Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization

Chiaki Ushijima11, Yoko Kumasako, Paul E. Kihaile, Keiko Hirotsuru and Takafumi Utsunomiya

St Luke Clinic, Oita, 870-0947, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is concern that intracytoplasmic sperm injection (ICSI) may lead to offspring with a high frequency of chromosomal abnormalities. Accordingly, we studied spermatozoa sampled from eight infertile men with oligoasthenoteratozoospermia (OAT) by multi-colour fluorescence in-situ hybridization (FISH), using DNA probes for chromosomes 13, 18, 21, X and Y. Results were compared with those of spermatozoa sampled from 10 healthy men with normal semen profiles. Analysis of the diploidy values was repeated twice in each of the 18 men. There was no significant difference in the two diploidy estimates; thus the FISH technique appeared to be accurate and reliable for determining aneuploidy in human spermatozoa. We found the average frequencies of disomy for chromosomes 13, 18, 21 and X or Y to be 0.13, 0.12, 0.24 and 0.59% respectively for the OAT group and 0.09, 0.13, 0.19 and 0.38% respectively for the control group. The diploidy rate was 0.29% in the OAT group, and 0.16% in the control group. Thus, the OAT group showed a significantly higher frequency of disomy for chromosomes 13 (P < 0.001), 21 (P < 0.05), sex (P < 0.001), and diploidy (P < 0.005) than the control group. This finding suggests there may be some risk of aneuploidy in the offspring conceived by the ICSI technique.

Key words: aneuploidy/chromosomal abnormality/FISH/infertile men/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) has overcome the problem of male infertility in the past few years. By this technique, even spermatozoa from men with an extremely low sperm count, and with poor sperm motility and morphology can be successfully used to fertilize the oocyte. However, there is concern that these spermatozoa may have a higher frequency of chromosomal abnormalities, so that ICSI may lead to a higher frequency of offspring with chromosomal abnormalities. Some recent reports on the prenatal diagnosis of ICSI pregnancies indicate an increased risk of sex chromosomal abnormalities in ~1% of cases (Liebaers et al., 1995Go). These were shown to be of paternal origin (Van Opstal et al., 1997Go).

Aneuploidy studies in human spermatozoa have been performed by the zona-free hamster egg penetration system (Rudak et al., 1978Go; Martin et al., 1982Go). However, this method is time-consuming and expensive. In addition, there are practical limitations as to the number of metaphases that can be studied. Furthermore, spermatozoa of certain subgroups of infertile men show an impaired capacity to penetrate eggs. The relative ability of genetically abnormal spermatozoa to penetrate hamster eggs has not been extensively studied.

Recently, fluorescence in-situ hybridization (FISH) has been used to study aneuploidy rates in human spermatozoa (Williams et al., 1993Go; Miharu et al., 1994Go; Martin et al., 1996Go; Lähdetie et al., 1997Go). This highly sensitive and specific method permits the study of larger numbers of spermatozoa than other existing techniques by using a chromosome-specific DNA probe that can be detected by fluorescence microscopy in cells in the interphase as well as the metaphase. We used FISH to investigate the rates of aneuploidy in infertile men with oligoasthenoteratozoospermia (OAT) and compared them with a control group of men with normal sperm profiles.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semen samples
Semen samples were obtained from eight Japanese men with OAT and from 10 healthy normal Japanese men (controls) with normal semen profiles. The age of both OAT men and controls ranged from 26 to 39 years. Five of the control subjects were of proven fertility whereas the other five controls were the healthy husbands with normal semen parameters and endocrine profiles. Their five female partners of the control group also underwent fertility work-up and they showed normal hormone profile, uterus, tubes, and therefore they were diagnosed as unexplained infertility. Informed consent was obtained from each subject.

Semen parameters are shown in Table IGo. Normal values for semen variables were adopted from WHO guidelines (WHO, 1992), and included the following: sperm concentration, 20x106 spermatozoa/ml or more; morphology, >=30%; motility, >=50% with forward progression within 60 min of ejaculation.


View this table:
[in this window]
[in a new window]
 
Table I. Sperm profiles for eight infertile men and control subjects
 
Furthermore, sperm normal morphology (SNM) was evaluated using the Tygerberg strict criteria as described (Kruger et al., 1988Go). The cut-off value used was >=14% normal morphology.

Preparation of sperm nuclei
Samples were washed three times in phosphate-buffered saline (PBS, pH 7.2) and centrifuged at 300 g for 10 min. The pellet was then carefully resuspended in 1 ml of fresh, cold fixative (methanol: acetic acid = 3:1) and stored at –20°C. The fixed sperm cell suspension was spread on a clean glass slide and air-dried.

Prior to hybridization, the slides were washed in 2xsaline sodium citrate (SSC) to remove excess fixative and were then incubated for 15–30 min in 1 mol/l Tris–HCl buffer (pH 8) that contained 20 mol/l dithiothreitol (DTT). After decondensation, the slides were washed twice in 2xSSC, dehydrated in an ethanol series (70:100%), and air-dried.

Two- and three-colour FISH
Multi-colour fluorescence in-situ hybridization studies were performed as described (McInnes et al., 1998Go). To determine the frequency of disomy for autosomes, two-colour FISH was conducted in which the probes for chromosomes 13 and 21 were hybridized simultaneously. We used a commercial dual probe mixture that contained a probe labelled with SpectrumGreenTM for chromosome 13 and a SpectrumOrangeTM labelled probe for chromosome 21 (Vysis, Downers Grove, IL, USA).

The hybridization solution was applied to a glass slide that contained the fixed sperm and covered with a coverslip. The slide was sealed with rubber cement and placed in a dark moist chamber at 37°C for 12 h. After hybridization, each slide was washed individually at 45°C in 50% formamide/2xSSC for 2 min, 2xSSC/0.1% Tween 20 for 10 min, and 2xSSC for 5 min. The slides were then mounted with 10 µl of 4',6-diamino-2-phenylindole (DAPI; Vysis) counterstain in anti-fade solution.

Three-colour FISH was performed to determine the frequency of disomy for the sex chromosomes using three direct-labelled probes: CEP 18 SpectrumAquaTM, CEP X SpectrumGreenTM, and CEP Y SpectrumOrangeTM. The protocol for hybridization was the same as that for two-colour FISH.

Scoring of sperm nuclei and analysis of preparations
Analyses of sperm probes were carried out without prior knowledge of the patients' sperm parameters. Only slides showing hybridization efficiencies >95% were evaluated. Sperm slides were scored according to previous recommendations (Williams et al., 1993Go). Sperm nuclei were scored when morphologically preserved, not clumping or overlapping, with well-defined outline and tail and sperm head decondensed to no more than twice the size of the normal non-decondensed spermatozoa. The presence of the tail was considered essential for a reliable evaluation.

FISH preparations were evaluated with a fluorescent microscope (Olympus BX60) at x1000 magnification. The filter used was the VysisTM Aqua/Green/Orange triple band-pass filter set and the VysisTM DAPI/Green/Orange triple band-pass filter set. A minimum of 5000 sperm nuclei per chromosome probe and 10 000 sperm nuclei per subject were examined.

Data were statistically analysed using Student's t-test and P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multi-colour FISH analysis was performed in the patients and the controls. Individual sperm profiles are shown in Table IGo. A minimum of 5000 spermatozoa per hybridization per subject were counted for a total of >140 000 spermatozoa analysed. The individual results for the frequency of diploidy for each hybridization are presented in Table IIGo. For each of the 18 men, the diploid value was analysed two times and there were no significant differences in the two diploidy values (P = 0.88). The mean diploidy rate was 0.16% in the control group, and 0.29% in the OAT group.


View this table:
[in this window]
[in a new window]
 
Table II. Frequency of diploid spermatozoa estimated by two different hybridization analyses
 
The disomy rates established for chromosomes 13, 18, 21, X and Y in the 18 men are summarized in Table IIIGo. The mean frequency of disomic sperm nuclei in the control group was 0.09% (range 0.06–0.14%) for chromosome 13, 0.13% (range 0.08–0.19%) for chromosome 18, 0.19% (range 0.12–0.25%) for chromosome 21, and 0.38% (range 0.32–0.46%) for sex chromosome (the sum of XX and XY and YY).


View this table:
[in this window]
[in a new window]
 
Table III. Frequency of disomy spermatozoa
 
In the OAT group, disomy rates were 0.13% (range 0.10–0.19%) for chromosome 13, 0.12% (range 0.08–0.18%) for chromosome 18, 0.24% (range 0.19–0.28%) for chromosome 21, and 0.59% (range 0.47–0.73%) for sex chromosomes. The frequency of disomy for chromosomes 13 (P < 0.001), 21 (P < 0.05), sex (P < 0.001), and that for diploidy (P < 0.005) showed a highly significant increase in the OAT group compared with the control group.

We observed a close, and statistically significant, inverse correlation (r = –0.730) between sperm morphology and sex chromosome abnormality (Figure 1Go).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 1. Correlation between sperm morphology and sex chromosomal abnormality in eight infertile patients with OAT and in 10 control men. Sperm morphology was evaluated by the Tygerberg strict criteria previously reported (Kruger et al., 1988Go).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present results demonstrate the suitability of FISH for studying aneuploidy in spermatozoa. Aneuploidy is responsible for a great deal of pregnancy loss and affects ~1 out of 300 livebirths (Jacobs, 1992Go). The FISH technique is relatively simple, and enables large numbers of spermatozoa to be examined at a time. Previous studies using FISH for disomy rates in spermatozoa have yielded a fairly wide range of values even for the same chromosome (Brianna et al., 1993; Miharu et al., 1994Go; Blanco et al., 1996Go; Egozcue et al., 1997Go; Guttenbach et al., 1997Go; Lähdetie et al., 1997Go; Pang et al., 1999Go). This variation can be accounted for by variations in technique, inadequate sample size, imprecise scoring criteria, and diploid sperm masquerading as disomic sperm when only single-colour FISH is utilized. Three-colour FISH is necessary for analysis of sex chromosomal aneuploidy to distinguish an XY spermatozoon that is diploid (two autosomal probe signals) from one that is disomic (one autosomal signal).

It has been observed (Downie et al., 1997Go) that aneuploidy estimates would not be accurate unless well designed studies were undertaken in which at least 10 000 spermatozoa per chromosome probe per man were assessed. However, it may be difficult to obtain sufficient spermatozoa from oligozoospermic men. The present study evaluated aneuploid frequencies for chromosome 13, 18, 21, X and Y obtained through the use of multi-colour FISH. More than 5000 spermatozoa per chromosome probe were analysed according to strict scoring criteria (Williams et al., 1993Go). We found no significant differences in the frequency of diploid spermatozoa when the same sperm sample from each of 18 men was scored on two separate occasions using different chromosome probes (Table IIGo). Our results indicate that the number of spermatozoa to be scored per individual and per set of probes are sufficient if more than 5000, and that the FISH technique is accurate and reliable for determining the frequency of aneuploidy in human spermatozoa.

Several different criteria are utilized for determining the morphology of spermatozoa. The most widespread are the Word Health Organization criteria (WHO, 1992) and Tygerberg strict criteria (Kruger et al., 1988Go). In the latter, all borderline forms are considered abnormal. It has been found (Enginsu et al., 1991Go) that evaluation according to strict criteria was more effective in predicting fertilization in vitro than the WHO criteria. A previous study in our laboratory (Takano et al., 1999Go) suggested that the fertilization rate in conventional IVF might be estimated from the normal morphology rate and cases in which the normal morphology rate is <6% should be treated by ICSI. In a study of meiotic segregation of 24 spermatozoa obtained from a 47,XXY male (Estop et al., 1998Go), 16 sperm cells presented with abnormal morphology by Kruger's strict criteria, and the rate of sex chromosome abnormality among spermatozoa with abnormal morphology was 61%.

It has not been possible to demonstrate any positive correlation between poor sperm morphology and an increased proportion of chromosomal abnormalities (Martin and Rademaker, 1988Go; Rosenbusch et al., 1992Go). However, other studies (Cohen et al., 1991Go; Parinaud et al., 1993Go) found that poor sperm morphology resulted in poor embryo quality, and there is speculation as to whether IVF with sperm samples of very poor morphology, apart from resulting in a poor fertilization rate, also produces a higher rate of spontaneous abortion (Oehninger et al., 1988Go).

In the present study, we observed a significant negative correlation between the percent of spermatozoa with a normal morphology and the incidence of chromosomal abnormality (r = –0.730) (Figure 1Go). Therefore further large studies are required to find out what the association is between normal morphology spermatozoa and chromosomal abnormality so that during ICSI only the normal morphology spermatozoa can be used.

Our data showed a significant increase (P < 0.05) in the frequencies of diploidy (0.29%), and both autosomal (13, 0.13%; 21, 0.24%) and sex chromosomal disomy in spermatozoa from OAT males. Recent studies showed significant increases in autosomal as well as sex chromosomal aneuploidy in spermatozoa from infertile patients (Moosani et al., 1995Go; Pang et al., 1999Go), and these results suggest that infertile men with poor sperm profiles have an increased frequency of chromosomal non-disjunction, resulting in spermatogenic disruption. However, these genetic mechanisms remain largely unknown. The incidence of aneuploidy was significantly greater in OAT patients than normal patients, which may suggest that chromosomal instability is a result of altered genetic control during mitotic or meiosis cell division and proliferation during spermatogenesis.

Several authors have described an increase in sex chromosome abnormalities in children who had been conceived by ICSI that used spermatozoa from fathers with normal chromosomes. Recently, an increase in chromosomal abnormalities was described in a subject with OAT who was selected for an ICSI programme (In't Veld et al., 1997Go). Our present study showed that in males with sex chromosome abnormalities, as well as in OAT males, the most common disomies were observed in the sex chromosomes. This fact could have a relationship with the increase of sex chromosome anomalies reported after ICSI (Liebaers et al., 1995Go; In't Veld et al., 1997Go).

In conclusion, our preliminary data thus showed that rates of disomy and diploidy were both increased in the majority of the subjects in the OAT group (eight) as compared with the control group (10). The analysis of sperm nuclei by multi-colour FISH in patients with severe oligozoospermia could be useful in estimating the risk of chromosomal anomalies in offspring conceived via ICSI. Because the increased frequency of chromosomal aneuploidy in OAT patients, we also recommend counselling before ICSI.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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]

Blanco, J., Egozcue J. and Vidal, F. (1996) Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization. Hum. Reprod., 11, 722–726.[Abstract]

Briana, J., Cynthia, A., Henry, E. et al. (1993) Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 1929–1936.[Abstract]

Cohen, J., Alikani, M., Malter, H. et al. (1991) Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil. Steril., 56, 696–706.[ISI][Medline]

Downie, S.E., Flaherty S.P. and Matthews C.D. (1997) Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization. Mol. Hum. Reprod., 3, 585–598.[Abstract]

Egozcue, J., Blanco, J. and Vidal, F. (1997) Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization. Hum. Reprod. Update, 3, 441–452.[Abstract/Free Full Text]

Enginsu, M.E., Dumoulin, J.C.M., Pieters, M.H.E.C. et al. (1991) Evaluation of human sperm morphology using strict criteria after Diff-Quick staining: correlation of morphology with fertilization in vitro. Hum. Reprod., 6, 854–857.[Abstract]

Estop, A.M., Munne, S., Cieply K.M. et al. (1998) Meiotic products of a Klinefelter, 47,XXY male as determined by sperm fluorescence in-situ hybridization. Hum. Reprod., 13, 124–127.[Abstract]

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

In't Veld, P.A., Broekmans, F.J., France, H.F. et al. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752–754.[Abstract]

Jacobs, P.A. (1992) The chromosome complement human gametes. In Milligan, S.R. (ed.), Oxford Reviews of Reproductive Biology, vol. 14. Oxford University Press, New York, pp. 47–72.

Kruger T.F., Acosta A.A., Simmons K.F. et al. (1988) Predictive value of abnormal sperm morphology in in-vitro fertilization. Fertil. Steril., 49, 112–117.[ISI][Medline]

Lähdetie, J., Saari, N., Ajosenpoa, M. et al. (1997) Incidence of aneuploid spermatozoa among infertile men studied by multicolour fluorescence in situ hybridization. Am. J. Med. Genet., 71, 115–121.[ISI][Medline]

Liebaers, I., Bonduelle, M., Van Assche, E. et al. (1995) Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet, 346, 1095.

Martin, R.H. and Rademaker, A. (1988) The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat. Res., 207, 159–164.[ISI][Medline]

Martin, R.H., Lin, C.C., Balkman, W. et al. (1982) Direct chromosomal analysis of human spermatozoa: preliminary results from 18 normal men. Am. J. Hum. Genet., 34, 459–468.[ISI][Medline]

Martin, R.H., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225 846 sperm from 10 normal men. Biol. Reprod., 54, 394–398.[Abstract]

McInnes, B., Rademaker, A. and Martin, R. (1998) Donor age and the frequency of disomy for chromosomes 1, 13, 21 and structural abnormalities in human sperm using multicolour FISH. Hum. Reprod., 13, 2489–2494.[Abstract]

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., Pattinson, H.A., Cater, 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]

Oehninger, S., Acosta, A.A., Morshedi, M. et al. (1988) Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil. Steril., 50, 283–287.[ISI][Medline]

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]

Parinaud, J., Mieusset, R., Vieitez, G. et al. (1993) Influence of sperm parameters on embryo quality. Fertil. Steril., 60, 888–892.[ISI][Medline]

Rosenbusch, B, Strehler, E. and Sterzik, K.(1992) Cytogenetics of human spermatozoa: correlations with sperm morphology and age of fertile men. Fertil. Steril., 58, 1071–1072.[ISI][Medline]

Rudak, E., Jacobs, P.A. and Yanagimachi, R. (1978) Direct analysis of the chromosome constitution of human spermatozoa. Nature, 274, 911–913.[ISI][Medline]

Takano,Y., Kihaile, P.E., Hirotsuru, K. and Utsunomiya, T. (1999) The effect of sperm morphology evaluation using strict criteria. Jpn J. Fertil. Steril., 44, 1–6.

Van Opstal, D, Los, F.J., Ramlakhan, S. et al. (1997) Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum. Reprod., 12, 682–686.[Abstract]

Williams, B.J., Ballenger, C.A. and Malter, H.E. (1993) Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 1929–1936.[Abstract]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK, pp. 43–44.

Submitted on September 21, 1999; accepted on January 25, 2000.