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BOR - Papers in Press, published online ahead of print April 4, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.057778
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BIOLOGY OF REPRODUCTION 77, 336–342 (2007)
DOI: 10.1095/biolreprod.106.057778
© 2007 by the Society for the Study of Reproduction, Inc.

Evaluation of Chromosomal Risk Following Intracytoplasmic Sperm Injection in the Mouse

Hiroyuki Tateno 1, and Yujiroh Kamiguchi 

Department of Biological Sciences, Asahikawa Medical College, Asahikawa 078-8510, Japan

ABSTRACT

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5–2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2–2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%–28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.

assisted reproductive technology,, gamete biology,, in vitro fertilization,, sperm,, toxicology

INTRODUCTION

Intracytoplasmic sperm injection (ICSI) into mouse metaphase II (MII) oocytes has enabled us to observe chromosomes of immotile (dead) mouse [1–7], whale [8], and human [9–11] spermatozoa, as well as morphologically abnormal mouse [12, 13] and human spermatozoa [14, 15], although the conventional in vitro fertilization (IVF) technique is not traditionally used for such purposes. These studies have yielded useful information on the chromosome damage of such defective spermatozoa. However, risk factors of generating chromosome aberrations occurring with ICSI are still a concern. As far as the results of chromosome analysis of mouse one-cell embryos produced by ICSI technique with normal (control) spermatozoa have shown [3–6, 13, 16], the incidence of embryos with structural chromosome aberrations (4%–9%) is high compared with one-cell embryos produced by conventional IVF technique (1%–2%) [17–21], although the genotypes of the IVF embryos are not identical to those of the ICSI embryos. Nevertheless, conclusive evidence of the increased incidence of chromosome aberrations associated with ICSI technique should be based on results of systematic investigation, because oocyte genotypes have a decisive impact on the repair of DNA lesions in spermatozoa [22]. Furthermore, chromosomal technique and criteria for chromosome aberrations can influence the incidence of chromosome aberrations [23, 24].

In this study, chromosomes of one-cell mouse embryos produced by conventional IVF and ICSI techniques were analyzed. In ICSI experiments, spermatozoa were incubated in five different media for various times before being injected into MII oocytes to determine in vitro sperm chromosome aberrations. Based on the chromosome analysis results of one-cell embryos, an attempt was made to examine the developmental potency of embryos and analyze the chromosomes of live fetuses developed from these embryos.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Nacalai Tesque Inc. (Kyoto, Japan) unless specifically stated. Lipid-rich BSA (AlbuMax; GibcoBRL, Auckland, New Zealand) was used instead of conventional fraction V albumin. Polyvinyl alcohol (PVA; cold water soluble; Sigma-Aldrich, St. Louis, MO) was appropriately used throughout the experiments. Enzymes included bovine testicular hyaluronidase (Type S-I; Sigma-Aldrich) and protease (commercially available as actinase E; Kaken Pharmaceuticals, Tokyo, Japan). Hyaluronidase was dissolved in Hepes-buffered modified CZB (mentioned later as H-mCZB) at a concentration of 0.1% (w/v), subdivided into a small volume (200 µl) in microtubes, and stored at –20°C. Protease was prepared in Ca2+ and Mg2+-free Dulbecco PBS at a concentration of 0.5% (w/v), filtrated with syringe filters (pore size of 1.2 µm), subdivided into 5-ml plastic tubes, and stored at –20°C. Vinblastine sulfate (Sigma-Aldrich) was used as an antimitotic agent, dissolved in distilled water at concentration of 4 µg/ml, and then stored in a refrigerator. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich. Antibiotics and paraffin oil (Art. 1.07162.1000) were from Meiji Seika (Tokyo, Japan) and from Merck Japan (Tokyo, Japan), respectively.

In fetal skin cell cultures, FBS was the product of Cell Culture Technologies (Lugano, Switzerland). Colcemid (GibcoBRL) was used as an antimitotic agent for fetal chromosome analysis.

Media

Five different media—TYH [25], Hepes-buffered TYH (H-TYH), CZB [26] modified by addition of 5.56-mM D-glucose (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 [27]—were used for sperm preparation. The media formulations are shown in Table 1. The pH of both H-TYH and H-mCZB were adjusted to approximately 7.4 through the addition of 1 N HCl. H-TYH, H-mCZB, and PB1 media were used under 100% air, and TYH and mCZB media were used under 5% CO2 in air. Medium for skin cell culture of fetuses was Dulbecco modified Eagle medium (D-MEM; Sigma-Aldrich).


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  		TABLE 1. Media used for preparation of spermatozoa and oocytes, and culture of embryos.

Animals

Hybrid mice B6D2F1 (C57BL/6Cr x DBA/2Cr; Japan SLC Inc., Hamamatsu, Japan) were used to collect oocytes and spermatozoa. Random-bred CD-1 (ICR) females mated with vasectomized males of the same strain were used as embryo recipients. They were maintained in a temperature- and light-controlled room (23 ± 2°C, 14 h light from 0500 h to 1900 h). Food and water were given ad libitum. All experiments were performed according to the Guidelines for Animal Experiments of Asahikawa Medical College.

Preparation of Oocytes and Spermatozoa for ICSI

Female mice 7–12 wk of age were superovulated by an intraperitoneal injection of 8–10 IU eCG (Teikokuzoki Pharmaceuticals, Tokyo, Japan), followed 48 h later with an injection of 8–10 IU hCG (Mochida Pharmaceuticals, Tokyo, Japan). Approximately 16 h after hCG injection, oocytes at MII were collected from the oviducts. They were freed from cumulus cells by treatment with 0.1% hyaluronidase for 3–5 min at room temperature. The cumulus-free oocytes were washed with mCZB and temporarily kept in a droplet (100 µl) of mCZB medium under paraffin oil at 37°C.

Mature spermatozoa were collected from the cauda epididymides of male mice 7–12 weeks of age and were incubated at concentration of 2 to 4 x 107/ml per droplet (100 µl) of five different media under paraffin oil at 37°C for 0.5 h, 2–2.5 h, or 6 h. Spermatozoa highly maintained their motility in these media even after 6 h of incubation.

ICSI Procedure

A small amount (2–3 µl) of the above sperm suspension was transferred into a droplet (approximately 10 µl) of medium containing 10%–12% PVP under paraffin oil, which was prepared in a plastic chamber (a cover of plastic dish; 100 mm in diameter; Falcon). When spermatozoa were incubated in either TYH or H-TYH medium, they were transferred into a droplet of H-TYH medium containing PVP. Once spermatozoa were incubated in either mCZB or H-mCZB medium, they were transferred into a droplet of H-mCZB medium containing PVP. The spermatozoa that had been incubated in PB1 medium were then transferred into a droplet of PB1 medium containing PVA and PVP instead of BSA.

Just before ICSI, oocytes were transferred from mCZB medium into a droplet (approximately 10 µl) of H-mCZB medium under the paraffin oil. This droplet was set aside, with the droplet for spermatozoa in the same plastic chamber. A highly motile spermatozoon with a morphologically normal head was selected, and the head was separated from the tail by applying a few piezo pulses. Immediately, the sperm heads were individually injected into oocytes using piezo micromanipulator, as described by Kimura and Yanagimachi [28]. In every operation, 9–25 oocytes were used, and the injection of sperm heads into these oocytes was finished within 30 min. Within approximately 1 h, the operation was run two or three times. Following the injection of sperm heads, oocytes were washed thoroughly with mCZB medium and transferred into a droplet (100 µl) of the same medium under the paraffin oil at 37°C for cultivation.

Procedure of Conventional IVF

As described earlier, mature spermatozoa were collected from the cauda epididymides. Spermatozoa were incubated in a droplet (100 µl) of TYH medium under the paraffin oil for 1.5–2 h to allow them to capacitate. Cumulus masses, including oocytes, were retrieved from the oviducts of superovulated females and transferred into a droplet (100 µl) of TYH medium under the paraffin oil. The cumulus masses were immediately inseminated with spermatozoa at approximately 105/ml and kept for 2 h at 37°C. The oocytes were adequately washed with mCZB medium and cultured in a droplet of the same medium under the paraffin oil at 37°C.

Chromosome Preparation and Analysis of One-Cell Embryos

Six to eight hours after ICSI or IVF procedure, eggs were transferred into a droplet of mCZB medium containing 0.02 µg/ml vinblastine sulfate and cultured until they reached the first cleavage metaphase. Between 17 and 20 h after the operation, eggs were treated with 0.5% protease to digest the zona pellucida and then placed in hypotonic solution (1:1 mixture of 1% sodium citrate and 30% FBS) for 8 min at room temperature. Chromosome slides of eggs were made by the gradual fixation-air drying method [29]. The chromosome slides were stained with 2% Giemsa (Merck) in phosphate-buffered solution (pH 6.8) for 8 min for conventional chromosome analysis to detect achromatic lesion due to gap, acentric fragment due to breakage, and rearrangements, including ring, translocation, and chromatid exchange. Subsequently, centromeric heterochromatin was stained by C-banding to detect dicentric chromosome, as described elsewhere [3]. Polyspermic eggs in IVF experiment and triploid eggs due to suppression of the second polar body in ICSI experiments were eliminated from the chromosome analysis.

Embryo Transfer, Postimplantation Development of Embryos, and Fetal Chromosome Analysis

Two- or four-cell embryos developed from oocytes following IVF and ICSI procedures were transferred into oviducts of CD-1 females, 8–12 wk of age, on the first day of pseudopregnancy. In the transfer of IVF embryos, the diploidy was confirmed by the presence of two pronuclei and a second polar body 8 h after the sperm insemination. Surrogate females were killed on Day 16 of pregnancy, and the number of implantation sites and live fetuses were recorded. Furthermore, live fetuses were externally examined. Chromosome slides of the live fetuses were prepared from skin cell culture. In brief, a piece of skin was cultured in D-MEM containing 20% FBS under 5% CO2 in air at 37°C. Two to five days later, the cultures were treated with colcemid (0.05 µg/ml) for 2–3 h, and then cells were harvested using trypsin (0.05%)-EDTA (0.53 mM) solution, treated with hypotonic solution (0.075 M KCl), and fixed with methanol-acetic acid (3:1) mixture. Chromosome slides were stained with Giemsa for conventional chromosome analysis. For precise karyotyping, the Giemsa bands (G-bands) of chromosomes were obtained by the routine trypsin digestion method. The G-banded karyotype was determined according to the nomenclature of mouse chromosomes [30].

Statistical Analysis

The chi-square test or Fisher's exact probability test was used to compare the percentage of embryos with chromosome aberrations. Individual group comparisons of mean number of structural chromosome aberrations were performed by the nonparametric Kruskal-Wallis test and Bonferroni/Dunn posthoc test. The chi-square test and Fisher's exact probability test were also used in an embryonic development assay to compare the percentages of implantation, live fetuses, and fetuses with chromosome aberrations. Differences were considered significant at P < 0.05.

RESULTS

Chromosome Analysis of One-Cell Embryos

In each ICSI experiment, 210–250 oocytes were successfully injected with sperm heads. Lack of oocyte activation following injection and failure in male pronucleus formation in activated ooplasm were found in fewer than 1% of the oocytes, regardless of medium type and length of sperm incubation. Apart from triploid embryos due to suppression of the second polar body, more than 200 diploid embryos were produced, and more than 95% of them were chromosomally analyzed. Results of chromosome analysis at the first cleavage metaphase of mouse embryos produced by IVF and ICSI techniques are summarized in Table 2 and Figure 1.


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  		TABLE 2. Chromosome analysis at the first cleavage metaphase of mouse embryos produced by IVF and ICSI.


Figure 01
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  		FIG. 1. Comparison of percentage of one-cell embryos with chromosome aberrations. The figure was made with the data in Table 2. Error bars represent standard deviations for different replicates in each experimental group.

In B6D2F1 mice, incidence of IVF embryos with structural chromosome aberrations was 2%, similar to previously reported results [17–21]. On the other hand, incidence of ICSI embryos with structural chromosome aberrations was dependent on the kind of medium used for sperm incubation and the time for sperm incubation. The aberration rates in ICSI embryos of all groups, except the groups with the incubation for 2–2.5 h and 6 h in TYH medium, were significantly higher than in IVF embryos. Interestingly, the aberration rate in ICSI embryos derived from spermatozoa that underwent incubation in TYH medium was reduced when the incubation time was 2–2.5 h (4%) and 6 h (4%) compared with when the incubation time was 0.5 h (7%). When mCZB medium was used for sperm preparation, there was little change in the incidence of chromosomally abnormal embryos with the time of sperm injection. In contrast, there was a time-dependent increase in incidence of structural chromosome aberrations found in embryos derived from spermatozoa when incubated in H-TYH, H-mCZB, and PB1 media. Surprisingly, the aberration rate increased to 28% in embryos derived from spermatozoa incubated for 6 h in PB1 medium. Thus, there was a great difference among media with respect to the incidence of ICSI embryos with structural chromosome aberrations when spermatozoa were incubated for longer than 2 h, although no such medium-dependent difference was found when spermatozoa were used after 0.5 h incubation.

Figure 2 presents all types of structural chromosome aberrations observed in ICSI embryos, and Figure 3 shows the number of each type of structural chromosome aberration per embryo in each experiment. Break and gap of chromosome type and dicentric were evident in ICSI embryos. Compared with IVF embryos, frequency of the break significantly increased when spermatozoa were incubated in H-TYH medium for 6 h (P < 0.001), H-mCZB medium for 6 h (P < 0.001), and PB1 medium for 2 h or more (P < 0.001). A significant increase in the frequency of the gap was found when spermatozoa were incubated in mCZB medium for 0.5 h (P < 0.05) and 6 h (P < 0.001), and in PB1 medium for 6 h (P < 0.001). There was a significant increased frequency of dicentric aberrations when spermatozoa were incubated in H-TYH medium for 2–2.5 h and 6 h (P < 0.001), and in H-mCZB and PB1 media in spite of the incubation time (P < 0.05–0.001).


Figure 02
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  		FIG. 2. Types of structural chromosome aberrations found in ICSI embryos. A) Two breaks of chromosome type. Arrows indicate derivative acentric fragments. B) A gap of chromosome type (arrow). C) A dicentric chromosome (long arrow) and a derivative fragment (short arrow) detected by C-band staining. D) A translocation detected by C-banding. There is a submetacentric chromosome (long arrow) formed by transition of a long arm of another chromosome (short arrow). E) An acentric ring (arrow). F) A break (long arrow) and a gap (short arrow) of chromatid type. G) A quadriradial exchange of chromatid type (arrow). Bar = 10 µm.


Figure 03
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  		FIG. 3. Distribution of each type of structural chromosome aberration found in IVF embryos and ICSI embryos derived from spermatozoa incubated for various times in different media. cte, Exchange of chromatid type; ctg, gap of chromatid type; ctb, break of chromatid type; r, ring; t, translocation; dic, dicentric; csg, gap of chromosome type; csb; break of chromosome type.

Incidence of aneuploid embryos in the ICSI groups ranged between 0% and 3%, but was only 1% in the IVF group. There was no significant difference between the two artificial fertilization methods.

Postimplantation Development of Embryos and Chromosome Analysis of Live Fetuses

As shown in Table 2, the minimal chromosome aberration rate (4%) of ICSI embryos was obtained when spermatozoa were incubated in TYH medium for 2–2.5 h, and the maximal aberration rate (28%) was obtained when spermatozoa were incubated in PB1 for 6 h. The former was not significantly different from IVF embryos (2%), whereas the latter was much higher than IVF embryos. Accordingly, performance of postimplantation embryo development was measured in these three groups (Table 3). There was no significant difference in rates of implantation and live fetuses between IVF-TYH and ICSI-TYH groups. In the ICSI-PB1 group, however, implantation rate was significantly lower than in the ICSI-TYH group, and the rate of live fetuses was obviously low compared with those of IVF-TYH and ICSI-TYH groups. In addition, there was a fetus with polydactyly in the ICSI-PB1 group (Fig. 4).


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  		TABLE 3. Postimplantation development of two- or four-cell embryos produced by IVF and ICSI techniques (examination on Day 16 postcoitum).


Figure 04
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  		FIG. 4. Polydactyly (arrows) in left hand of a Day 16 fetus developed from an ICSI embryo that was produced by a spermatozoon incubated in PB1 for 6 h. Bar = 1 mm.

Live fetuses obtained in postimplantation development assay were chromosomally analyzed (Table 4). Karyotyping of one fetus in the IVF-TYH group and one in the ICSI-TYH group could not be accomplished because of accidental contamination during cell culture. Only one fetus with chromosome anomaly was found in the ICSI-TYH group. It was a mosaic consisting of normal cells (45 [75%] of 60 cells: 40,XY) and hyperploid (trisomy) cells with a Robertsonian translocation (15 [25%] of 60 cells). The G-band patterns revealed that the extra chromosome was chromosome 16 and the translocation was between chromosome 3 and chromosome 16 (i.e., 40,XY,der(3;16)+16; Fig. 5). The fetus with polydactyly found in ICSI-PB1 group had a normal female karyotype.


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  		TABLE 4. Chromosome analysis of live fetuses (Day 16) developed from two- or four-cell embryos produced by IVF and ICSI techniques.


Figure 05
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  		FIG. 5. Chromosome spread of a hyperploid (16 trisomy) cell with a Robertsonian translocation (thick arrow) between chromosomes 3 and 16 in a chromosomally mosaic fetus that was developed from an ICSI embryo produced by a spermatozoon following incubation in TYH for 2–2.5 h. A long arrow and two short arrows indicate normal chromosome 3 and normal chromosome 16, respectively (inset: relevant chromosomes at higher magnification). (X), X chromosome; (Y), Y chromosome. Bar = 10 µm.

DISCUSSION

The present results demonstrate that the ICSI technique does not necessarily generate chromosome damage, because the chromosome damage of ICSI embryos derived from spermatozoa incubated in TYH medium for 2–2.5 h and 6 h was similar to that of IVF embryos. Thus, when using the mouse ICSI technique, spermatozoa should be used at least 2 h after incubation in TYH medium, unless otherwise specified. Furthermore, the use of TYH medium can protect spermatozoa against generation of chromosome damage due to in vitro aging. In addition, these results remove all doubt surrounding artificial separation of the sperm head from the tail by piezo pulses, a mechanical puncture of the oolemma, and small amounts of PVP injected with the sperm heads, inducing chromosome aberrations in the mouse ICSI technique.

The present findings help identify potential chromosomal risk factors in the mouse ICSI procedure. It should be noted first that the aberration rate in ICSI embryos was lower when spermatozoa were incubated in TYH medium for 2 h or more compared with spermatozoa incubated in the same medium for 0.5 h. TYH medium has the ability to effectively promote capacitation in mouse spermatozoa following incubation for 90 min or more [25, 31]. When spermatozoa undergo in vitro capacitation, a large amount of cholesterol is lost from the sperm plasma membrane [32, 33]. This triggers the acrosome reaction. Therefore, the decreased incidence of chromosome aberrations in ICSI embryos derived from spermatozoa incubated in TYH medium for 2 h or more may be related to the dissociation of cholesterol from the plasma membrane and/or the exocytosis of acrosome enzymes prior to injection. Conversely, the increased incidence of chromosome aberrations in ICSI embryos derived from spermatozoa after incubation in TYH medium for 0.5 h and other culture media may be due to insufficient or incomplete sperm capacitation. Our preliminary experiment has provided supportive evidence that ICSI embryos derived from spermatozoa without incubation in TYH medium had more chromosome lesions compared with ICSI embryos derived from spermatozoa that were incubated in the medium for 0.5 h. The appropriate concentrations of protein sources, HCO3– and Ca2+, are essential to in vitro capacitation [34]. Unlike TYH medium, however, mCZB medium includes a calcium-chelating agent, EDTA. Both H-TYH and H-mCZB media include no BSA and a low level of HCO3–, and PB1 medium has no HCO3– (Table 1). It is probable that mouse spermatozoa incompletely undergo in vitro capacitation in these media. The previous results also indicate that the increased incidence of structural chromosome aberrations is associated with the uncapacitated state of spermatozoa; there is a trend toward a higher incidence of aberrations (6%–9%) in ICSI embryos derived from spermatozoa after short incubation (5–15 min) in H-mCZB medium [4–6, 13] compared with ICSI embryos (4%) derived from spermatozoa after a long incubation (~90 min) in TYH medium [16].

It has been reported that the acrosome can alter sperm chromatin remodeling in the ooplasm following ICSI in the mouse [35], the pig [36], and the rhesus monkey [37–39]. Morozumi and Yanagimachi [40] showed that mouse acrosome enzymes can potentially induce deformation and degeneration of oocytes. More recently, Morozumi et al. [41] demonstrated that removal of sperm plasma membrane and acrosome before ICSI improved embryonic development in the mouse. It has been suggested that the topologic rearrangements take place in the DNA during sperm chromatin remodeling, because exposure of the remodeled sperm chromatin to topoisomerase II inhibitors caused DNA nicks [42] and structural chromosome aberrations [43] in male pronuclei of mouse one-cell embryos. Therefore, when uncapacitated spermatozoa are injected, the sperm plasma membrane cholesterol, acrosome, or its enzymes may affect sperm chromatin remodeling, thus leading to DNA damage. It remains to be investigated whether artificial removal of sperm plasma membrane cholesterol and acrosome can reduce DNA damage in ICSI embryos in spite of the type of sperm incubation medium used.

Also, the fact that there was a time-dependent increase of aberration rates in ICSI embryos derived from spermatozoa following the incubation in H-TYH, H-mCZB, and PB1 media is worth noting. This result supports previous findings that in vitro aging of mouse and human spermatozoa affects its own chromosomes. In chromosome analysis of mouse IVF embryos derived from spermatozoa that were stored in unsupplemented Tyrode (T6) medium at room temperature under 5% CO2 in air, the incidence of structural chromosome aberrations of paternal origin increased from 1% in the control to 6% after 6 h of aging [44] and 12% after 12 h of aging [45]. Chromosome analysis of human spermatozoa using zona-free hamster eggs demonstrated that the incidence of structural chromosome aberrations was elevated 3.3-fold when spermatozoa were stored in unsupplemented BWW medium for 24 h at room temperature under 5% CO2 in air prior to capacitation [46]. Using sperm chromatin structure assay, Estop et al. [44] found that in vitro incubation of mouse spermatozoa in unsupplemented T6 medium changed sperm chromatin structure, speculating that this makes DNA susceptible to denaturation, thus leading to structural chromosome aberrations. Another possibility is that the excess generation of reactive oxygen species (ROS) in spermatozoa may be involved in chromosome damage, because it has been suggested that intracellular ROS can impair DNA integrity in human spermatozoa without reduced motility [47]. As shown in the present and previous studies, chromosome damage in aged spermatozoa occurred entirely when spermatozoa were incubated in media without either serum albumin or HCO3–. Therefore, there is a possibility that oxidation of plasma membrane cholesterol of uncapacitated spermatozoa by ROS links to impairment of DNA integrity in spermatozoa.

In chromosome analysis of one-cell embryos, only a small number of reciprocal translocations could be found. However, there is little doubt that this type of aberration has been generated in similar proportions to the dicentric, because the reciprocal translocation is an exchange between terminal portions of two broken chromosomes, whereas the dicentric is an exchange between centromeric pieces of two broken chromosomes. Therefore, it is possible the actual chromosomal risk is greater than that calculated from data presented in Table 2 and Figures 1 and 3, when H-TYH (in instances of incubation for 2 h or more), mCZB, H-mCZB, and PB1 media were used for sperm preparation.

Imbalanced chromosome aberrations such as break, gap, dicentric, ring and chromatid exchange are the major causes of birth defects and congenital anomalies. This is exemplified by the fact that developmental efficiency of ICSI embryos was significantly reduced when spermatozoa were incubated in PB1 for 6 h (Table 4). There is additional concern of whether the resultant live fetuses are chromosomally normal, because balanced chromosome aberrations, such as reciprocal translocation and inversion, can be inherited by offspring but usually obstruct the gametogenesis. As far as the present results went, there was no fetus with reciprocal translocation or inversion, although one case was a mosaic of normal cells and trisomic cells with a Robertsonian translocation. More data are needed to accurately evaluate the inheritable risk of chromosome aberrations in mouse offspring produced by ICSI technique. Concurrently, the identification of chromosomal risk factors in mouse ICSI technique and the understanding of how these factors can cause chromosome aberrations are fundamental to further improvements in assisted reproduction of humans and other animal species.

Correspondence: 1Hiroyuki Tateno, Department of Biological Sciences, Asahikawa Medical College, 2-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan. FAX: 81 166 68 2783; e-mail: htateno{at}asahikawa-med.ac.jp

Received: 28 September 2006.

First decision: 23 October 2006.

Accepted: 1 April 2007.

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