Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI

Ervin Macas1, Bruno Imthurn and Paul J. Keller

Clinic of Endocrinology, Department of Gynaecology and Obstetrics, University Hospital Zurich, Frauenklinikstrasse 10, CH-8091 Zurich, Switzerland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The potential risk of transmitting chromosomally abnormal spermatozoa from infertile males into oocytes through intracytoplasmic sperm injection (ICSI) has prompted us to investigate the male pronuclei of tripronuclear zygotes (3PN) obtained after ICSI. To specify the type of anomalies, we used triple colour fluorescent in-situ hybridization (FISH) with three specific probes for chromosomes X, Y and 18. From a total of 163 paternal complements of ICSI-3PN zygotes, 90 (55.2%) had Y-chromosome signals. Eighty-three of these were normal, four had the disomy XY and three were diploid. In the remaining 73 ICSI-3PN zygotes without Y-chromosome signals, the origin of paternal pronuclei was extrapolated through chromosome constitution of the first polar body. Five anomalies were found in this group of zygotes, giving a total rate of numerical chromosome aberrations for fertilizing spermatozoa of 7.4%. In contrast to ICSI, only two disomies (1.5%) were found in the control group of IVF-3PN zygotes. Compared with the incidence of chromosome anomalies between paternal-derived pronuclei of ICSI- and IVF-3PN zygotes, the difference was statistically significant (P < 0.025). This study provides the first direct evidence of a higher incidence of numerical chromosome anomalies in sperm-fertilized human oocytes after ICSI.

Key words: chromosome anomalies/FISH analysis/ICSI/male infertility/tripronuclear zygotes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The potential risk of chromosome defects in the offspring resulting from intracytoplasmic sperm injection (ICSI) has recently necessitated intense cytogenetic investigation of spermatozoa from males with fertility problems. For example, a significantly higher incidence of XY disomy and disomy for chromosome 1 in the spermatozoa of infertile males has been observed (Moosani et al., 1995Go). More recently, the refined fluorescent in-situ hybridization (FISH) technique has been successfully applied by several research groups to demonstrate disomy rates for almost all chromosomes in men with fertility problems (Bernardini et al., 1997Go; Storeng et al., 1998Go; Aran et al., 1999Go; Colombero et al., 1999Go; Pang et al., 1999Go; Pfeffer et al., 1999Go).

However, genetic screening of spermatozoa from infertile males before ICSI does not give a definitive answer concerning the extent to which abnormal spermatozoa might contribute to chromosome aberrations after ICSI. This is supported by the observation that the incidence of aneuploidy in sperm cells of infertile men reported in the literature is the subject of considerable controversy. Hence, no difference was found in the incidence of aneuploidy for the sex chromosomes when comparing sperm cells from infertile with fertile males (Miharu et al., 1994Go; Guttenbach et al., 1997Go). In contrast, Pfeffer et al. and Pang et al. found incidences of aneuploidy for sex chromosomes in infertile men of 2.2 and 3.9% respectively, but only 0.7 and 0.4% in fertile males (Pang et al., 1999Go; Pfeffer et al., 1999Go). In our experience, an additional problem for assessing a potential risk of ICSI is the relatively high heterogeneity observed between infertile males screened for sperm chromosome anomalies. Thus, depending on the group investigated, the disomy rate for X/Y chromosomes ranged from 1.6 to 4.9%; and, for chromosome 18, from as low as 0.4% to a significant 3.1% (Pang et al., 1999Go). Moreover, the incidence of diploidy, one of the most frequently observed anomalies in sperm cells from infertile males, ranged from 0.3 to 1.4% (Pfeffer et al., 1999Go), from 0.2 to 1.3% (McInnes et al., 1998Go) and from 0.4 to 9.6% (Pang et al., 1999Go).

These various methodological problems stimulated us to look for improved possibilities to investigate the true contribution of spermatozoa from infertile males to chromosomally abnormal conceptuses after ICSI. Ideally, the spermatozoa themselves should be screened when fertilization is achieved by ICSI. One possibility to do so is to analyse sperm-derived pronuclei of tripronuclear (3PN) zygotes arising after ICSI (Macas et al., 1996Go).

In the present study, therefore, in the attempt to obtain an insight into the chromosome constitution of fertilizing spermatozoa from infertile patients, the male-derived genomes of ICSI-3PN zygotes were analysed for disomy of chromosomes X, Y and 18, as well as for the incidence of diploidy, using the three-colour FISH technique. The rationale for also investigating the incidence of disomy for chromosome 18 was because this anomaly, like sex chromosome disorders, might be also associated with male infertility (Colombero et al., 1999Go; Pang et al., 1999Go; Pfeffer et al., 1999Go).

Furthermore, we investigated in parallel the chromosome constitution of 3PN zygotes arising after IVF. In contrast to ICSI, the IVF male patients are recruited from a normozoospermic group of men. We postulated that there should be a lower incidence of chromosome aberrations in the sperm-derived pronuclei of 3PN zygotes arising after conventional in-vitro insemination.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 183 ICSI- and 74 IVF-3PN zygotes were obtained during a 2 year period of performing the assisted procreation programme at our department. The use of human 3PN zygotes for this project was approved by the ethical committee at the University Hospital of Zurich.

Assessment of male infertility
Semen of each patient was assessed on several occasions before admission to the treatment cycle. According to the quality of the semen, the male partners were divided into groups with normozoospermia (n = 60), oligoasthenoteratozoospermia (OAT, <=20x106/ml, <=50% motile, and/or <=30% normal sperm forms; WHO, 1992; n = 90) and cryptozoospermia (<=0.1x10 6/ml of motile spermatozoa after microcentifugation of entire ejaculate; n = 28). Furthermore, when no spermatozoa were present because of obstructive or non-obstructive azoospermia, these patients were treated either by micro-epididymal sperm aspiration (MESA; n = 2) or by testicular sperm extraction (TESE, n = 18).

Once male patients were definitively identified as possible candidates for ICSI, their peripheral karyotypes were checked for the presence of possible chromosomal abnormality. Only men with a normal 46,XY karyotype were included in the ICSI programme.

Zygote fixation and staining
Each 3PN zygote found was examined under an inverted microscope using interference optics (Hoffman optic; Nikon, Tokyo, Japan) to ascertain the exact number of pronuclei and polar bodies. Tripronuclear zygotes derived either from ICSI or IVF treatment cycles were transferred immediately to equilibrated culture medium containing 1 µg/ml of colcemid (Gibco, Paisley, UK), incubated for 14–16 h and then submitted to cytogenetic analysis. The methods for zygote fixation have been previously described (Macas et al., 1996Go). After fixation and drying for 24 h, slides were stained with 5% Giemsa for 10 min and examined under a light microscope using a x40 PlanApo objective lens (Nikon). For each intact zygote, a small diagram was made to check exactly the position and a ploidy status (N or 2N) was registered for each individual metaphase before FISH. Slides were stored at –20°C for up to 3 months or were processed immediately for FISH.

FISH procedures
A slight modification of the FISH procedures described previously was used for this study (Harper et al., 1994Go). Briefly, fixed zygotes were dehydrated through an ethanol series prior to pretreatment with 100 µg/ml pepsin in 0.01 N HCl for 20 min at 37°C. The slides were washed twice in bi-distilled water and phosphate-buffered saline (PBS), fixed in ethanol/acetic acid (3:1) washed again in PBS and bi-distilled water and dehydrated through an ethanol series. After drying for 10 min the probes (DXZ1 locus, SpectrumGreen® for chromosome X; D18Z1 locus, SpectrumAqua® for chromosome 18; DYZ1 locus, SpectrumOrange® for chromosome Y; Vysis Inc., Downers Grove, IL, USA) and hybridization mixture were added under a glass coverslip (diameter 12 mm, Mencel, Germany), and the target and probes were co-denaturated at 80°C for 1 min using the hot plate (Hybrite; Vysis). The slides were then incubated in a moist chamber at 42°C for a minimum of 4 h or overnight at 37°C.

Post-hybridization washes consisted of 2 min in 0.4xSSC at 75°C and a 1 min wash in 2xSSC/0.1% Nonidet P-40 (Sigma) at room temperature. Metaphases were counterstained with 15 µl of DAPI in antifade solution (0.5 mg/ml; Vysis), covered with a coverglass, and assessed at x1000 magnification with an epifluorescence microscope (Nikon EFD-3) equipped with a single bandpass filter for fluorescein isothiocyanate (FITC, Nikon), tetramethylrhodamine isothiocyanate (TRIC, Nikon), aqua (Vysis) and DAPI (Nikon).

Images were recorded using a black and white CCD (charge coupled device) camera (COHU, Inc., Electronics Division, San Diego, CA, USA) controlled by a Pentium EISA computer using Lucia Image (Nikon) and FISH analysis software.

Scoring criteria
The presence of well spread metaphases significantly facilitated the identification of anomalies in male and female meioses. Otherwise, in cases when metaphases were not well spread, the analysis of FISH signals was performed according to previously published criteria (Hopman et al., 1988Go) subsequently adapted (Munné et al., 1994Go).

Determination of gametic origin of individual pronuclei after ICSI
The following criteria were used to determine the paternal origin of metaphases after ICSI: (i) only zygotes with three discrete chromosome complements were selected for the FISH analysis; (ii) the paternal origin of individual metaphases was detected directly by the presence of a Y-chromosome signal; (iii) when no Y-chromosome signal was present, the origin of the paternal genome was extrapolated through chromosome constitution of the first polar body. For example, when one X-chromosome signal was present per each metaphase analysed, it was considered that the male pronucleus had a normal genetic constitution for the X chromosome. Secondly, when disomy XX was found in one metaphase, it was considered that this anomaly occurred in the male meiosis when a first polar body complement and two additional, presumably maternal, chromosome complements contained a single X-chromosome signal. Thirdly, when disomy 18 was found in one metaphase, it was considered a that this anomaly occurred in the male meiosis when the polar body complement and two additional, presumably maternal, metaphases contained single signal for chromosome 18. Finally, when a diploid metaphase was found, it was considered that this metaphase belonged to the sperm-derived pronucleus if the first polar body and two additional maternal metaphases possessed the normal number of signals for both the X chromosome and chromosome 18.

Determination of the gametic origin of individual pronuclei after IVF
The same criteria were used to determine the origin of male pronuclei in a group of IVF zygotes, with the following exception: when no Y-chromosome signals were found, both polar bodies were carefully investigated to reconstitute maternal and then paternal chromosome composition.

Statistical analysis
The results were analysed by the {chi}2-test with the Yates correction.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic constitution of paternally derived pronuclei after ICSI
From a total of 183 ICSI-3PN zygotes, 15 were immediately discarded from study because 12 had chromosomes scattered within the cytoplasm and three were in the stage of syngamy. Based on criteria given previously, a total of 168 ICSI zygotes with three individual chromosome complements were selected for this study. Five of these were subsequently discarded as no chromosome signals were seen following in-situ hybridization.

Since ICSI zygotes, due to their digynic origin, contain one paternal and two maternal pronuclei, 163 out of a total of 489 metaphases found after FISH were calculated to belong to fertilizing spermatozoa. In 90 cases the presence of Y-chromosome signals pointed directly to the paternal origin of attendant metaphases (55.2%). Eighty-three of these metaphases displayed normal chromosome complements (Y/18), four had the XY disomy (XY/18, Figure 1aGo) and three were diploid (YY/1818; XY/1818; XY/1818).



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Figure 1. Four types of chromosome anomalies found in four paternal complements of tripronuclear zygotes derived from intracytoplasmic sperm injection: (a) disomy XY, (b) diploidy, (c) nullisomy for sex chromosomes, and (d) disomy XX. The red arrow identifies chromosome Y, the green arrow chromosome X, and the blue arrow chromosome 18. Scale bar = 3 µm.

 
The origin of male pronuclei in the second group of 73 zygotes having no Y-chromosome signals was extrapolated through genomic constitution of the first polar body. Informative FISH results were obtained from 64 zygotes having first polar body chromatin and six zygotes having polar body chromosomes. Three zygotes in this group did not contain the polar body chromatin. Thus, based on the polar body FISH analysis, we found abnormal chromosome complements for another five paternally derived pronuclei: two nullisomies, one for chromosome 18 and one for X chromosome (X/0; 0/18, Figure 1cGo), two disomies for chromosome X (XX/18, Figure 1dGo), and one case of diploidy (XX/1818, Figure 1bGo). In all these abnormal cases, two identical paired signals (dyads) were observed on the polar body, indicating the normal distribution of chromosome X and 18 between two corresponding maternal complements. Taking together all 12 cases with abnormal chromosome constitution, the total rate of chromosome anomalies transmitted by fertilizing spermatozoa was 7.4%.

When the incidence of chromosome anomalies found in male pronuclei was analysed in relation to the type of male infertility, however, no statistical difference was observed between men with OAT, cryptospermia or azoospermia (Table IGo).


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Table I. The incidence of chromosome anomalies after applying the fluorescent in-situ hybridization technique on tripronuclear (3PN) zygotes obtained after intracytoplasmic sperm injection (ICSI) and IVF
 
Genetic constitution of paternally derived pronuclei after IVF
A total of 74 3PN zygotes resulting from IVF were prepared for cytogenetic investigation, and FISH analysis was possible in 68 of these (91.9%). Six zygotes were unsuitable for FISH because the chromosomes either were in the stage of syngamy (n = 2), lost during the fixation (n = 3), or no chromosome signals were found following in-situ hybridization (n = 1).

For the remaining 3PN zygotes, we focused on the determination of the number of polar bodies in an attempt to exclude digyny as one of two possible modes in the generation of triploidy after IVF. Thus, based on cytogenetical investigations, we were able to rule out this phenomenon in 66 out of 68 IVF-3PN zygotes possessing the first and second polar body chromatins. One zygote showed one, and one cell had no detectable polar body but both of them had three individual haploid chromosome complements. Therefore, due to dispermic origin, 136 of a total of 204 chromosome complements found in 68 IVF-3PN zygotes were calculated to belong to fertilizing spermatozoa. In 54 cases (39.7%) the presence of Y-chromosome signals pointed directly to the paternal origin of these metaphases. Except one abnormal case with Y-chromosome disomy (YY/18), all other zygotes in this group had normal chromosome complements (Y/18). Furthermore, comparing the number of Y-bearing metaphases after IVF with the number of Y-bearing metaphases after ICSI, the difference was statistically significant ({chi}2 = 4.3; P < 0.05).

In the remaining 82 complements without Y-chromosome signals, the determination of the genetic constitution of paternal genomes facilitated the following finding: with the exception of only one abnormal case for disomy 18 found in the paternal genome, all other metaphases (maternal and paternal) had a balanced number of fluorescent signals for the particular chromosomes investigated. The FISH observations after conventional in-vitro insemination are summarized in Table IGo. When the rate of chromosomal aberrations occurring in ICSI zygotes (7.4%) was compared with anomalies occurring after IVF (1.5%), the difference was statistically significant ({chi}2 = 5.95, P < 0.025).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite increased evidence that abnormal spermatozoa from infertile men can be a risk factor in ICSI treatment, the extent to which these anomalies might contribute to chromosome irregularities at the time of conception is still unknown. Although some FISH analyses on early ICSI conceptuses were carried out before this investigation (Grossmann et al., 1997Go; Staessen and Van Steirteghem, 1997Go), the purpose of performing those studies differed from our objective. For example, Grossmann et al. (1997), studying 3PN zygotes at the interphase stage, primarily intended to determine the genetic origin of triploidy and not to evaluate the chromosome constitution for male genomes participating in the fertilization process after ICSI. Our report is the first to provide direct information on the incidence of chromosome anomalies in human spermatozoa at the time of conception after ICSI.

Methodological advantages
Two methodological prerequisites were of key importance for achieving the objective for this study. The first was the use of FISH. We applied this technique because, compared with some classical staining methods, it is a more accurate approach for the determination of chromosome anomalies at the first cleavage division. This technique enabled us to analyse 3PN zygotes for chromosome anomalies with an efficiency of >97% irrespective of the chromosome morphology and degree of metaphase spreading. The lack of fluorescent signals in a few zygotes was, in our view, primarily due to some mistakes occurring in the preparative procedure and not to the inability of probes to bind the respective chromosomes during in-situ hybridization.

The second important technical requirement was the polar body FISH analysis which helped us to determine the origin of anomalies for all 3PN zygotes without Y-chromosome signals. Of particular interest was the finding that the majority of polar bodies had single paired signals for chromosome X and chromosome 18, while <20% showed balanced pre-division of dyads for respective chromosomes. This finding was unexpected, as other studies dealing with polar body analysis found a higher incidence of balanced pre-division of dyads, suggesting that the human first polar body undergoes a rapid degeneration during a prolonged time of in-vitro incubation (Munné et al., 1995Go; Dyban et al., 1996Go). We speculate that some specific extrinsic factors, for example, different types of stimulation regime or patients groups used, time in culture or use of incubation in colcemid medium for 14–16 h, could be responsible for the better preservation of the genetic integrity of polar bodies in the present study.

Clinical implications
Although the rate of numerical chromosomal aberrations of 7.4% observed in the present study might be a relatively good indicator for assessing the genetic risk of ICSI at the time of conception, this incidence still does not give complete information on numerical chromosomal anomalies transmitted by injection of spermatozoa from infertile men. Certainly, the application of the three-colour FISH technique for chromosomes X, Y and 18 provides limited scope for the investigation of this problem, and additional chromosome pairs would need to be selected in future studies in order to estimate the global frequency of aneuploidy after ICSI.

The second and more specific question is the extent to which the chromosomal anomalies transmitted by fertilizing spermatozoa might affect normal embryo development at the later stages of pregnancy. Since a certain number of embryos with severe chromosome disturbance are more likely to be eliminated before or soon after implantation, the further development of some anomalies encountered in the present study can be consequently predicted in a similar fashion. For example, the four anomalies where diploid spermatozoa participated in the fertilization process might have led to the formation of an embryo with a triploid number of chromosomes. Having two sets of paternal chromosomes, these embryos might have developed into partial hydatidiform moles or, because of the very low developmental potential, died soon after implantation.

A similar negative consequence on further embryo development might apply to the single case of nullisomy for chromosome 18, as it is known that autosomal monosomies, in contrast to sex chromosome monosomies, are usually lethal and can be only seen accidentally in abortuses.

Unfortunately, some unbalanced anomalies, as for example the six cases of sex chromosome disomies and one case of sex chromosome nullisomy found in the present study, might have escaped all barriers of the natural selection mechanism after conception and could only have been detected later at the time of performing prenatal diagnosis. The results of recent surveys conducted perinatally on children conceived after ICSI might partially support this assumption. Bonduelle et al. (1999), after karyotyping more than 1000 fetuses after ICSI, found an increased incidence of chromosome anomalies of almost 2%. Interestingly, apart from trisomies for chromosomes 18 and 21, the majority of these de-novo anomalies were sex chromosome aneuploidies, again demonstrating the dominating role of the paternal factor on the induction of chromosome anomalies after ICSI.

Sex ratio and sex chromosome disorders
An interesting finding in this study was the excess of Y- over X-chromosome signals in ICSI- compared to IVF-3PN zygotes. This finding can be potentially explained by the observations that the natural sex ratio among X- and Y-bearing spermatozoa can be influenced by differences in sperm preparation methods. For example, sperm fractions collected following the Percoll method have shown a higher proportion of X- over Y-bearing spermatozoa. In contrast, Y-bearing spermatozoa were selectively expressed in spermatozoa obtained using a swim-up procedure (Samura et al., 1997Go). Since in the present study spermatozoa for ICSI were prepared using Percoll and not the swim-up method, the true reasons for the discrepancy between our and other studies remain unknown.

Nevertheless, if we take into account only the anomalies caused by sex chromosome disorders, the increased prevalence of disomy over nullisomy for sex chromosomes (6 versus 1) is surprising, since equal frequencies were expected following errors in the male meiosis. The possibility that a certain number of nullisomic complements were discarded as FISH failures is less likely, because only 3PN zygotes with complete lack of fluorescence signals were excluded from this study. We believe that the observed differences were caused in a random way. However, further studies with larger number of 3PN zygotes will be required to address the relationship between gonosomal disomy and nullisomy after ICSI.

In addition, the 4.3% (7/163) incidence of sex chromosome disorders was considered very high, since only one chromosome pair contributed to these anomalies. For example, in contrast to our observation, Martin et al. (1991) found a total incidence of ~5% numerical chromosomal anomalies in spermatozoa from men with normal semen profiles. Taken together, our findings suggest that apart from total numerical chromosomal anomalies, there is also a higher risk of sex chromosome anomalies at the time of fertilization after ICSI.

Despite this higher incidence of sex chromosome anomalies, we were not able to show significant differences for sex chromosome anomalies in paternal genomes of 3PN zygotes obtained following ICSI and IVF (4.3 versus 0.7%). The lack of significance was surprising since a significantly high number of sex chromosome anomalies have consistently been found in spermatozoa from infertile men (Storeng et al., 1998Go; Pang et al., 1999Go; Pfeffer et al., 1999Go; Gil-Salom et al., 2000Go; Vegetti et al., 2000Go). Because, in contrast to our study, the level of significance in the above studies was obtained using a large population of spermatozoa, additional studies involving a greater number of 3PN zygotes are required to obtain a statistically higher incidence of sex chromosome aneuploidy at the time of conception after ICSI.


    Acknowledgments
 
We thank Dr R.Dubey for helpful reading of the manuscript and Ms M.Borsos for expert technical assistance. This study was financially supported in part by of the EMDO Stiftung, Zurich, No. 23.2105.


    Notes
 
1 To whom correspondence should be addressed. E-mail: Ervin.Macas{at}fhk.usz.ch Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aran, B., Blanco, J., Vidal, F. et al. (1999) Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program. Fertil. Steril., 72, 696–701.[ISI][Medline]

Bernardini, L., Martini, E., Geraedts, J.P.M. et al. (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol. Hum. Reprod., 3, 431–438.[Abstract]

Bonduelle, M., Camus, M., De Vos, A. et al. (1999) Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum. Reprod., 14 (Suppl. 1), 243–264.[ISI][Medline]

Colombero, L.T., June, M.D., Hariprashad, J.J. et al. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 90–96.[ISI][Medline]

Dyban, A.P., Freidine, M., Severova, E. et al. (1996) Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization. J. Assist. Reprod. Genet., 13, 73–78.[ISI][Medline]

Gil-Salom, M., Rubio, C., Vidal, F. et al. (2000) Correlation between severity of oligozoospermia and the incidence of sperm chromosomal abnormalities in a risk population. Hum. Reprod., 15, (Abstract Bk. 1), Abstr. O-126, pp. 50.

Grossmann, M., Calafell, J.M., Brandy, N. et al. (1997) Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum. Reprod., 12, 2762–2765.[Abstract]

Guttenbach, M., Martinez-Expósito, M.-J., Michelmann, H.W. et al. (1997) Incidence of diploid and disomic sperm nuclei in 45 infertile men. Hum. Reprod., 12, 468–473.[ISI][Medline]

Harper, J.C., Coonen, E., Ramaekers, C.S. et al. (1994) Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes. Hum. Reprod., 9, 721–724.[Abstract]

Hopman, A.H.N., Ramaekers, F.C.S., Raap, A.K. et al. (1988) In situ hybridization as a tool to study numerical chromosome aberrations in solids bladder tumours. Histochemistry, 89, 307–316.[ISI][Medline]

Macas, E., Imthurn, B., Rosselli, M. and Keller, P.J. (1996) The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection. Hum. Reprod., 11, 2496–2501.[Abstract]

Martin, R.H., Ko, E. and Rademaker, A. (1991) Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes. Am. J. Med. Genet., 39, 321–331.[ISI][Medline]

McInnes, B., Rademaker, A., Greene, C.A. et al. (1998) Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum. Reprod., 13, 2787–2790.[Abstract/Free Full Text]

Miharu, N., Best, R.G. and Young, S.R. (1994) Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in-situ hybridization. Hum. Genet., 93, 502–506.[ISI][Medline]

Moosani, N., Pattison, H.A., Carter, M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in-situ hybridization. Fertil. Steril., 64, 811–817.[ISI][Medline]

Munné, S., Grifo, J., Cohen, J. and Weiner, H.U.G. (1994) Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am. J. Hum. Genet., 55, 150–159.[ISI][Medline]

Munné, S., Dailey, T., Sultan, K.M. et al. (1995) The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum. Reprod., 10, 1014–1020.[Abstract]

Pang, M.G., Hoegerman, S.F., Cuticchia, A.J. et al. (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 1266–1273.[Abstract/Free Full Text]

Pfeffer, J., Pang, M-G., Hoegerman, S.F et al. (1999) Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil. Steril., 72, 472–478.[ISI][Medline]

Samura, O., Miharu, N., He, H. et al. (1997) Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods. Hum. Reprod., 12, 2437–2442.[Abstract]

Staessen, C. and Van Steirteghem, A.C. (1997) The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intacytoplasmic sperm injection and conventional in-vitro fertilization. Hum. Reprod., 12, 321–327.[Abstract]

Storeng, R.T., Plachot, M., Theophile, D. et al. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191–197.[ISI][Medline]

Vegetti, W., Van Assche, E., Frias, A. et al. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum. Reprod., 15, 351–365.[Abstract/Free Full Text]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.

Submitted on June 26, 2000; accepted on October 10, 2000.