Department of Anatomy, Hirosaki University School of Medicine, 5 Zaifucho, Hirosaki 0368562, Japan. e-mail: sage{at}cc.hirosaki-u.ac.jp
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Abstract |
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Key words: chromosome aberrations/human sperm/ICSI/mouse oocytes
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Introduction |
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Recently, the success rate of human sperm karyotyping was improved by combining the ICSI method of Kimura and Yanagimachi with the gradual fixationair-drying method, which is an efficient chromosome preparation technique for early mammalian embryos (Mikamo and Kamiguchi, 1983). In this assay system, furthermore, no ageing-related increase of chromosome aberrations occurs in either sperm or oocyte, providing the injection is finished within 3 h after oocyte collection (Watanabe and Kamiguchi, 2001a
). It therefore satisfies the conditions for efficient cytogenetic analysis of human sperm using ICSI. In this study, the aim was to determine the risk of chromosome abnormalities in micro-manipulated human sperm. To rectify the defect of sample size in the previous ICSI studies, therefore, a large quantity of fresh and frozenthawed human sperm were injected into mouse oocytes, and cytogenetically analysed in detail. Then, the result in the present ICSI study was compared with the result in our previous IVF study. At the same time, since mouse chromosome complements were also analysable in the 1-cell hybrid oocytes, the effects of micro-manipulation on female chromosome complements, for which information was not available, was additionally estimated.
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Materials and methods |
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Collection of human sperm
Human semen samples used for this study were obtained from a fertile donor showing normozoospermia according to World Health Organization (1999) criteria. Fresh semen was liquefied for 30 min at 37°C in air. The sample was washed twice by centrifugation (700 g for 5 min) along with 6 ml HEPESBiggersWhittenWhittingham medium containing 0.3% bovine serum albumin (BSA) (Watanabe and Kamiguchi, 2001a). After the sperm were suspended in 5% polyvinylpyrrolidone (PVP) dissolved in Dulbeccos phosphate-buffered saline (Dulbecco and Vogt, 1954
), the sperm suspension was placed in a manipulation chamber for sperm selection.
Some of the semen samples were used after being frozen and thawed (Kobayashi et al., 1991). The frozen samples were prepared in the same manner as the fresh samples after thawing at 37°C.
Preparation of manipulation chamber
For the injection of human sperm into mouse oocytes, a manipulation chamber was prepared by placing four kinds of droplets in a line on the cover of a 10 cm plastic dish (Falcon Plastics, USA) and covering them with mineral oil. The first droplet was 5 µl of 10% PVP for washing the injection pipette. The second droplet was 10 µl of sperm suspension. The third droplet was 5 µl of 10% PVP for the immobilization of sperm. The fourth droplet was 20 µl of HEPESCZB medium for the oocytes. The injection chamber was prepared immediately before use and placed on the cooling microplate (Kitazato supply, Tokyo) of the inverted microscope with Hoffmans modulation contrast optics. The temperature of the microplate was maintained at 1718°C during micro-manipulation (Kimura and Yanagimachi, 1995).
Injection of human sperm into mouse oocytes
Human sperm were injected into mouse oocytes using a piezo-driven micromanipulator according to Kimura and Yanagimachi (1995). Only motile human sperm with normal-shaped heads (35 µm in length and 3 µm in width; Menkveld et al., 1990
) were selected for ICSI, to exclude chromosome aberrations implicated in morphological abnormality or immotility. Motile sperm were transferred from the sperm droplet into the 10% PVP droplet, and piezo-pulses were applied a few times at the midpiece of the sperm to immobilize them. The sperm thus immobilized were injected into metaphase II mouse oocytes with the first polar body in the HEPESCZB droplet. Since the meiotic spindle of mouse oocytes was indicated by a hump in the cortex, it was possible to inject the sperm so as to prevent damaging this spindle (Kimura and Yanagimachi, 1995
). The hybrid oocytes were transferred into the culture dishes which had been prepared by placing CZB medium droplets (0.2 ml) on the 35 mm dish and covering them with mineral oil. All oocytes were used for ICSI within 3 h after collection.
Preparation of chromosome slides
The hybrid oocytes were incubated in the culture dish for 6 h and were then transferred into the CZB medium droplets (0.2 ml) containing 0.006 µg/ml vinblastin to block karyogamy and mitotic spindle formation. When the hybrid oocytes reached the first cleavage metaphase, 1624 h after the micro-injection, they were prepared for chromosome slides. After zona pellucida had been removed by 5 min treatment of 0.5% actinase E (Kaken Pharmac., Japan), the hybrid oocytes were treated with hypotonic solution (0.5% sodium citrate containing 15% BSA) for 10 min at room temperature. For the preparation of chromosome slides, the gradual fixationair-drying method was used (Mikamo and Kamiguchi, 1983). The analysis of the chromosome slides was carried out twice after successive staining with 2% Giemsa and C-banding (Figure 1). Those hybrid oocytes that contained one, two or three visible pronuclei 24 h after ICSI were also prepared in the same way.
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Results |
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Along with human sperm chromosome complements, 615 mouse oocyte chromosome complements were analysed to evaluate the effects of micro-manipulation on female chromosomes in this study (Table III). The incidence of mouse chromosome complements with aneuploidy was 4.0%. There was no discernible evidence of mouse chromosomes in 2.0% of the oocytes. The incidences of diploidy and structural chromosome aberrations were 5.4 and 1.3% respectively.
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Discussion |
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It has not been possible to estimate exactly the genetic constitution of human sperm injected into mouse oocytes, because the incidences and types of chromosomally abnormal sperm varied widely among the previous ICSI studies in the motile human sperm with morphologically normal heads (Table IV). This wide variation could be due to spontaneous variation in sperm from donor to donor, as found in a previous IVF study using hamster oocytes (Kamiguchi et al., 1994). However, infertility might also be one of the factors affecting the incidence of aberrant sperm, since the incidence of aneuploidy was comparably higher in an infertile donor (4.9%) who was used by Rybouchkin et al. (1997
) than fertile donors (0.02.4%) used by the other authors (Rybouchkin et al., 1996a
;b
; Lee et al., 1996
). Moreover, it is also possible that the small sperm number in the previous ICSI studies is the cause of this wide variation, although the sperm number was adequate in each case for the authors to elucidate the correlation between chromosome abnormalities and sperm phenotypes (morphology or motility). Rybouchkin et al. (1997
) actually detected no structural chromosome aberration in sperm (Table IV), although such a case was never found in the cytogenetic data of 51 fertile donors gathered by Kamiguchi et al. (1994
) using IVF between hamster oocytes. To settle the problem of small sample and obtain further information about the risk of chromosome aberrations in micro-manipulated human sperm, a detailed cytological and cytogenetic analysis was performed on a large number (a total of 618 cells) of human sperm injected into mouse oocytes in the present study. Before comparing this result with those in the previous ICSI studies (Table IV), it is important to determine whether our result using sperm from the same individual reflects typical normal variation, or results from spermatogenesis unique to the subject. Our previous IVF study on the sperm from this donor (Watanabe and Kamiguchi, 2001b
), which is shown in Table II, is a clue to help clarify this. The cytogenetic result in our IVF study is directly comparable with those from 51 donors in the IVF study of Kamiguchi et al. (1994
), since both studies were performed using the same method and the same equipment. According to Kamiguchi et al., sperm samples from 51 normal donors contained aneuploidy ranging from 0 to 4% (1.4 ± 1.0% average) and structural chromosome aberrations ranging from 3.6 to 24.6% (14.1 ± 4.1% average). In our IVF study, the incidences of those types of aberrations were 2.6 and 10.2% respectively and these values were within the range of normal variations of aneuploidy and structural chromosome aberrations shown by Kamiguchi et al. (1994
). Therefore, it is concluded that there is no variation caused by individual characteristics in the spermatogenesis of the particular donor.
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In this study, no diploidy was found in 618 motile human sperm with normal heads which were obtained from a fertile donor. The same was also true of the previous ICSI studies (Table IV). Paradoxically, this fact strongly suggests the correlation between diploidy and sperm head abnormality. It is easy to think of large-headed sperm as the morphological abnormality related to diploidy. However, Lee et al. (1996) reported that there were no diploid cells in 11 large-headed human sperm injected into mouse oocytes. Furthermore, Calogero et al. (2001
) did not refer to the correlation between diploidy and large sperm head in the semen samples from the patients with oligoasthenoteratozoospermia, which show a significantly higher incidence of diploid sperm in a fluorescence in-situ hybridization method, and the average rate of diploidy among the patients was extremely low (0.09%). These findings indicate that large sperm heads do not necessarily contain a diploid nucleus, although this does not mean that diploid sperm have large heads. On the other hand, the possibility has been reported that a diploid sperm without any morphological abnormality was contained in ejaculate from patients with oligoteratozoospermia (Rosenbush et al., 1998
). Therefore, diploidy may not be implicated in this particular morphological abnormality.
In the present study, the first attempt at cytogenetic analysis of mouse chromosome complements was made after ICSI into mouse oocytes. These data were compared with the data in previous mouse in-vivo and in-vitro studies (Table V) to estimate the risk of chromosome abnormalities in female chromosome complements after micro-manipulation. The incidences of structural chromosome aberrations in female chromosome complements did not differ between our ICSI study and the previous studies. This result may be explained by the fact that the meiotic spindle of mouse oocytes locates right under the cortex where a hump is formed (Kimura and Yanagimachi, 1995). Since the hump was easily confirmed under a light microscope, human sperm could be injected into the mouse oocyte without any direct damage to mouse meiotic chromosomes. The aneuploidy rate in this study (4.0%) was significantly higher than the rates reported by Fraser and Maudlin (1979
) (0.9 or 1.5%, P < 0.001) and higher (but not significantly so) than the rate reported by Martin-Deleon and Boice (1983
) (2.2%). Santalo et al. (1986
) did not count hypoploidy cells. However, when the rate in in-vitro study of them (1.2%) is doubled on the assumption that hyper- and hypoploidy are induced at 1:1 ratio, the resultant rate (2.4%) is lower (but not significantly so) than our rate (4.0%). Remarkably, ICSI oocytes with no mouse chromosomes accounted for a half of our aneuploidy rate (Tables III and V). When the rate of this type of aberration (2.0%) was subtracted from the overall rate of aneuploidy (4.0%), the remainder (2.0%), which consisted of hyper- and hypoploidy, did not differ from the aneuploidy rates in the previous studies. This type of aberration, to which none of the authors has referred in the previous reports shown in Table V, is probably induced by micro-manipulation. The most probable mechanism causing the loss of female chromosome complements is extrusion of the whole meiotic chromosomes into the second polar body. The diploidy rate was also significantly higher in ICSI than in-vivo and in-vitro fertilization (5.4 versus 0.32.5%, P < 0.01), suggesting that micro-manipulation induces diploidy in female chromosome complements. Since the MII mouse oocytes with the first polar body were selected for injection in this study, the diploid mouse chromosome complements must have been attributed to failure to expel the second polar body. Macas et al. (1996
) has reported that most human multipronuclear ICSI oocytes contained the diploid female chromosome complements which resulted from failure to extrude the second polar body. Although the number of pronuclei was not counted before chromosome preparation in this study, six of 33 diploid mouse oocytes contained two individual mouse metaphases which probably originated from two individual pronuclei. Moreover, 19 tripronuclear hybrid oocytes were found in 59 hybrid oocytes arrested at the pronuclear stage after injection of the fresh or frozenthawed human sperm (Table I). It is therefore considered that multipronuclei were formed in some ICSI mouse oocytes owing to failure to extrude the second polar body. Failure of the second meiosis described above indicates that micro-manipulation has a harmful effect on the oocyte cytoskeleton system. Macas et al. (1996
) proposed the possibility that external calcium ions injected along with sperm disorganized microtubules constructing the meiotic spindle. This may be the cause of frequent hyper- and hypoploidy in human tripronuclear ICSI oocytes observed by Macas et al. (1996
), since an increase of hyper- and hypoploidy has been reported in hamster primary oocytes exposed to colchicine, suggesting that depolymerization of microtubules is implicated in the induction of hyper- and hypoploidy (Sugawara and Mikamo, 1980
). Moreover, Harderson et al. (2000
) examined meiotic spindles of human oocytes visualized with microtubule immunostaining, and found that the position of the meiotic spindle cannot be predicted by the location of the first polar body. However, human sperm are traditionally injected into human oocytes on the assumption that meiotic spindles are located under the cortex close to the first polar body, although the spindles are actually invisible under the light microscope. Hence, there is the possibility in human oocytes that the meiotic spindle is exposed to calcium ions originating from the injection pipette, the tip of which is inserted close to the spindle. In contrast to the result in human ICSI oocytes, no significant increase of hyper- and hypoploidy was observed in mouse chromosome complements, although the complete loss of mouse chromosome complements was frequently induced in this study. Similarly, the incidence of diploidy was extremely low in hamster primary oocytes exposed to colchicine. Moreover, the possibility that the meiotic spindle was exposed to calcium ions seems to be comparably low in mouse oocytes, the spindles of which could easily be identified under a microscope. These facts suggest that the induction of diploidy may be attributed to a different mechanism from the one inducing hyper- and hypoploidy. The possible explanation may be that the stretching of the oocyte plasma membrane by micro-injection disturbs the microfilament network underneath oocyte plasma membrane, which plays a role in the formation of the contractile ring where the site of cleavage is determined (White and Borisy, 1983
; Rappaport, 1986
; Tolle et al., 1987
). To resolve this problem, further experiments on the alteration of the cytoskeleton network during the extrusion of the second polar body are needed.
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In this study, detailed cytogenetic data were collected from a large number of human sperm using ICSI. On the basis of the results, further detailed investigations are being conducted to assess the risk of chromosome abnormalities in the morphologically abnormal human sperm and immotile human sperm, for which information remains inadequate.
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Acknowledgements |
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References |
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Submitted on May 7, 2002; resubmitted on January 17, 2003; accepted on February 3, 2003.