Frequent structural chromosome aberrations in immotile human sperm exposed to culture media
Seiji Watanabe1
Department of Anatomy, Hirosaki University School of Medicine, 5 Zaifucho, Hirosaki 036-8562, Japan
1 To whom correspondence should be addressed. e-mail: sage{at}cc.hirosaki-u.ac.jp
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Abstract
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BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbeccos phosphate-buffered saline) for 1.52.5 h at 18°C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozenthawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.332.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozenthawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.
Key words:
chromosome aberrations/ICSI/immotile sperm/mouse oocytes
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Introduction
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ICSI is the only means of treatment for infertile patients showing severe asthenozoospermia. Numerous pregnancies and healthy births have been reported using intracytoplasmic injection of immotile sperm (Kahraman et al., 1996
; Nijs et al., 1996
; Barros et al., 1997
; Vandervorst et al., 1997
; Ved et al., 1997
; Wang et al., 1997
; Nagy et al., 1998
; Sallam et al., 2001
), although cases have also been reported that failed to obtain fertilized oocytes despite repeated attempts. Moreover, it is known that the fertilization and pregnancy rates are lower than for ICSI using motile sperm (Nagy et al., 1998
; Shulman et al., 1999
). Frequent structural chromosomal abnormality is one of the explanations for the problems of ICSI using immotile sperm. Rybouchkin et al. (1997)
conducted chromosome analysis of immotile sperm from infertile patients and demonstrated that the incidences of structural chromosome aberrations were in proportion to the incidences of dead immotile sperm. In contrast, when our group attempted a preliminary cytogenetic analysis of immotile human sperm obtained from a healthy man, there was no significant increase in structural chromosome aberrations as compared with motile sperm (S.Watanabe, unpublished data). This observation may have resulted from the fact that the immotile sperm were obtained from a fertile donor, but we hypothesized that the difference in the results between the previous report and this observation could be attributed to whether immotile sperm were exposed to culture media before microinjection. To confirm this, in this study immotile sperm were cytogenetically analysed using injection into mouse oocytes before and after exposure to several kinds of culture media.
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Materials and methods
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Collection of mouse oocytes
B6D2F1 female mice, 611 weeks old, were induced to superovulate by i.p. injection of 7 IU pregnant mares serum gonadotrophin followed by i.p. injection of 7 IU HCG 48 h later. Oocytecumulus cell complexes were collected from oviducts 16 h after the HCG injection. The oocytes were freed from cumulus cells by 5 min treatment of 0.1% hyarulonidase dissolved in HEPES-CZB medium (Kimura and Yanagimachi, 1995
) containing 0.3% bovine serum albumin (BSA; Sigma, Carlsbad, CA, USA). The oocytes were washed twice with fresh HEPES-CZB medium and were then incubated up to 3 h in CZB medium containing 0.3% BSA (Chatot et al., 1989
; 1990
) at 37°C, under 5% CO2 in air before micromanipulation.
Collection of human sperm
Human semen samples were obtained from two fertile donors (donor I and II) showing normozoospermia according to WHO criteria (WHO, 1999
). All the semen samples from donor I were liquefied for 30 min at 37°C in air, and were immediately used for ICSI. All the samples from donor II were used after cryopreservation according to a previously described method (Watanabe and Kamiguchi, 2001a
). During the experiment, the fresh or frozen sperm samples were pretreated according to the manner shown in Table I.
Preparation of injection chamber
For ICSI, an injection chamber was prepared by placing four kinds of medium droplets on a cover of 10-cm plastic dish and covering them with mineral oil. The first was a sperm droplet (5 µl) for sperm selection. The second droplet (5 µl) was seminal plasma or one of four kinds of culture media [HEPES-BWW, HEPES-BWW modified with 20 mmol/l NaCl, Dulbeccos phosphate-buffered saline (PBS), modified-human tubal fluid (HTF)] for sperm storage. HEPES-BWW modified with 20 mmol/l NaCl, modified-HTF and Dulbeccos PBS will hereafter be abbreviated as mBWW, mHTF and PBS, respectively. Compositions of the four kinds of media are shown in Table II. The third droplet (5 µl) was 10% polyvinylpyrrolidone (PVP) dissolved in PBS for sperm immobilization. The fourth droplet (20 µl) was HEPES-CZB containing 0.3% BSA for mouse oocytes. The injection chamber was placed on the microcool plate (Kitazato supply, Tokyo, Japan) of the inverted microscope with Hoffmans modulation contrast optics. The temperature of the microcool plate was maintained at 1718°C (Watanabe, 2003
).
Experiment 1: injection of initially immotile human sperm
Injection of immotile human sperm into mouse oocytes was performed as described previously (Watanabe, 2003
). To avoid contamination of the secondary immotile sperm, which probably resulted from centrifugation or dilution with culture media, liquefied fresh semen was used for the sperm droplet (the first droplet). The immotile human sperm were sucked into an injection pipette, and were then transferred into the 10% PVP droplet (the second droplet). As soon as the immotile sperm were freed from sticky seminal plasma as much as possible by repetitive gentle pipetting, a few Piezo-pulses were applied to the cells, taking into consideration that this process was indispensable to immobilization of the motile sperm used in a control group. The sperm were sucked into the injection pipette again and were then injected into mouse oocytes that had been placed in the HEPES-CZB droplet (the fourth droplet). Within 2 h,
2030 immotile sperm were injected into mouse oocytes. As the control group, initially motile sperm were also injected into mouse oocytes in the same manner as for the immotile sperm.
Experiment 2: the effects of culture media on immotile human sperm
The immotile human sperm were injected into mouse oocytes after being exposed to culture media for 1.52.5 h. About 40 immotile sperm were transferred from the semen droplet into the medium droplet (HEPES-BWW, mBWW, mHTF or PBS). Along with the sperm,
40 pl of seminal plasma was unavoidably taken into the medium droplet, resulting in 120 times dilution. Then, the immotile sperm were stored for 1.52.5 h at 1718°C in air. After storage, the immotile sperm were subjected to the same immobilization procedures as motile sperm and injected into mouse oocytes in the manner described in Experiment 1. As a control group, the immotile sperm were injected into mouse oocytes after storage in seminal plasma, which was obtained by filtering fresh semen with 0.8 µm diameter membrane filter.
Experiment 3: the effect of centrifugation on immotile human sperm
In a previous ICSI study using immotile human sperm, semen samples were diluted and centrifuged along with culture media (Rybouchkin et al., 1997
). Using the same conditions, 6 ml of HEPES-BWW was added to semen sample (23 ml) and centrifuged at 700 g for 5 min. The sperm pellet was suspended in 1 ml of HEPES-BWW, and the sperm suspension was applied to a 080% continuous Percoll density gradient centrifugation procedure according to our previous report (Watanabe and Kamiguchi, 2001a
). After centrifugation at 600 g for 20 min, 1 ml of sperm suspension was collected from upper layer of the Percoll gradient column, where the proportion of the immotile sperm was higher than at the bottom. The sperm suspension was diluted with HEPES-BWW and centrifuged at 700 g for 5 min to remove Percoll. After the sperm pellet was resuspended in 1 ml HEPES-BWW, the suspension was placed in the injection chamber for the sperm droplet. ICSI was performed as described in experiment 1.
Experiment 4: injection of frozenthawed immotile human sperm
The frozen semen sample was thawed at 37°C. To remove cryoprotectant medium, the sample was centrifuged at 700 g along with 5 ml of seminal plasma that had been stored at 80°C after filtering with a membrane (0.8 µm). After centrifuging, 1 ml of seminal plasma was added to the sperm pellet. The sperm suspension was used for the sperm droplet. The following ICSI operation was carried out as described in experiment 1. The frozenthawed immotile human sperm washed with HEPES-BWW medium were also used for comparison.
Incubation of ICSI oocytes
After microinjection, ICSI oocytes were transferred into CZB medium containing 0.3% BSA and were covered with mineral oil in a 35-mm culture dish. After 6 h incubation at 37°C under 5% CO2 in air, the oocytes were transferred into CZB medium containing 0.006 µg/ml vinblastin to block karyogamy and spindle formation. When the ICSI oocytes reached first cleavage metaphase 1624 h after the microinjection, they were prepared for chromosome slides.
Preparation of chromosome slides
The ICSI oocytes at first cleavage metaphase were treated with 0.5% actinase E to remove zona pellucidae, and were then placed in the hypotonic solution (0.5% sodium citrate supplemented with 15% BSA) for 10 min at room temperature. Chromosome slides were prepared using the gradual fixation/air drying method (Mikamo and Kamiguchi, 1983
). Chromosome analysis was carried out twice after 2% giemsa and C-band staining (Figure 1).

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Figure 1. A typical C-banding pattern in human sperm (left) and an example of the structural chromosome aberrations observed in an immotile human sperm (right). The C-banding method stained thickly centromere regions of chromosomes. In the right photograph, a dicentric chromosome with two dark spots (arrow heads) and a chromosome fragment without a dark spot (arrow) are seen.
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Live and dead sperm viability test
The membrane integrity of immotile sperm was assessed by a live/dead sperm viability test (WHO, 1999
). The fresh semen samples from donor I were stained with 0.5% eosin solution for 30 s. Under the microscope,
100 sperm were observed, and the frequency of dead immotile sperm, which were stained red, was calculated.
Measurement of osmotic pressure of media
Osmotic pressures of HEPES-BWW, mBWW, mHTF, PBS and seminal plasma were measured with Osmometer Mark3 (Fiske Associates, Norwood, MA, USA).
Statistical evaluation
The
2-test was used and differences were considered significant at P < 0.05 level.
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Results
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Experiment 1
In the assessment of liquefied semen samples using a live/dead sperm viability test, 48% of immotile sperm were stained red, indicating that half of the immotile sperm were dead cells. None of the motile sperm from the same samples stained red. The success rate of ICSI is summarized in Table III. In group A, 239 mouse oocytes were injected with fresh immotile human sperm. The oocyte survival rate was 84.6% (202/239). Of the 202 oocytes that survived ICSI, 198 were successfully prepared for chromosome slides, but the rest burst in a hypotonic solution. Oocyte activation, which was assessed by the completion of the second meiosis resulting in the presence of mouse pronucleus (PN) or mitotic chromosomes, was recognized in 98.5% (195/198) of the oocytes prepared. The rest (1.5%) were not activated, showing meiotic mouse chromosomes along with swollen human sperm head (SH) or prematurely condensed human chromosomes (PCC). The oocytes activated were classified into three categories depending on the sperm transformation (Watanabe, 2003
). The majority (89.2%, 174/195) of them developed to first cleavage metaphase, where both human sperm and mouse oocyte chromosome complements were contained. In the second category (6.2%, 12/195), two or three PN were contained. In the third group (4.6%, 9/195), sperm remained SH or PCC, although mouse nuclei formed PN or mitotic chromosomes. In group B, 201 mouse oocytes were injected with the fresh motile human sperm. The oocyte survival rate (84.5%, 170/201) and the incidence of ICSI oocytes at the first cleavage metaphase (84.2%, 139/165) did not differ significantly from those in group A, suggesting that there was no relationship between sperm motility and activation or early development of ICSI oocytes.
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Table III. Activation and development of mouse oocytes injected with immotile or motile human sperm collected from the liquefied semen samples (donor I)
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The results of sperm chromosome analysis in ICSI oocytes at the first cleavage metaphase are shown in Table IV. There was no difference in the incidences of aneuploidy (1.3% versus 0.9%), diploidy (0% versus 0%) and structural chromosome aberrations (4.5% versus 3.4%) between the groups A and B, suggesting that the risk of chromosome aberrations did not differ between immotile and motile sperm.
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Table IV. Chromosome aberrations in immotile and motile human sperm collected from the liquefied semen samples (donor I)
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Experiment 2
The success rates of ICSI (Table V) and the results of cytogenetic analysis (Table VI) were compared among the immotile sperm exposed to culture media (groups C-F) or seminal plasma (group G) for 1.52.5 h. Only PBS (group F), the chemical composition of which is the simplest of the media used in this study, caused a significant decrease in the oocyte activation rate (from 97.9% to 90.2% P < 0.05) as compared with seminal plasma (group G). In all culture medium groups (groups C-F), the arrest of ICSI oocyte development was frequently caused at SH stage or polyPN stage, resulting in the decrease of the ICSI oocytes at first cleavage metaphase (from 81.3% to 83.6%). This result suggests that arrest of ICSI oocytes is attributable mainly to the exposure of sperm to culture media. The difference in osmotic pressure between the media used in this study did not influence the incidences of ICSI oocytes at the first cleavage metaphase. The results of cytogenetic analysis (Table VI) showed a significant increase in structural chromosome aberrations in groups C-E (P < 0.001) and an increasing tendency in group F (P = 0.07) compared with group G (seminal plasma).
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Table V. Activation and development of mouse oocytes injected with immotile human sperm that were exposed to culture media (donor I)
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Experiment 3
The effects of sperm centrifugation on the development of ICSI oocytes and sperm chromosomes are summarized in Tables VII and VIII, respectively. In group H, the incidence of ICSI oocytes at first cleavage metaphase was significantly lower (P < 0.01) than the incidence in group C (Table VII). The difference resulted from an increased incidence of ICSI oocytes arrested at the PN stage in group H. Also, the results of cytogenetic analysis (Table VIII) indicated a significant increase of structural chromosome aberrations (48.1%, P < 0.01) in group H as compared with group C (25.8%). This result suggests that immotile sperm were vulnerable to the centrifugation.
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Table VII. The effects of sperm centrifugation on the activation and development of mouse oocytes after ICSI (donor I)
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Table VIII. Chromosome aberrations in the immotile human sperm with or without centrifugation along with HEPES-BWW (donor I)
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Experiment 4
The cytological and cytogenetic data for the immotile sperm from frozenthawed semen samples are shown in Tables IX and X, respectively. When frozenthawed semen samples were used, centrifugation was necessary to remove the cryoprotective medium. When motile and immotile sperm were obtained from semen samples centrifuged along with seminal plasma, there was a tendency (P = 0.06) for a decrease in the incidence of ICSI oocytes to be found at first cleavage metaphase in the immotile sperm group (group J) compared with the motile sperm group (group I). When centrifugation of sperm was carried out along with HEPES-BWW, a significantly decreased incidence of ICSI oocytes at first cleavage metaphase was found with immotile sperm (group K, P < 0.005). Similarly, the difference in the incidences of structural chromosome aberrations was significant between groups I and K (P < 0.001), and an increasing tendency (P = 0.06) was seen between groups I and J (Table X). It is remarkable that the number of structural chromosome aberrations per sperm in group K was 12 times greater than in group J (2.595 vs 0.203), suggesting that sperm DNA lesions caused by centrifugation with HEPES-BWW were transformed into multiple structural chromosome aberrations.
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Table IX. Activation and development of mouse oocytes injected with frozenthawed immotile human sperm that were centrifuged with seminal plasma or HEPES-BWW (donor II)
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Table X. Chromosome aberrations in the frozenthawed immotile human sperm that were centrifuged with seminal plasma or HEPES-BWW (donor II)
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Discussion
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In this study, the mechanisms causing increases in structural chromosome aberrations in immotile human sperm were examined by confronting the sperm with culture media or centrifugation. The results of all experiments performed are summarized in Figure 2. In fresh samples from donor I, there was no difference in the incidences of chromosome aberrations between the motile (group A) and the immotile (group B) human sperm, which were handled without culture media and centrifugation before ICSI, although 48% of the immotile sperm stained red with eosin Y and were therefore presumed to be dead. However, when the immotile sperm were exposed to culture media before ICSI, the incidence of ICSI oocytes at first cleavage metaphase decreased, and the incidence of structural chromosome aberrations was significantly increased (compare groups C, D, E or F with group G). In addition, the detrimental effect of culture medium on the immotile sperm increased in combination with the centrifugation (compare groups C, G and H). In frozenthawed semen samples from donor II, a significant increase in structural chromosome aberrations in the immotile sperm was seen with the combination of centrifugation and HEPES-BWW medium (compare group J with K), although there was no opportunity to analyse fresh samples from donor II before cryopreservation. These results lead to the conclusion that the majority of immotile sperm from the two donors used in this study initially had no DNA legions, while the DNA of the immotile sperm were vulnerable to culture media. This conclusion indicates the possibility that the significant increase in structural chromosome aberration in immotile sperm from infertile patients that was reported previously (Rybouchkin et al., 1997
) was attributable to the fact that sperm samples were centrifuged along with culture medium. This suggestion is reinforced by a common observation between the present and previous studie; specifically, the incidence of structural chromosome aberrations in the immotile sperm centrifuged with HEPES-BWW (48.1%) is almost identical to the rate of dead cells (48%) obtained from a live/dead sperm viability test in this study, which is similar to the previous finding in immotile sperm from patients with severe asthenozoospermia (Rybouchkin et al., 1997
). Both results strongly suggest that the structural chromosome aberrations observed were mainly caused in dead immotile sperm, which have damaged plasma membranes. It is therefore highly probable that the majority of immotile sperm from infertile patients do not have DNA lesions initially, although further studies are needed in the immotile sperm from unwashed semen samples of infertile patients to prove this.

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Figure 2. The incidences of ICSI oocytes at first cleavage metaphase (gray bars) and sperm with structural chromosome aberrations (black bars) obtained from experiments 14. Refer to Table I for ICSI conditions in groups AH.
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This observation suggests that the hypo-osmotic swelling test (HOST), which distinguishes live from dead sperm, would cause an effective decrease in the risk of structural chromosome aberrations in ICSI using immotile sperm. However, it is probable that such a risk of aberrations increases even in immotile sperm with intact plasma membrane on exposure to the hypotonic solution used for HOST. In our previous ICSI study using motile sperm from donor I (Watanabe, 2003
), the incidence of structural chromosome aberrations was 8.8% (42/477) after centrifugation with HEPES-BWW. The value was significantly higher (P < 0.05, Fishers t-test) than the incidence (3.4%, 4/116) in the motile sperm that were not centrifuged and exposed to culture medium in this study (Table IV, group B), suggesting that some living sperm were damaged by centrifugation or culture medium. Using a fluorescence in situ hybridization method, Zeyneloglu et al. (2000)
directly examined nuclei of the immotile human sperm with intact plasma membranes that were selected by the HOST. Regrettably, however, the authors seem to have focused on the number of chromosome aberrations. Therefore, the risk of structural chromosome aberrations in immotile sperm with intact plasma membrane exposed to HOST medium is not clear. As a mechanism inducing DNA lesions in sperm with intact plasma membrane, Munne and Estop reported a decrease in the number of disulfoxide bonds in sperm nuclei, which corresponded to an increase in structural aberrations after incubation for 12 or 24 h (Munne and Estop, 1991
; 1993
).
Among the four kinds of culture media used for the storage of immotile human sperm (groups C-F), the osmotic pressure of mHTF was lowest (277 mOsm) and induced the most frequent structural chromosome aberrations in immotile human sperm (32.6%, group E). This result suggests that osmotic pressure may be one of the factors involved in an increase in structural chromosome aberrations in immotile human sperm. However, the role of water on the induction of structural chromosome aberrations does not seem to have been so important, because irrespective of whether or not the plasma membrane is intact, water moves into sperm whenever the cells are transferred into a more hypotonic condition. Moreover, structural chromosome aberrations in the immotile sperm that were exposed to mBWW were still frequent, even though there was no difference in osmotic pressure between mBWW and seminal plasma. On the other hand, if the sperm have a damaged plasma membrane, some chemicals seem to move into the cells down their concentration gradient along with water. Tateno et al. (2000)
reported that sodium ions caused DNA lesions in mouse sperm when their plasma membranes were artificially damaged by sonication. In the present study and that of Rybouchkin et al. (1997)
, DNA of the dead cells might have been selectively injured by sodium ions. However, mBWW and PBS, which contain a comparably higher sodium concentrations than mHTF (Table II), showed significantly lower incidences of structural chromosome aberrations compared with mHTF (Table VI, compare groups D, E and F). Moreover, no component showing distinct correlation with the incidences of structural chromosome aberrations was found by comparing the compositions of the media used (Table II). In addition to osmotic pressure and sodium ions, therefore, some factors may affect the incidences of structural chromosome aberrations in immotile sperm in a complex manner.
Paradoxically, the harmful effect of culture media described above shows that seminal plasma strongly inhibits the increase of DNA lesions in immotile human sperm. Barrios et al. (2000)
reported that in ram sperm, the cooling-induced damage of plasma membrane was reversed by
20 kDa seminal plasma proteins. It is still not known whether human seminal plasma contains the proteins responsible for the restoration of the plasma membrane. However, similar mechanisms may be implicated in the inhibition of DNA lesions in immotile human sperm. Consequently, injection of immotile sperm stored in seminal plasma seems to be useful to decrease the risk of fertilization of chromosomally aberrant sperm, if no motile sperm is obtained from the patients with severe asthenozoospermia for ICSI treatment. However, a drawback in the present study is that it is not certain whether seminal plasma was completely removed from sperm surface by repetitive pipetting in 10% PVP solution. The effect of seminal plasma on the oocyte development was not determinable. This problem has to be overcome before the technique of using unwashed immotile sperm can be included in the treatment of male infertility.
After cryopreservation, the incidence of structural chromosome aberrations was significantly higher in the immotile sperm (group I) than in the motile sperm (group J) from donor II (17.2% versus 4.0%), although such a significant difference was not observed between motile (group A) and immotile sperm (group B) from donor I before cryopreservation. Since frozenthawed semen from donor I or fresh semen from donor II was not analysable in this study, it cannot be ruled out that this inconsistency was dependent on differences between individual spermatogenesis of the donors used. However, the freezingthawing operation was possibly the main cause of the increase in structural chromosome aberrations in the frozenthawed immotile sperm. Even after addition of a cryoprotectant medium, it is inevitable that immobilization by ice crystals to some initially motile sperm occurs during the freezing operation. DNA of the accidentally immobilized sperm was probably damaged as DNA of the sperm unprotected with cryoprotectant medium was chipped by a freezing and thawing operation (Rybouchkin et al., 1996
).
In this study, it was concluded that almost all immotile sperm contained in ejaculates of fertile men were genetically normal, and that some of them lost their integrity due to culture media used for sperm preparation. It is unfortunate that the production of hybrid embryos between human and other species was legally prohibited in Japan before more donors were compared allowing us to assess the influence of different culture media and sperm preparation procedures. However, the present results from a large number of immotile sperm from two fertile donors is valuable, since no similar chromosome study has been reported since Rybouchkin et al. (1997)
attempted a cytogenetic analysis in
40 immotile spermatozoa from three infertile donors. Furthermore, sperm samples of one donor used in this study have been intermittently examined in previous reports (Watanabe and Kamiguchi, 2001a
; b
; Watanabe, 2003
), confirming that the incidences of chromosomally aberrant sperm were stable and within normal variations. This use of sperm from a well-studied donor gave extra validity to the present results and allowed a more comprehensive interpretation as described above.
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Acknowledgements
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I would like to express my sincere appreciation to Professor Y.Kamiguchi (Department of Biological Sciences, Asahikawa Medical college) for guiding me during this work. Thanks are also due to Professor T.Kachi for giving me the chance to prepare this manuscript. I am grateful to Mr S.N.Bayley for his assistance in preparing this manuscript. This study was supported by Grant-in-Aid for Scientific Research (B) (no. 10470339) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Submitted on September 16, 2003;
accepted on December 3, 2003.