physical considerations will be published elsewhere). The final outcome is the essentially similar results, in terms of sperm motility, fertilization ability, and DNA integrity, observed after instant vitrification by direct plunging into liquid nitrogen and relatively slow cooling in liquid nitrogen vapor.

The first is the successful cryoprotectant-free vitrification of frog spermatozoa in 1938 [57]. The second is that there are reports of an effective relatively slow-freezing protocol for mouse spermatozoa not requiring any permeable CPAs [58–60]. In both cases, spermatozoa even larger than human were used, so for small human sperm, the beneficial effect of cell size would be even more pronounced. The third finding supporting our hypothesis is the fact that we were not able to achieve cell survival after vitrification without CPAs using large cells such as embryos and oocytes (unpublished data).

Recently, simplified cryoprotectant-free freezing methods of mouse spermatozoa was reported. Authors examined the fertilization capacity of the warmed spermatozoa. Because mouse spermatozoa is a specific and noncryostabile object, after cryopreservation, spermatozoa were immotile and could fertilize oocytes only after ICSI [61]. Our method allows restoring motile properties of human spermatozoa after cryopreservation, which is important for use of conventional fertilization in artificial reproductive technologies.

As the founder of modern cryobiology, Luyet, in 1937 [17], emphasized that devitrification and the growth of ice crystals formed during cooling could be a key factor promoting cell damage during rewarming and thawing procedures. Herein, we directly melted the specimens in a warm solution, ensuring a very high rate of warming. In this process, the probability of substantial devitrification (recrystallization) of the vitrified intracellular solution and regrowth of large lethal intracellular crystals is low due to the high speed and very short time of warming. Our estimations showed that, in general, during warming/resuscitation, the small specimen size, high viscosity of the freezing medium and intracellular matrix, very high speed of warming, small size of the cells, and their low water content and high degree of compartmentalization should, to a large extent, avoid devitrification (especially intracellular) [44, 62].

In conclusion, our results point to the feasibility of the cryoprotectant-free cryopreservation of human spermatozoa by fast or relatively slow cooling, respectively, achieved by direct plunging into liquid nitrogen (vitrification) or freezing in liquid nitrogen vapor beforehand, followed in both cases by rapid thawing. The DNA integrity of sperm cryopreserved using both regimes is comparable with that of fresh sperm.


   ACKNOWLEDGMENTS
 
The authors are very grateful to Ms. A. Burton for reviewing the English.


   FOOTNOTES
 
1 Correspondence: Vladimir Isachenko, Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. FAX: 0049 228 2875795; vovaisachenko{at}yahoo.com Back

2 Current address: Reproductive Medicine and Gynecological Endocrinology, Endokrinologikum Hamburg, 22767 Hamburg, Germany Back

Received: 23 February 2004.

First decision: 22 March 2004.

Accepted: 27 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bernstein AD, Petropavlovski VV. Influence of nonelectrolytes on viability of spermatozoa. Buleten Experimentalnoi Biologii i Medicini 1937 III 21-25
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  3. Parkes AS. Preservation of human spermatozoa at low temperatures. Br Med J 1945 2:212-213[Free Full Text]
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  6. Smirnov IV. Preservation of domestic animal semen by deep cooling. Sovetskaja Zootechnia 1949 4:63-65
  7. Sanger WG, Oslon JH, Sherman JK. Semen cryobanking for men with cancer—criteria change. Fertil Steril 1992 58:1024-1027[Medline]
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