Archives of Andrology: Journal of Reproductive Systems, 53:275284, 2007 Copyright # Informa Healthcare USA, Inc.

ISSN: 0148-5016 print=1521-0375 online DOI: 10.1080/01485010701569874

Research Article

Freezing-Free Preservation of Human Spermatozoa A Pilot Study

Jonathan M. Riel Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI Thomas T. F. Huang Department of Obstetrics and Gynecology, John A. Burns School of Medicine, University of Hawaii and Pacific IVF Institute, Kapiolani Medical Center, Honolulu, HI

Monika A. Ward Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI

This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and infertile patients with a new method that can simplify sperm preparation for ICSI, assisting men who are unable to provide semen on the day of assisted fertilization.
KEYWORDS ICSI, sperm chromosomes, sperm motility, sperm preservation

INTRODUCTION
Since its introduction in 1992 [Palermo et al. 1992], intracytoplasmic sperm injection (ICSI) has revolutionized treatment of severe male factor infertility. In contrast to in vitro fertilization (IVF), where the concentration of spermatozoa required for fertilization is high, ICSI requires only one spermatozoon per oocyte to effect fertilization. When a couple is scheduled for ICSI (or IVF), the male partner usually provides a semen sample on the day of the females oocyte retrieval. This is sometimes difficult to achieve and could be overcome with the use of cryopreserved sperm. However, spermatozoa from male patients, for which ICSI is recommended, are often of low quality and do not tolerate cryopreservation well. A method for maintaining sperm for several days without freezing would avoid cryodamage permitting the ejaculate to be obtained in advance. Ion channels are important for exchange of information between cells and their environment. Several sperm features, including sperm motility,
275

Abbreviations: ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; ART: assisted reproduction technologies; HTF: human tubal fluid. Received 04 February 2007; accepted 01 April 2007. Address correspondence to Dr. Monika A. Ward, Institute for Biogenesis Research, University of Hawaii, 1960 East-West Rd, Honolulu, HI, 96822. E-mail: mward@hawaii.edu

depend on ion permeability changes modulated by environmental cues [Darszon et al. 2006]. Sodiumpotassium-dependent ATPase maintains intracytoplasmic ion concentration and cell volume by active transfer of sodium and potassium ions using energy produced by the hydrolysis of adenosine triphosphate. This ATPase is highly sensitive to hypothermia and at low temperatures reduction of its activity inhibits activity of a sodium pump. This results in the uptake of sodium from extracellular milieu which is detrimental to the nucleus and DNA [Suzuki et al. 1998; Tateno et al. 2000]. It likely hampers cell preservation at low temperatures. Thus, depleting a preservation solution of ions may inhibit cell damage. Recently, a method based on depleting the preservation medium of ions, that upon entering cells cause their rupture, has been developed for maintaining human spermatozoa without freezing [Saito et al. 1996; Saito et al. 1999]. Here we tested this method for subsequent use in ICSI. We preserved human sperm in an electrolyte-free solution for a prolonged time and tested sperm motility and viability during storage, sperm ability to participate in early post-fertilization events after ICSI, and paternal chromosome integrity. Using a mouse

model we analyzed the ability of preserved sperm to participate in normal embryogenesis.

RESULTS Human sperm preserved in electrolyte-free medium maintain motility for up to 28 days
Individual samples from 12 ejaculates (with assigned ejaculate ID 112, listed in Table 1) were analyzed for changes in motility. Three ejaculates (1, 2, and 5) were analyzed daily for 14 days to determine the pace with which motility decreased (Fig. 1). The remaining ejaculates were evaluated every other day (occasionally every 3 days) for up to 21 days, and at 7 day intervals after that, or until motility ceased. Sperm motility decreased over time (Fig. 1). Nevertheless, after 7 days of storage, motile spermatozoa were present in all but one sample and after 14 days of storage in two-thirds of the samples (Table 1). Motile sperm could also be found after 21 days (ejaculate 2 and 7, 8% and 1%, respectively), and after 28 days (ejaculate 2, 1%). Thus, some motile sperm were present after 4 weeks of storage.

TABLE 1 Human Sperm Motility and Viability After Storage in Electrolyte-Free Solution
Motility (%) After storage for After Percoll separation 92 87 67 75 91 56 31 55 58 41 11 17 After Percoll separation 75 61 n=a 81 71 80 64 97 68 61 12 53 Viability (%) After storage for

Ejaculate ID 1 2 3 4 5 6 7 8 9 10 11 12

Initial 67 68 60 67 45 46 34 38 43 21 7 12

7d 46 37 12 20 6 10 12 0 20 3 2 3

14d 7 21a 3 3 1 1 4b 0 0 1 0 0

Initial 87 73 n=a 78 60 56 68 87 n=a n=a n=a 52

7d 66 39 47 62 40 24 19 32 41 57 8 16

14d 43 33 41 24 45 10 12 34 32 27 1 12

21d 30 11 n=a 19 33 17 14 27 6 n=a n=a 1

28d 28 19 n=a 4 36 5 1 n=a 1 n=a n=a 0

35d 28 5 n=a 0 26 0 0 n=a 2 n=a n=a 0

42d 29 3 n=a 0 15 0 0 n=a 0 n=a n=a 0

Ejaculates were classified according to initial motility as normal ( >50% motile sperm; ejaculates 14), mild asthenozoospermia (2050% motile sperm, ejaculates 510), and severe asthenozoospermia (<20% motile sperm, ejaculates 11 and 12) a Sample evaluation after 21 and 28 days demonstrated 8% and 1% motile sperm, respectively b Sample evaluation after 21 days demonstrated 1% motile sperm n=a not analyzed due to lack of samples available for analysis or other reasons

Riel et al.

276

FIGURE 2 Human sperm chromosomes visualized after injection of sperm into mouse oocytes sperm were preserved in electrolyte-free solution for 14 days.

FIGURE 1 Decrease in motility and viability of human sperm

preserved in electrolyte-free solution. Each point represents a mean from measures of three ejaculates (ejaculates: 1, 2 and 5). Error bars are standard deviations.

Human sperm preserved in electrolyte-free medium maintain viability for up to 42 days

Sperm viability was tested using Live=Dead Sperm Viability Kit. Three ejaculates (1, 2, and 5) were analyzed daily for 14 days to determine the rate with which viability decreased (Fig. 1). The remaining ejaculates were evaluated every other day (occasionally every 3 days) for up to 21 days, and at 7 day intervals after that, or until viability ceased. In some cases the analyses were terminated earlier while viable sperm were still present because of insufficient number of samples initially prepared (ejaculate 3, 8, 10, 11, Table 1). Sperm viability decreased steadily over time but at a much slower rate than motility (Fig. 1 and Table 1). After 14 days viable spermatozoa could be found in all samples. After 42 days of storage three ejaculates (1, 2, and 5) yielded viable spermatozoa (Table 1). Samples 1 and 5 had 15% and 29% viability, respectively, suggesting that it is likely that viable sperm would be present even after longer storage.

oocytes were injected. Seventy one (69%, 71=103) survived. The majority of oocytes that survived (89%, 63=71) extruded 2nd polar bodies and developed normal pronuclei, an indication of successful fertilization. Sixty-three fertilized oocytes were used for chromosome analysis and 49 yielded chromosome complements for analysis (Fig. 2). Forty seven sperm complements (96%, 47=49) were scored as normal. The proportion of normal karyoplates after ICSI with sperm stored for 14 days in electrolyte-free solution was not different (P > 0.05) than that obtained after ICSI with fresh human sperm (97%, 31=32).

Mouse sperm preserved in electrolyte-free medium yield developmentally competent embryos and viable offspring
The mouse model was used to test sperm competence in normal embryogenesis. Because the number of sperm in the mouse is lower than in a human

Human sperm preserved in electrolyte-free medium fertilize oocytes in vitro and maintain its genetic integrity
Motile spermatozoa from two ejaculates preserved in electrolyte-free medium for 14 days were used for ICSI into mouse oocytes. Finding motile sperm during ICSI was not difficult. In two experiments 103
277

FIGURE 3 Decrease in motility of mouse sperm preserved in

electrolyte-free solution. Two mouse sperm samples (Sample 1 and Sample 2) were analyzed.

Preservation of Human Spermatozoa Without Freezing

FIGURE 4 Development of embryos produced by ICSI with mouse sperm stored in electrolyte-free solution for 7 days. (A) blastocyst stage embryos after in vitro culture for 96 hours, (B) term fetuses delivered at Day 20 of gestation.

($ 1020 106 sperm in epididymides from normal B6D2F1 male) we pooled epididymal sperm from two males for each Percoll separation. We first prepared two individual mouse sperm samples and tested if mouse sperm can maintain motility when stored in electrolyte-free solution similarly as human sperm. The individual samples of the same preparations were evaluated daily for motility. In both preparations, sperm motility was maintained for 21 days (Fig. 3). To examine two developmental milestones: in vitro development to blastocyst and development to live offspring after embryo transfer we prepared one 7-day stored mouse sperm sample for ICSI into mouse oocytes. When mouse sperm preserved for 7 days were injected into mouse oocytes, more than 50% of 2-cell embryos cultured in vitro developed to normal blastocysts (56%, 27/48, combined data from two replicates). When twenty-five 2-cell embryos produced with sperm stored in electrolyte-free solution were transferred into 2 pseudo-pregnant females, 75% of embryos implanted (19/25) and 36% (9/25) developed to term yielding normal offspring (Fig 4). Fetal development was not significantly different (P > 0.05) when compared to development of fetuses produced by IVF in our past study (Szczygiel et al. 2002b).

and when sperm chromosomes were examined after storage, an increased frequency of structural aberrations was reported [Martin et al. 1990; Munne and Estop 1993]. Moreover, the use of egg yolk was troublesome because it promoted the growth of bacteria and the use of antibiotics to prevent it decreased sperm motility [Jaskey and Cohen 1981]. In the initial phase of this study we tried to store unprocessed ejaculates, either at room temperature (25C) or at 4C. Sperm motility ceased by day 3, and viability by day 7 regardless of storage temperature (Table 2). We also attempted to maintain sperm suspended in a regular sperm handling medium containing Hepes. This approach allowed preserving sperm motility and viability for longer periods than the unprocessed ejaculate (data not shown).
TABLE 2 Motility and Viability Analysis of Human Sperm from
Unprocessed Ejaculates Stored at 25C and 4C

Storage Days of storage temperature Ejaculate ID (C) Initial 1 2 3 4 5 6 Motility 6 7 4 Viability 6 7 4

DISCUSSION
We describe the freezing-free maintenance of human sperm for subsequent use in ICSI. Prior attempts to maintain human spermatozoa without freezing were based mostly on short-term (up to 96 hours) storage in TEST-yolk buffer [Bolanos et al. 1983; Jaskey and Cohen 1981; Kesseru and Carrere 1984]. In this approach, sperm motility began to decline after 24 hours [Kesseru and Carrere 1984]
Riel et al.

4 25 4 25 4 25 4 25 4 25 4 25

46 34 67

3 9 5 3 11 9 29 35 55 43 58 47

0 2 2 1 1 1 12 24 42 24 24 20

0 0 0 0 0 0 10 13 18 11 4 4

0 0 0 0 0 0 12 0 5 9 1 1

n=a 0 0 0 0 0 8 8 0 0 0 0

n=a n=a n=a n=a n=a n=a 0 2 0 0 0 0

n=a n=a n=a n=a n=a n=a 0 0 0 0 0 0

56 68 78

Numbers shown represent % of motile or viable sperm. n=a not analyzed.

278

However, considering that sperm storage under such conditions is known to be detrimental to DNA and causes structural chromosome aberrations in humans [Munne and Estop 1993] and in mice [Munne and Estop 1991], we did not continue this path. Previous attempts demonstrated that human sperm stored in electrolyte-free solution for 1 week maintained much higher motility (43.4%) than sperm stored in modified human tubal fluid (HTF) medium (9.5%) [Quan et al. 2002]. This prompted us to focus on testing sperm maintained in an electrolyte-free environment. Previous attempts to preserve human sperm in electrolyte-free medium were directed towards the goal of repeated insemination. Although sperm motility was maintained for up to 4 weeks [Kanno et al. 1998; Saito et al. 1996], the total number of motile sperm after storage was not assessed. Compared to ICSI, inseminations and IVF require high numbers of sperm to achieve fertilization. Taking into consideration that the proportion of motile sperm after storage was very low, it is doubtful whether these spermatozoa would succeed in fertilizing oocytes in conventional IVF. Perhaps this is the reason why no follow up took place and there are no reports of the actual artificial insemination by donor, artificial insemination by husband, or IVF cases with sperm preserved in electrolyte-free solution available. Introduction of ICSI re-opened the chance to incorporate freeze-free sperm storage into assisted reproduction technology (ART) procedures. Intracytoplasmic sperm injection requires only one spermatozoon to fertilize one oocyte. Thus, even if the overall number of motile sperm is low, it is still sufficient for achieving fertilization. Although in this study the majority of ejaculates had relatively high sperm motility, we also examined two ejaculates with the initial motility below 15% and were able to find motile sperm after 1 week of storage. When we stored sperm with initial motility of 12%, we noted a motility decrease to 3% after 1 week. When the same semen was cryopreserved, sperm motility decreased to below 1%. Sperm viability in these poor quality samples was maintained longer than motility (Table 1). Immotile but viable sperm can be used for ICSI when used in conjunction with the hypoosmotic swelling test [Jeyendran et al. 1984]. Thus, we anticipate that sperm from men with severe asthenozoospermia can also be successfully
279

preserved in electrolyte-free solution for subsequent use in ICSI. To date no attempts have been made to evaluate the genetic integrity of either human or mouse sperm stored in electrolyte-free solution. The examination of sperm DNA is recognized as a measure of sperm quality that has diagnostic and prognostic capabilities, supplemental to standard sperm parameters [Agarwal and Said 2003]. Testing DNA integrity may help select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception. In turn, this may alleviate the financial, social and emotional problems associated with failed ART. Sperm chromatin is highly condensed and difficult to analyze. Only after fertilization does it unfold making the evaluation possible. Using human oocytes is impractical due to the low number of oocytes available and creates ethical problems. Rodent oocytes provide an alternative environment that unfolds human sperm chromatin making it accessible for analysis. For example, the hamster test was used to visualize human sperm chromosomes after IVF [Rudak et al. 1978; Yanagimachi et al. 1976] and provided insights into the frequency of structural sperm chromosome aberrations in infertile and fertile men [Martin et al. 1983; Martin and Rademaker 1987; Moosani et al. 1995; Rosenbusch et al. 1992]. When ICSI became available, human spermatozoa unable to fertilize in vitro were injected into oocytes from other species [Araki et al. 2004; Lee et al. 1996; Rybouchkin et al. 1995; Rybouchkin et al. 1996; Terada et al. 2004]. The ability to activate oocytes, from pronuclei, then condense into normal chromosomes was examined and subsequently used as a basis for recommendation for ART [Kim et al. 2001; Nardo et al. 2002; Zeyneloglu et al. 2002]. Here, we injected human spermatozoa preserved in electrolyte-free solution into mouse oocytes and demonstrated that there was no chromosome damage after 2 weeks of storage. The significance of this study for ART in humans is multifold. First, the new method offers scheduling flexibility. The male partner is not required to provide a semen sample the day of assisted fertilization. This is of special importance for men whose job requires long absences from home and for those who find it difficult to ejaculate under stress. Second, sperm preparation for storage in electrolyte-free solution is simple and less time consuming than
Preservation of Human Spermatozoa Without Freezing

FIGURE 5 The effect of cryopreservation on sperm motility and viability. Fresh and cryopreserved sperm from seven ejaculates (ejaculates: 3, 4, 6, 7, 9, 11, 12) were evaluated for motility (A) and viability (B). Fresh sperm motility assessments (all ejaculates) and fresh sperm viability assessments (ejaculates: 4, 6, 7, 12) were done within 1 hour after obtaining ejaculates. Fresh sperm viability assessments for ejaculate 9 and 11 were done after Percoll separation and for ejaculate 3 after Percoll separation and 4 days of storage in electrolyte-free solution.

cryopreservation. The method does not require any special investments. ART clinics can immediately incorporate this technology into their service. Moreover, maintaining sperm at 4C rather than in liquid nitrogen decreases the cost of storage. Third, the method offers an opportunity to collect several ejaculates over time, preserve without freezing, and pool sperm prior to fertilization. Such an approach would be of value for patients with severe oligozoospermia, when few spermatozoa can be found. Fourth, such preserved sperm samples are available for ICSI without any special pretreatments, just requiring a simple dilution with electrolyte-containing medium and a brief incubation at 37C. Finally,
Riel et al.

sperm preservation in electrolyte-free solution offers an alternative over conventional cryopreservation, and therefore eliminates the danger of cryodamage. Although cryopreservation is commonly used in human ART, it has been reported to result in a significant decrease in sperm quality and pregnancy rates [Alfredsson et al. 1983; Steinberger and Smith 1973]. In our experience, human sperm motility after cryopreservation decreased from 1.7 to 12 times, and there was great variability between individual ejaculates (Fig. 5). Loss of viability after cryopreservation in the same ejaculates ranged from 1.1 to 4.0 times (Fig. 5). Recently it was shown that in addition to the known cryopreservation effects, there are also hidden effects such as disturbed plasma membranes not detectable by supravital staining and changed patterns of intracellular enzyme activities [Glander and Schaller 2000]. It cannot be excluded that when low quality, fragile ejaculates (common to infertile men with spermatogenic defects) are cryopreserved, some DNA damage occurs. Such damage can result in pre- and post-implantation embryo loss, and decrease the chance of successful infertility treatment. We have shown that human spermatozoa can maintain motility for 4 weeks and viability for 6 weeks when stored in an electrolyte-free solution. After two weeks of storage, motile spermatozoa could be easily found in the sample and used for ICSI. Sperm DNA analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage did not negatively affect sperm chromosome integrity. Finally, when the same storage method was applied to mouse sperm, normal embryos and viable offspring were obtained after ICSI. The results from this study provide the clinicians working in ART with a new method that would simplify sperm preparation for ICSI for certain classes of patients.

MATERIALS AND METHODS Chemicals

Mineral oil was purchased from Squibb and Sons (Princeton, NJ) and pregnant mares serum gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise stated.
280

Animals
Mice were obtained at 6 weeks of age from the following vendors: B6D2F1 (C57BL=6J DBA=2) from National Cancer Institute (Raleigh, NC) and CD-1 mice from Charles River (Wilmington, MA). Mature oocytes for ICSI were obtained from 8 to 12 weeks old B6D2F1 females. Eight- to 16-week-old CD-1 females were used as surrogate mothers for embryo transfer. Sperm were obtained from 816-week-old B6D2F1 males. The mice were fed ad libitum with a standard diet and maintained in a temperature and light-controlled room (22C, 14 h light=10 h dark), in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and guidelines presented in National Research Councils (NCR) Guide for Care and Use of Laboratory Animals published by Institute for Laboratory Animal Research (ILAR) of the National Academy of Science, Bethesda, MD, 1996. The protocol for animal handling and treatment procedures was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.

CZB medium (HEPES-CZB), [Kimura and Yanagimachi 1995]. Sperm-injected oocytes and embryos were cultured in CZB medium [Chatot et al. 1989]. Human tubal fluid (HTF) medium [Quinn et al. 1985] was as used to revitalize human sperm samples after storage in the electrolyte-free solution and to resuspend sperm after cryopreservation. CZB and HTF media were maintained in an atmosphere of 5% CO2, and HEPES-CZB in air.

Sperm Storage in Electrolyte-Free Solution

The method of sperm preparation for storage in electrolyte-free solution was adapted from that described [Saito et al. 1996]. Briefly, an electrolytefree, three-layer discontinuous Percoll gradient was prepared in a 15 mL conical tube. The gradient consisted of: 1 mL of 66% (vol=vol) Percoll, 0.33 M glucose, 0.5% BSA; 4 mL of 44% (vol=vol) Percoll, 0.33 M glucose, 1.5% BSA; and 22% (vol=vol) Percoll, 0.33 M glucose, 2.5% BSA. One milliliter of liquefied human semen or epididymal mouse sperm suspended in HEPES-CZB was placed on the top of the Percoll gradient, and the column was centrifuged for 20 minutes at 400 g at room temperature. After centrifugation, the pelleted sperm were resuspended in 0.2 mL of 0.33 M glucose containing 3% BSA. Ten mL aliquots of sperm suspension were transferred into 0.2 mL tubes, and the tubes were immediately placed in a refrigerator. To revitalize sperm sample an equal volume (10 mL) of electrolyte-containing HTF medium was added to the tube, and sperm were incubated for 10 minutes at 37C. The analysis of sperm motility and viability was carried out immediately after incubation.

Human Sperm Samples

Sperm samples in this study were discarded ejaculates after semen analysis. The ejaculates were obtained from twelve men presenting for infertility treatment at the Pacific IVF Institute at Kapiolani Medical Center, Honolulu. Hawaii. Basic seminological analysis was performed according to the WHO criteria for normal semen [World Health Organization 1999]. All ejaculates had normal sperm number (> 20 106) and morphology (> 30% normal forms). In eight ejaculates sperm motility was below the norm (< 50% of motile sperm) and these ejaculates were classified as asthenozoospermic. Two had motility below 15% and were classified as severely asthenozoospermic. Each ejaculate was assigned an ID number (listed in Table 1). The ejaculates were processed on discontinuous density gradient and stored in electrolyte-free solution at 4C for up to 6 weeks, and used for assays. A portion of some ejaculates prior to Percoll separation was also cryopreserved to test the effect of cryopreservation on sperm motility and viability.

Sperm Cryopreservation
Sperm were cryopreserved in a cryoprotectant routinely used by our group for human sperm cryopreservation (100 mM NaCl, 5.365 mM KCl, 2.721 mM CaCl2, 0.492 mM MgCl2 6H2O, 12.856 mM sodium lactate, 0.321 mM NaH2PO4 2H2O, 30.949 mM NaHCO3, 20 mM Hepes, 50 mM sucrose, 5.506 mM glucose, 50 U Penicillin, 50 mg Streptomycin, 15% glycerol, 0.4% human serum albumin; pH 7.3). Aliquots of liquefied semen were mixed 1:1 with
Preservation of Human Spermatozoa Without Freezing

Media
Oocyte collection and subsequent manipulation, including microinjections used in HEPES-buffered
281

cryoprotectant and transferred into cryovials. Semen samples were slowly frozen in liquid nitrogen vapors for 15 minutes and then stored in liquid nitrogen. To thaw, samples were transferred from liquid nitrogen to a room temperature water bath until melted, and then incubated for 10 minutes at 37C. To remove cryoprotectant, sperm samples were washed (1000 g, 10 minutes, room temperature) and sperm pellets were resuspended in HTF medium. Sperm samples were then incubated at 37C for 10 minutes then immediately processed for motility and viability assessment.

Collection of Mouse Epididymal Sperm

The caudae epididymides were removed and the epididymal fluid recovered by squeezing on the bottom of the 1.5 mL tube containing 1.2 mL of HEPESCZB. Spermatozoa were allowed to swim up for 5 minutes at room temperature and 1 mL was collected from the top and used for Percoll separation.

Intracytoplasmic Sperm Injection (ICSI)

ICSI was performed as described by Szczygiel and Yanagimachi [2003], with modifications for human sperm. A small drop of revitalized sperm suspension was mixed thoroughly with an equal volume of HEPES-CZB containing 12% (w=v) polyvinylpyrrolidone (PVP, Mr 360 kDa) immediately before ICSI. Injections were performed using Eppendorf Micromanipulators (Micromanipulator TransferMan, Eppendorf, Germany) with a Piezo-electric actuator (PMM Controller, model PMAS-CT150, Prime Tech, Tsukuba, Japan). For injecting human sperm, the injection pipette had an external diameter 6 mm and for mouse ICSI 7.58.0 mm. A single spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. When injecting human sperm one Piezo pulse was applied to induce some membrane damage that immobilized sperm and facilitated further sperm decondensation inside the oocyte. The entire human spermatozoon was immediately injected into an oocyte. During mouse sperm injection the head was separated from the midpiece by applying one or more piezo pulses. After discarding the midpiece and tail, the head was redrawn into the pipette and injected immediately into the oocyte. ICSI was performed in HEPES-CZB within 1 hour of oocyte collection and sperm revitalizing. Sperm-injected oocytes were transferred into CZB medium for culture. The oocytes were examined $ 6 hours after ICSI to assess survival and activation. The oocytes with two well-developed pronuclei and a distinct second polar body were recorded as activated. Activated oocytes were used for chromosome analysis (human sperm) or for culture and embryo transfer (mouse sperm).
282

Sperm Motility and Viability Analysis

Sperm motility analysis was performed according to the protocol recommended by WHO [World Health Organization 1999]. Our goal was to use preserved sperm for ICSI, not for IVF or insemination. Thus, the total number of motile sperm was defined rather than differentiating between a, b, c, and d grades of motility. Sperm motility was used as a marker of unquestionably live sperm, and not a factor enabling successful fertilization. The motility was assessed manually by an experienced sperm analyst. Three independent scorings per sample were done and the final result was expressed as the mean. Sperm viability was assessed using Live=Dead Sperm Viability Kit (Invitrogen Molecular Probes, Chicago, IL; cat. no L-7011). Viable and non-viable sperm were differentiated based on their ability of binding SYBR 14 and Propidium Iodide. Live sperm with intact cell membranes fluoresced bright green while cells with damaged cell membranes fluoresced red.

Oocyte Collection
Female mice were induced to superovulate with injections of 5 iu eCG and 5 iu hCG given 48 h apart. Oviducts were removed 1415 hours after the injection of hCG and placed in PBS, in a Petri dish. The cumulus-oocyte complexes were released from the oviducts into 0.1% of bovine testicular hyaluronidase (300 USP units=mg) in HEPES-CZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with HEPES-CZB medium and used immediately for ICSI.
Riel et al.

Sperm Chromosome Analysis

Fertilized oocytes were transferred after 68 hours of culture in CZB containing 0.006 mg=mL vinblastine. Vinblastine was added to inhibit syngamy. Between 19 and 21 hours after ICSI the oocytes were treated with 1% pronase (1000 tyrosine units=mg, Kaken Pharmaceuticals, Tokyo, Japan) for 5 minutes at room temperature to soften the zonae pellucidae. Then the oocytes were treated with hypotonic solution (1:1 mixture of 1% sodium citrate and 30% fetal bovine serum) for 5 minutes at 37C or 10 minutes at 25C. Chromosomes were spread on to clean glass slides by the gradual fixation=airdrying method [Mikamo and Kamiguchi 1983]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in PBS (pH 6.8) for 10 minutes for chromosome analysis. Human sperm chromosomes were easily distinguished from the mouse oocyte chromosomes on the basis of their morphology. We previously used 1-cell embryo chromosome analysis as a measure for sperm genetic integrity [Kusakabe et al. 2001; Szczygiel et al. 2002a].

significance was noted when at least one of the three tests showed P 0.01 or P 0.05. The computations were done using KyPlot version 2.0 beta 13 software (developed by Koichi Yoshioka and available online: http://www.woundedmoon.org/win32/kyplot.html).

REFERENCES
Agarwal, A. and Said, T. M. (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9:331345. Alfredsson, J. H., Gudmundsson, S. P. and Snaedal, G. (1983) Artificial insemination by donor with frozen semen. Obstet Gynecol Surv 38:305313. Araki, Y., Yoshizawa, M., Abe, H. and Murase, Y. (2004) Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection. Zygote 12:111116. Bolanos, J. R., Overstreet, J. W. and Katz, D. F. (1983) Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 degrees C to 5 degrees C. Fertil Steril 39:536541. Chatot, C. L., Ziomek, C. A., Bavister, B. D., Lewis, J. L. and Torres, I. (1989) An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 86:679688. Darszon, A., Acevedo, J. J., Galindo, B. E., Hernandez-Gonzalez, E. O., Nishigaki, T., Trevino, C. L., Wood, C. and Beltran, C. (2006) Sperm channel diversity and functional multiplicity. Reproduction 131: 977988. Glander, H. J. and Schaller, J. (2000) Hidden effects of cryopreservation on quality of human spermatozoa. Cell Tissue Bank 1:133142. Jaskey, D. G. and Cohen, M. R. (1981) Twenty-four to ninety-six-hour storage of human spermatozoa in test-yolk buffer. Fertil Steril 35:205208. Jeyendran, R. S., Van Der Ven, H. H., Perez-Pelaez, M., Crabo, B. G. and Zaneveld, L. J. (1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 70:219228. Kanno, H., Saito, K., Ogawa, T., Takeda, M., Iwasaki, A. and Kinoshita, Y. (1998) Viability and function of human sperm in electrolyte-free cold preservation. Fertil Steril 69:127131. Kesseru, E. and Carrere, C. (1984) Duration of vitality and migrating ability of human spermatozoa cryopreserved at 4 degrees C. Andrologia 16:429433. Kim, S. T., Cha, Y. B., Park, J. M. and Gye, M. C. (2001) Successful pregnancy and delivery from frozen-thawed embryos after intracytoplasmic sperm injection using round-headed spermatozoa and assisted oocyte activation in a globozoospermic patient with mosaic Down syndrome. Fertil Steril 75:445447. Kimura, Y. and Yanagimachi, R. (1995) Intracytoplasmic sperm injection in the mouse. Biol Reprod 52:709720. Kusakabe, H., Szczygiel, M. A., Whittingham, D. G. and Yanagimachi, R. (2001) Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci U S A 98:1350113506. Lee, J. D., Kamiguchi, Y. and Yanagimachi, R. (1996) Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Human Reproduction 11:19421946. Martin, R. H., Balkan, W., Burns, K., Rademaker, A. W., Lin, C. C. and Rudd, N. L. (1983) The chromosome constitution of 1000 human spermatozoa. Hum Genet 63:305309. Martin, R. H. and Rademaker, A. W. (1987) The effect of age on the frequency of sperm chromosomal abnormalities in normal men. Am J Hum Genet 41:484492. Martin, R. H., Templado, C., Ko, E. and Rademaker, A. (1990) Effect of culture conditions and media on the frequency of chromosomal

Mouse Embryo Culture and Transfer

After ICSI, the oocytes were placed in 50 mL drops of CZB medium pre-equilibrated overnight in humidified 5% CO2 air. The culture drops were contained in plastic culture dishes (Falcon, Bedford, MA) and overlaid with mineral oil. Cultured embryos were evaluated for developmental progress at 24, 48, 72 and 96 hours post fertilization. Some of the 2-cell stage embryos were transferred to the oviducts (5 to 10 per oviduct) of CD-1 females which had been exposed to a vasectomized male the night before. Live, full-term fetuses were retrieved via caesarian section at Day 20 of gestation. Caesarian section was performed to avoid offspring being cannibalized upon delivery and to assess the number of implantation sites to determine the extent of embryonic loss after implantation.

Statistics
Chi-Square, Likelihood Ratio, Fishers Exact Probability tests were used for analyzing all responses. Lack of statistical significance was reported when all tests gave P > 0.05. The presence of statistical
283

Preservation of Human Spermatozoa Without Freezing

abnormalities in human sperm chromosome complements. Mol Reprod Dev 26:101104. Mikamo, K, and Kamiguchi, Y. (1983) A new assessment system for chromosomal mutagenicity using oocytes and early zygotes of the Chinese Hamster. In: Radiation-Induced Chromosome Damage in Man, Inshihara, T. and Sasaki, M.S. (Eds.), New York: Alan R. Liss; pp 411432. Moosani, N., Pattinson, H. A., Carter, M. D., Cox, D. M., Rademaker, A. W. and Martin, R. H. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil Steril 64:811817. Munne, S. and Estop, A. (1991) The effect of in-vitro ageing on mouse sperm chromosomes. Hum Reprod 6:703708. Munne, S. and Estop, A. M. (1993) Chromosome analysis of human spermatozoa stored in vitro. Hum Reprod 8:581586. Nardo, L. G., Sinatra, F., Bartoloni, G., Zafarana, S. and Nardo, F. (2002) Ultrastructural features and ICSI treatment of severe teratozoospermia: Report of two human cases of globozoospermia. Eur J Obstet Gynecol Reprod Biol 104:4042. Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A. C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:1718. Quan, S., Zhou, H. K., Shuji, Y., Hisayo, N. and Toshihiro, A. (2002) Fertilizing capacity of human sperm preserved in cold electrolyte-free solution. Di Yi Jun Yi Da Xue Xue Bao 22:928930. Quinn, P., Kerin, J. F. and Warnes, G. M. (1985) Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 44:493498. Rosenbusch, B., Strehler, E. and Sterzik, K. (1992) Cytogenetics of human spermatozoa: correlations with sperm morphology and age of fertile men. Fertil Steril 58:10711072. Rudak, E., Jacobs, P. A. and Yanagimachi, R. (1978) Direct analysis of the chromosome constitution of human spermatozoa. Nature 274: 911913. Rybouchkin, A., Dozortsev, D., De Sutter, P., Qian, C. and Dhont, M. (1995) Intracytoplasmic injection of human spermatozoa into mouse oocytes: A useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa. Hum Reprod 10:11301135. Rybouchkin, A., Dozortsev, D., Pelinck, M. J., De Sutter, P. and Dhont, M. (1996) Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod 11:21702175.

Saito, K., Kinoshita, Y., Kanno, H., Iwasaki, A. and Hosaka, M. (1996) A new method of the electrolyte-free long-term preservation of human sperm at 4 degrees C. Fertil Steril 65:12101213. Saito, K., Kinoshita, Y., Yumura, Y., Iwasaki, A. and Hosaka, M. (1999) Effects of extracellular ions on the reactivation of human spermatozoa preserved in electrolyte-free solution. Andrologia 31:211215. Steinberger, E. and Smith, K. D. (1973) Artificial insemination with fresh or frozen semen. A comparative study. Jama 223:778783. Suzuki, K., Yanagida, K. and Yanagimachi, R. (1998) Comparison of the media for isolation and storage of round spermatid nuclei before intracytoplasmic injection [published erratum appears in J Assist Reprod Genet 1998 Jul;15(6):408]. J Assist Reprod Genet 15:154 157. Szczygiel, M. A., Kusakabe, H., Yanagimachi, R. and Whittingham, D. G. (2002a) Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa. Biol Reprod 67:12781284. Szczygiel, M. A., Kusakabe, H., Yanagimachi, R. and Whittingham, D. G. (2002b) Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: A study with various mouse strains. Biol Reprod 67:287292. Szczygiel, M. A. and Yanagimachi, R. (2003) Intracytoplasmic sperm Injection. In: Cold Spring Harbor Laboratory Press Book. Intracytoplasmic Sperm Injection. Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. R., (Eds). pp. 585597 Tateno, H., Kimura, Y. and Yanagimachi, R. (2000) Sonication per se is not as deleterious to sperm chromosomes as previously inferred. Biol Reprod 63:341346. Terada, Y., Nakamura, S., Morita, J., Tachibana, M., Morito, Y., Ito, K., Murakami, T., Yaegashi, N. and Okamura, K. (2004) Use of Mammalian eggs for assessment of human sperm function: Molecular and cellular analyses of fertilization by intracytoplasmic sperm injection. Am J Reprod Immunol 51:290293. World Health Organization, W. (1999) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Yanagimachi, R., Yanagimachi, H. and Rogers, B. J. (1976) The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biology of Reproduction 15:471476. Zeyneloglu, H. B., Baltaci, V., Duran, H. E., Erdemli, E. and Batioglu, S. (2002) Achievement of pregnancy in globozoospermia with Y chromosome microdeletion after ICSI. Hum Reprod 17:18331836.

Riel et al.

284