Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men

Walter Vegetti1,4, Elvire Van Assche2, Alexandra Frias2, Greta Verheyen3, Massimiliano M. Bianchi1, Maryse Bonduelle2, Inge Liebaers2 and André Van Steirteghem3

1 First Department of Obstetrics and Gynaecology, University of Milan, Via della Commenda 12, 20122 Milan, Italy, 2 Centre for Medical Genetics and 3 Centre for Reproductive Medicine, University Hospital, Dutch-speaking Free University of Brussels (Vrije Universiteit Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = –0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.

Key words: aneuploidy/chromosomes/FISH/male infertility/reciprocal translocation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of intracytoplasmic sperm injection (ICSI) for treatment of infertility due to severe male factor has improved the chances of achieving pregnancy in many infertile couples (Van Steirteghem et al., 1993Go, 1996Go). Nevertheless, concerns have been raised about the possible high risk of chromosomal aneuploidies from paternal origin in the concepti, especially since natural sperm selection does not occur in the ICSI procedure (Chandley and Hargreave, 1996Go). This potential hazard seems to be confirmed by reports of a higher incidence of sex chromosomal aneuploidies of paternal origin and structural de-novo chromosomal abnormalities in children conceived after ICSI, compared to the general population (In't Veld et al., 1995Go; Liebaers et al., 1995Go; Bonduelle et al., 1998Go).

Since men with abnormal sperm parameters, and especially those with severe oligozoospermia, make up the majority of ICSI candidates, it is of great interest to study the chromosomal constitution in their spermatozoa. After the first attempts to study chromosomal constitution of human spermatozoa using differential staining techniques (Pearson and Brobow, 1970Go; Brobow et al., 1972Go; Geraedts and Pearson, 1973Go), in 1976 the zona-free hamster oocyte–sperm fusion technique was introduced (Yanagimachi et al., 1976Go). Two years later, this technique was applied for the first time to visualize and analyse the chromosomal constitution of human spermatozoa (Rudak et al., 1978Go). Since then, many studies have been published (reviewed by Guttenbach et al., 1997a): more than 20 000 sperm chromosome complements from healthy, normal donors and spermatozoa from about 50 men with constitutional chromosome aberrations have been analysed (reviewed by Guttenbach et al., 1997a). The majority of these latter patients were carriers of reciprocal translocations. Although this technique provides valuable information on the whole chromosomal complement of spermatozoa, it is cumbersome, time-consuming, expensive and has been applied by few laboratories. Another drawback of this technique is the possibility to analyse the chromosomal complements in only a limited number of spermatozoa able to fertilize the hamster eggs. The primed-in-situ (PRINS) labelling technique is another tool that has been applied to detect chromosomal aneuploidies in human spermatozoa by the French group in Montpellier (Pellestor et al., 1996aGo,bGo). In the last 10 years, non-isotopic in-situ hybridization (ISH) and fluorescent in-situ hybridization (FISH) have provided a reliable and simple tool for cytogenetic studies. Using whole chromosome painting probes or locus-specific probes it is possible to study numerical and structural chromosomal abnormalities on metaphase chromosomes (Pinkel et al., 1988Go; Schröck et al., 1996Go). Centromeric or locus-specific probes make chromosome enumeration in interphase nuclei possible and since 1990 this technique has been applied to investigate sperm chromosomal aneuploidies. After the first reports using an isotopic ISH technique (Joseph et al., 1984Go; West et al., 1989Go), many reports using non-isotopic ISH (Guttenbach and Schmid, 1990Go; Guttenbach et al., 1994aGo,bGo) and FISH (reviewed by Downie et al., 1997; by Guttenbach et al., 1997a and by Egozcue et al., 1997) have been published to study numerical chromosomal abnormalities in human sperm nuclei from normal fertile men. These results have demonstrated the reliability of this technique in studying human sperm aneuploidies and are comparable, at least in normal men, with data obtained using sperm fusion with the zona-free–hamster egg technique (Martin et al., 1996Go; Estop et al., 1997aGo; Guttenbach et al., 1997aGo). Major advantages of the FISH technique versus the zona-free hamster egg–human sperm fusion technique are that it is simpler, faster and allows for the analysis of thousands of spermatozoa (except in patients with very low sperm count). However, an important disadvantage of the FISH technique applied on interphase nuclei, is that chromosomal structural abnormalities are impossible to detect (except for an attempt to study telomeric deletion and duplication with centromeric and telomeric probes: Van Hummelen et al., 1996). Another disadvantage of applying FISH on interphase nuclei is that so far, it has been impossible to study more than five chromosomes simultaneously (and not more than three in the smaller sperm nuclei).

Using dual and triple colour FISH techniques, some authors recently reported a higher frequency of chromosomal aneuploidies in spermatozoa of infertile men compared to normal fertile donors, at least for some chromosomes (Moosani et al., 1995Go; Pang et al., 1995Go; Colombero et al., 1997Go; Bernardini et al., 1997Go; Lahdethie et al., 1997; Martin et al., 1997Go; Rives et al., 1998aGo; Storeng et al., 1998Go; Pang et al., 1999Go). Conversely, in other reports no differences between infertile and fertile men were found (Miharu et al., 1994Go; Guttenbach et al., 1997bGo). Results from these (or from some of these) previous studies will be discussed later on.

In this study, using dual and triple colour FISH techniques, we analysed the rates of aneuploidy for chromosomes 13, 18, 21, X and Y in 32 infertile patients and in 13 controls with normal semen parameters. We decided to study these chromosomes because trisomies 13, 18 and 21, together with the sex chromosome aneuploidies, are the most common numerical chromosomal abnormalities in human live births (Nielsen and Wohlert, 1991Go). The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes, 13 were infertile men with abnormal karyotypes and normal or abnormal semen, and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an ICSI cycle. We compared the aneuploidy rates of the different chromosomes among the three patient groups and controls. We also evaluated the interchromosomal variation in aneuploidy rates and investigated the correlation between frequencies of aneuploidy and different abnormal semen parameters (sperm concentration, total progressive motility and normal morphology).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Thirty-two patients attending the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University were recruited. Samples were provided by 15 infertile men with normal karyotype and abnormal semen (group 1), 13 infertile men with abnormal karyotype, 11 of whom had abnormal semen (group 2), four infertile men with abnormal semen who fathered a child (or a fetus) with abnormal karyotype following an ICSI procedure (group 3). We also recruited 13 healthy controls with normal peripheral blood karyotype and normal semen analysis. All the controls were selected from the semen donor programme. Characteristics of the analysed patients (n = 32) and controls (n = 13) are detailed in Table I.

Karyotype analysis
Blood (10 ml) was drawn into tubes containing heparin to prevent clotting. Metaphase spreads were made from phytohaemagglutinin-stimulated peripheral lymphocytes using standard cytogenetic techniques (Rooney and Czepulhowski, 1992Go).

Semen preparation and fixation
All the semen samples were first analysed to evaluate concentration, motility, according to the WHO parameters (WHO, 1992), and morphology according to strict criteria (Kruger et al., 1986Go). Semen samples with volume >=2 ml, sperm concentration >=20x106/ml, percentage of spermatozoa with total progressive motility >=50% and with normal morphology >=14% were regarded as normal. Except for 15 samples which were cryopreserved in liquid nitrogen and then thawed and fixed after a few months, all remaining samples were fixed immediately after collection according to the following procedure. The sperm samples were washed three times in phosphate-buffered saline (PBS), pH 7.2, centrifuged at 280 g for 10 min and the sediment was then fixed in methanol/acetic acid (3:1). The fixed specimens were stored at –20°C until further processing. The fixed spermatozoa were spread on Silane-coated slides (Silane-Prep; Sigma Diagnostics, St Louis, MO, USA) and kept at room temperature for 1–3 days. At least two slides were prepared for each patient.

Decondensation treatment
Slides were washed in 2x standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol (DTT) (Martini et al., 1995Go). After decondensation, the slides were washed once in 2xSSC, once in 1xPBS and finally dehydrated through an ethanol series and air-dried. This decondensation treatment is simple and fast and allows the maintenance of sperm structure, including the tail. It also makes the differentiation between spermatozoa and other cells present in the ejaculate easier and unequivocal.

DNA probes
The commercially available kit AneuscreenTM (Vysis, Downers Grove, IL, USA) was used. AneuscreenTM consists of two separate probe mixtures in hybridization buffer.

Probe mixture no. 1 consists of three repetitive DNA sequence centromeric probes (chromosome enumeration probe, CEP®) for chromosome 18 (D18Z1), chromosome X (DXZ1) and chromosome Y (alpha satellite region, DYZ3), directly labelled respectively with the Spectrum AquaTM, Spectrum GreenTM and Spectrum OrangeTM fluorophores.

Probe mixture no. 2 consists of unique sequence DNA locus specific identifier probes (LSITM) specific for chromosome 13 (Retinoblastoma gene, RB-1, located at region 13q14) and 21 (Loci D21S259, D21S341 and D21S342, located at region 21q22.13–q22.2), directly labelled with the Spectrum GreenTM and the Spectrum OrangeTM fluorophores respectively.

FISH procedure
The FISH procedure was performed according to the protocol recommended by Vysis: slides were denatured for 5 min in a 70% formamide 20xSSC solution pre-warmed at 73 ± 1°C in a waterbath. Slides were then dehydrated through an ethanol series and air-dried. Ten µl of each probe mixture was added to each slide (one slide per probe set and two slides per patient) and covered with an 18 mm square glass coverslip. The coverslips were sealed with rubber cement and hybridization took place overnight in a humidified chamber at 37°C. Post-hybridization washings were carried out as follows: coverslips were removed and the slides were immersed immediately in a 0.4xSSC/0.3% NP-40 solution at 73 ± 1°C for 2 min in a waterbath and then in a 2xSSC/0.1% NP-40 solution for 1 min at room temperature. Finally the slides were mounted in Vectashield antifade medium (Vector Laboratories Inc., Burlingame, CA, USA) containing 450 ng/ml 4',6-diamidino-2-phenyl-indole (DAPI) counterstain.

Microscopy and scoring criteria
Slides were observed using a Zeiss-Axioscope fluorescence microscope with the appropriate filter sets (Vysis, Downers Grove, IL, USA): single band pass filter (DAPI), triple band pass filter (DAPI/Orange/Green), dual band pass filter (Aqua/Orange) and a single band pass filter (Green). Only slides with a hybridization rate >=98% were analysed and, with few exceptions, at least 2000 sperm nuclei per slide (4000 per patient) were scored.

For slide scoring we applied the stringent scoring criteria (Williams et al., 1993Go). Only intact spermatozoa bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping spermatozoa were excluded from analysis. Spermatozoa were regarded as abnormal if they presented two (or more) distinct hybridization signals for the same chromosome, each equal in intensity and size to the single signal found in normal monosomic nuclei. We considered only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head. Divided (splitted) signals were not scored erroneously as disomies. Since we performed dual and triple colour FISH procedures, we were also able to evaluate nullisomies: spermatozoa were scored as nullisomic if they did not show any signal of the investigated chromosomes and the signal(s) of the other tested chromosome(s) was present. Finally, a spermatozoon was considered diploid if it exhibited two signals for each tested chromosome and if the tail was evident, as well as the normal oval shape of the head.

Statistical analysis
Student's t-test was used to test the homogeneity of mean ages between patients and controls. Differences in aneuploidy rates for each chromosome among groups were analysed by Wilcoxon score (rank sums), and Spearman correlation coefficients were used to correlate total chromosomal abnormalities and sperm parameters (SAS System, SAS Institute Inc., Cary, NC, USA). The {chi}2-test was used to verify that the proportion of X- and Y-bearing spermatozoa did not deviate from the expected 1:1 ratio and to test the homogeneity of disomy rates for all the different analysed chromosomes among groups (SPSS for Windows, version 6.1.2, SPSS Inc., USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Age, karyotype, semen parameters and reproductive history of patients and controls are detailed in Table I. The mean age of the three groups of patients was not significantly different when compared to controls. Six patients in group 1, two patients in group 2 and three patients in group 3 showed oligoasthenoteratozoospermia (OAT); seven patients in group 1 and five patients in group 2 showed asthenoteratozoospermia; one patient in group 2 showed oligoteratozoospermia; two patients in group 1 and three patients in group 2 showed teratozoospermia only, one patient in group 3 showed asthenozoospermia only and two patients in group 2 showed a normal semen analysis as well as all the controls.

A total of 251 277 spermatozoa was scored: 89 828 in group 1, 51 974 in group 2, 23 906 in group 3 and 112 918 in the control group.

The total rate of chromosomally abnormal spermatozoa was 1.4% in group 1, 2.7% in group 2, 0.8% in group 3, 1.2% in group 1+3 and 0.46% in the control group. Pooling the data of the three patient groups (n = 32), a total of 1.7% of spermatozoa was found to be abnormal.

The number of spermatozoa scored, the nullisomy, disomy and diploidy rates for each patient and each control in the four study groups are reported in Tables IIGo (chromosome 13 and 21) and III (chromosome 18, X and Y). In the calculation of the total means shown in the tables, the data of chromosomes involved in a constitutional translocation were excluded: chromosome 21 in patient no. 13, chromosome 18 in patient no. 19, and chromosome 13 in patient no. 31. These data were also excluded from the final statistical analysis.


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Table II. Incidence of sperm nullisomy and disomy and diploidy for chromosomes 13 and 21 (values are expressed in %) in 35 patients and six controls
 
Chromosomes 13 and 21
The incidence of spermatozoa with nullisomy of chromosomes 13 or 21 was significantly higher compared to controls in group 1 (P = 0.0030 and P = 0.0015 respectively) and in group 2 (P = 0.0022 for both chromosomes) but not in group 3 (P = 0.1441 for both chromosomes). The incidence of disomy for chromosome 13 was significantly higher in group 1 and in group 2 (P = 0.0231 and P = 0.0022 respectively) but not group 3 (P = 0.4652). Similarly, the incidence of disomy for chromosome 21 was significantly higher in group 1 and in group 2 compared to controls (P = 0.0015 and P = 0.0029 respectively).

We also analysed the pooled data of patients from groups 1 and 3, because all these patients were infertile, had abnormal semen parameters and normal karyotypes while the karyotypes of the offspring were abnormal only in patients from group 3. Moreover, statistical analysis of patients from group 3 is influenced by the low number of subjects (n = 4).

After pooling these data, in the 19 patients from group 1 and 3, nullisomy rates for chromosomes 13 and 21 were significantly higher compared to controls (P = 0.0030 and P = 0.0015 respectively), as was the incidence of disomy for these two chromosomes (P = 0.0231 and P = 0.0015 respectively). There was no statistically significant difference between group 1 and group 3 with respect to aneuploidy rates for chromosomes 13 and 21.

The diploidy rate was only significantly higher compared to controls in patients from group 2 (P = 0.0019). Patients from groups 1 and 3, analysed separately and taken together, did not show a significantly higher rate of diploidy compared to controls (P = 0.0597, P = 0.4652 and P = 0.7150 respectively).

Chromosome 18 and sex chromosomes
The incidence of spermatozoa with nullisomy of chromosome 18 or with nullisomy of the sex chromosomes was significantly higher compared to controls in group 1 and in group 2 (group 1: P = 0.0015 and P = 0.0019 respectively; group 2: P = 0.0022 and P = 0.0015 respectively) but not in group 3 (P = 0.0679 and P = 0.0679 respectively). The incidence of disomy for chromosome 18 was significantly higher in group 1 and in group 2 (P = 0.0058 and P = 0.0022 respectively) but not in group 3 (P = 1.0000). For disomy of sex chromosomes we considered the incidence of XX, YY and XY disomies as well as the total sex chromosome disomy rate. XX, YY and XY disomy rates were significantly higher compared to controls in group 1 (P = 0.0071, P = 0.0030 and P = 0.0024 respectively) and also in group 2 (P = 0.0019, P = 0.0071 and P = 0.0019 respectively). In group 3, XX, YY and XY disomy rates were not statistically different when compared to controls. The total rate of disomy of sex chromosomes was significantly higher compared to controls in group 1 and in group 2 (P = 0.0019 for group 1, P = 0.0015 for group 2) but not in group 3 (P = 0.0679).

Analysing the pooled data from patients of groups 1 and 3, we obtained statistically significantly higher rates of nullisomy and disomy for all chromosomes: P = 0.0015 and P = 0.0058 for chromosome 18 nullisomy and disomy rates, P = 0.0019 for sex chromosomes nullisomy rate, P = 0.0071 for XX disomy rate, P = 0.0030 for YY disomy rate and P = 0.0024 for XY disomy rate and P = 0.0019 for the total sex chromosomes disomy rate. Comparing groups 1 and 3, we found no statistically significant difference between these two groups with respect to the aneuploidy rates of chromosome 18 and the sex chromosomes.

The diploidy rate was significantly higher in patients from group 1 and group 2 (P = 0.0058 and P = 0.0107 respectively). Patients from groups 1 and 3, taken together, also showed a significantly higher rate of diploidy compared to controls (P = 0.0058).

Interchromosomal variations
We analysed the disomy rates of the different chromosomes to evaluate if they were differently prone to meiotic non-disjunction. The disomy rate for chromosomes X and Y was significantly higher compared to the disomy rate for chromosome 18 in all three groups of patients and also in the control group (P < 0.001 for all groups). The disomy rate for chromosome 21 was significantly higher compared to the disomy rate for chromosome 13 only in group 1 (P = 0.025). In groups 2, 3 and in the control group there was no statistically significant difference in the disomy rates for chromosomes 13 and 21.

X- and Y-bearing spermatozoa
Among the normal spermatozoa, the percentages of X- and Y-bearing spermatozoa was respectively 49.74% and 50.26% in group 1, 49.31% and 50.69% in group 2, 49.89% and 50.11% in group 3 and 49.47% and 50.53% in group 4. Statistical analysis demonstrated that the proportion of X- and Y-bearing spermatozoa was not significantly different from the 1:1 ratio expected in all patients and controls (individual data are reported in Table IIIGo).


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Table III. Incidence of sperm nullisomy and disomy and diploidy for chromosomes 18, X and Y in 35 patients and six controls. Values are expressed as percentages
 
Correlation between aneuploidy rates and sperm parameters
We evaluated the correlation between the total abnormality rates (sum of nullisomies and disomies of all the investigated chromosomes and of diploidies) and three sperm parameters: concentration of spermatozoa (106/ml), percentage of spermatozoa with progressive motility and percentage of spermatozoa with normal morphology.

The total abnormality rates for chromosomes 13 and 21 and for chromosomes 18, X and Y were significantly and inversely correlated in group 1 with the concentration of spermatozoa (r = –0.907, P = 0.0001 for chromosome 13 and 21 total aneuploidies and –0.810, P = 0.0002 for chromosome 18, X and Y total aneuploidies) (Figures 1 and 2GoGo) and with the total progressive motility (r = –0.524, P = 0.0449 for chromosome 13 and 21 total aneuploidies and –0.556, P = 0.0313 for chromosome 18, X and Y total aneuploidies) (Figures 3 and 4GoGo). Such a correlation was not present in group 1 with the normal morphology and in the other groups with all the three sperm parameters considered (data not shown).



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Figure 1. Scatterplot of the sperm concentration versus the total number of abnormalities (chromosomes 13 and 21) in group 1 (15 patients) (Spearman correlation coefficient = –0.907, P = 0.0001).

 


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Figure 2. Scatterplot of the sperm concentration versus the total number of abnormalities (chromosomes 18, X and Y) in group 1 (15 patients) (Spearman correlation coefficient = –0.810, P = 0.0002).

 


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Figure 3. Scatterplot of the total progressive motility of spermatozoa versus the total number of abnormalities (chromosomes 13 and 21) in group 1 (15 patients) (Spearman correlation coefficient = –0.52415, P = 0.04).

 


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Figure 4. Scatterplot of the total progressive motility of spermatozoa versus the total number of abnormalities (chromosomes 18, X and Y) in group 1 (15 patients) (Spearman correlation coefficient = –0.55635, P = 0.0313).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since 1990, many studies have been published using FISH to investigate the prevalence of aneuploidy in human spermatozoa. In the first published studies, results were hampered by the use of a single probe technique. This approach allowed for the study of only a single chromosome: disomic from diploid spermatozoa could not be discriminated, and neither could nullisomic spermatozoa be differentiated from the ones with lack of hybridization. Applying two-colour FISH with two probes labelled with two different fluorochromes or three-colour FISH with three probes labelled with three different fluorochromes, it is possible to obtain reliable data on nullisomy, disomy and diploidy rates of the analysed spermatozoa. Not all the chromosomes have been studied so far: for example the aneuploidy rates for chromosomes 19 and 22 have been reported only by one group (Rives et al., 1998bGo).

The reported frequencies of disomy in normal fertile men range from 0.23 to 0.28% for chromosome 13, 0 to 0.39% for chromosome 18 and 0.1 to 0.48% for chromosome 21. With regard to sex chromosomes, the reported frequencies range from 0.018 to 0.7% for XX disomy, 0.009 to 0.6% for YY disomy and 0.062 to 0.42% for XY disomy; the diploidy rates range from 0.06 to 0.97% (see Downie et al., 1997 and Egozcue et al., 1997 for review). These results are often not homogeneous when several studies are compared. This variability may be explained in different ways: first, the patients belonged to various geographical areas, criteria for patient selection were dissimilar and the semen samples were probably obtained after variable periods of abstinence; second, FISH protocols were not homogeneous: decondensation procedures were different and probes with various hybridization efficiencies were used; third, the same scoring criteria were not always applied.

The decondensation treatment is crucial in order to obtain a proper hybridization efficiency: this makes it possible for the probes to access the extremely highly histamine-packed DNA of the spermatozoa. The decondensation procedure is one of the most frequent variables when comparing different studies. Taking into account these variables, and especially the stringent scoring criteria (Williams et al., 1993Go) applied in our study (which can explain our relatively low percentages of disomic and diploid spermatozoa), the results in our control group are comparable with results previously reported in literature for normal fertile men.

In 1994 the first report on spermatozoa from infertile men analysed by FISH was published (Miharu et al., 1994Go). Since then, more studies on infertile men with abnormal semen parameters have been published. At least 11 out of these reports analysed chromosomes 13, 18, 21, X and Y (Table IV) (Miharu et al., 1994Go; Moosani et al., 1995Go; Pang et al., 1995Go, 1999Go; Bernardini et al., 1997Go; Colombero et al., 1997Go; Estop et al., 1997aGo; Francisco et al., 1997Go; Guttenbach et al., 1997bGo; Storeng et al., 1998Go; Rives et al., 1998aGo). Another study (Lähdetie et al., 1997Go) investigated chromosomes 1 and 7 in infertile patients with normal or nearly normal semen parameters and in OAT patients. The reported findings are not unequivocal. In one study (Miharu et al., 1994Go), the rate of disomy for chromosomes 1, 16, X and Y in spermatozoa from nine fertile and 21 infertile subjects using FISH with single colour chromosome-specific probes and the rate of disomy for chromosomes 17 and 18 using FISH with a dual colour technique were investigated. They analysed a total of 450 580 spermatozoa and they did not observe differences between fertile and infertile men for either diploidy or disomy for the investigated chromosomes. On the contrary, another group (Pang et al., 1995Go), found a significant increase in the frequencies of diploidy, autosomal and sex chromosomal aneuploidies in sperm samples from nine OAT infertile patients, compared to four donor controls. They investigated chromosomes 7, 11, 12, 18, X and Y using a two-probe, two-colour FISH; 1000 spermatozoa for each probe set were scored and in OAT patients higher disomy rates for chromosome 18 and for the sex chromosomes, together with a higher diploidy rate, were found. In the same year, chromosomes 1, 12, X and Y using a dual-colour FISH were investigated (Moosani et al., 1995Go), together with a complete sperm karyotyping using the zona-free hamster egg–human sperm fusion technique. This group reported an increased frequency of chromosomal abnormalities in five men with OAT and infertility. In 1997, in three studies (Bernardini et al., 1997Go; Colombero et al., 1997Go; Francisco et al., 1997Go), using multiprobe, multicolour FISH, a higher frequency of autosomal and/or sex chromosomal disomies in OAT infertile men was demonstrated. In one of these studies (Francisco et al., 1997Go) it was also demonstrated that the swim-up semen preparation did not allow for the separation of haploid from aneuploid spermatozoa. In the same year another group (Guttenbach et al. 1997bGo) did not find any difference in disomy rates for chromosomes 1, 7, 10, X and Y between 45 infertile patients with varying degrees of semen abnormalities and six healthy controls of proven fertility.

Comparing semen samples from patients undergoing IVF and ICSI, a significantly higher incidence of sex chromosomal aneuploidies in the latter patients was found (Storeng et al., 1998Go). A French group (Rives et al., 1998aGo) investigated semen samples from 50 infertile males with semen abnormalities of various degrees and from 10 controls. Using whole chromosome paint probes for chromosomes 1, 13, 14, 18, 21, 22, X and Y they found an increased incidence of YY- and XY-bearing spermatozoa and a higher incidence of total autosomal aneuploidy in sperm nuclei from infertile OAT patients compared to control subjects with proven fertility. This study also showed a significant difference in the disomy frequencies between the different autosomes, with the highest frequency for chromosome 18 (0.51%) and the lowest for chromosome 1 (0.35%). More recently nine infertile patients undergoing ICSI were analysed for aneuploidies of chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y (Pang et al., 1999Go). The authors found significantly elevated frequencies of diploidy, aneuploidies of autosomes and of sex chromosomes and total aneuploidy in OAT patients. The frequencies per chromosome for disomy for autosomes and sex chromosomes in OAT patients were 0–5.4% versus 0.05–0.2% in controls. Interestingly six series of ICSI in five of these patients failed to gave a viable pregnancy and the authors therefore suggested that the elevated aneuploidy in spermatozoa of these patients can contribute to their infertility.

These different results, as reported for studies in fertile men, are most probably due to the different criteria for patient recruitment, the different decondensation treatment used, the different probes and FISH protocols applied and also the different scoring criteria adopted.

Nevertheless, our results are in agreement with the majority of the reported data. According to our findings, there seems to be a higher incidence of chromosomes 13, 18, 21, X and Y aneuploidies in infertile patients with abnormal semen and normal karyotype (group 1) and in pooled patients of group 1 and group 3, compared to normal controls. Moreover, in these patients an inverse correlation between aneuploidy rates and sperm parameters (concentration and total progressive motility) was found. Such a correlation was present for all the investigated chromosomes. On the contrary we did not find a significant correlation between chromosomal aneuploidies and abnormal morphology of spermatozoa, although this finding was reported by other authors (Yurov et al., 1996Go; Colombero et al., 1997Go; Estop et al., 1997aGo; In't Veld et al., 1997Go).

Infertile patients with abnormal karyotype and normal or abnormal semen were also investigated (group 2). Data from the literature regarding aneuploidy rates evaluated by FISH in spermatozoa from patients with abnormal karyotype are less numerous and semen samples from only a few patients have been analysed. These data are also difficult to compare among published studies because it is probable that the degree of semen parameter abnormalities might be conditioned by the different chromosomes involved in a chromosomal rearrangement and by the different breakpoints (Van Assche et al., 1996Go). To substain this hypothesis, it is interesting to note that two out of 10 infertile patients carrying a reciprocal translocation in our study presented normal semen analysis (patients no. 6 and 33).

Some other authors studied patients with reciprocal or Robertsonian translocation or inversion using FISH (Lu et al., 1994Go; Rosseaux et al., 1995aGo,bGo; Colls et al., 1997Go; Martini et al., 1998Go; Mercier et al., 1998Go).

However, these previously reported results are too scanty to draw any conclusion. In this study, we analysed by FISH semen samples from a group of 10 patients carrying a reciprocal translocation, two patients carrying a Robertsonian translocation and one patient with a 46,XY/47,XYY mosaic in order to evaluate the aneuploidy rates for chromosomes 13, 18, 21, X and Y. From our findings there appears to be a higher frequency of diploid spermatozoa and of aneuploidies for chromosomes 13, 18, 21, X and Y in patients carrying a translocation compared to controls. Therefore the hypothesis of an interchromosomal effect of a chromosomal rearrangement on meiotic pairing, as reported by other authors (Spriggs and Martin, 1994Go; Rousseaux et al., 1995a,b; Mercier et al., 1998Go), seems acceptable.

In group 2 a male carrier of a low grade 46,XY/47,XYY mosaicism was included. The mosaicism was found in one out of 200 metaphase nuclei and in four out of 200 interphase nuclei of peripheral lymphocytes by FISH. This patient (no. 26) presented a similar diploidy rate and disomy rates for chromosomes 13, 18, 21, X and Y compared to normal controls and an X- and Y-bearing spermatozoa ratio not different from 1:1. There are few data in the literature on FISH analysis on spermatozoa from patients carrying an extra sex chromosome. The majority of these studies suggest a higher incidence of aneuploid spermatozoa in 47,XYY males (Martini et al., 1996Go; Mercier et al., 1996Go; Blanco et al., 1997Go) and in a Klinefelter male (Estop et al., 1998Go). Two analysed Klinefelter mosaics (Chevret et al., 1996Go; Martini et al., 1996Go) showed increased levels of aneuploidies. All these patients were found able to produce mature normal haploid spermatozoa, with an X:Y ratio not significantly different from 1:1 (Martini et al., 1996Go; Blanco et al., 1997Go) or with an excess of Y-bearing spermatozoa (Mercier et al., 1996Go; Chevret et al., 1997Go) or an excess of X-bearing spermatozoa (Chevret et al., 1996Go).

The four infertile patients from group 3 were analysed separately because they fathered a child with an abnormal karyotype through an ICSI cycle: in patients no. 14 and 18, the paternal origin of the supernumerary Y chromosome is obvious, whereas the paternal origin of the supernumerary 21 and Y chromosomes in patient no. 20 was proven by molecular analysis. With regard to patient no. 17, no DNA was available from the miscarriage for molecular analysis, thus the origin of the supernumerary 16 chromosome could not be proven. The paternal origin of the majority of prenatally detected chromosome aberrations was also demonstrated in another ICSI series (six out of nine cases) (Van Opstal et al., 1997Go).

The mean age of these four patients was 33 years (range 31–36) and the mean age of their wives was 30.2 years (range 28–33). This is the first report on an aneuploidy study of spermatozoa from infertile men who fathered offspring with abnormal karyotype through an ICSI procedure. Probably due to the small number of these subjects (n = 4), in this group we did not observe a significantly higher frequency of aneuploidy rate for the analysed chromosomes compared to controls. These patients also did not show any significant difference in aneuploidy rates compared to patients of group 1 and therefore seem to have the same risk for aneuploidy in offspring as patients of group 1. According to our data, the frequency of abnormal spermatozoa seems to be influenced by semen parameters more than by the history of a conceptus carrying a chromosomal aneuploidy: the incidence of aneuploidy in spermatozoa of our patients is significantly correlated with the poor semen quality, distinctively with a low sperm concentration and a low total progressive motility and it is not higher in group 3 compared to group 1. Patients with the highest risk for aneuploidies in spermatozoa and in offspring therefore seem to be men with severe oligoasthenozoospermia. Our findings in severe OAT patients are in agreement with other reports which show that the frequency of non-disjunction of autosomes and gonosomes increases when the sperm count decreases (Rives et al., 1998aGo; Storeng et al., 1998Go). These data are also confirmed by the results of a follow-up study (Bonduelle et al., 1998Go) on children conceived after ICSI: patients at a higher risk of conceiving a child with an abnormal karyotype are males with severe OAT.

On the other hand, about 90% of trisomies are maternal in origin (Koehler et al., 1996Go) and studies on oocytes of similar age groups (30–39 years) have showed an aneuploidy rate above 20% (Dailey et al., 1996Go; Martini et al., 1997Go). These data suggest that the increase in aneuploidy rate in oligoasthenozoospermic men is of limited clinical relevance, when compared to the maternal contribution to chromosomal abnormalities of the embryos. It has also been demonstrated that there is no difference in chromosome abnormalities between embryos obtained after ICSI and IVF (Gianaroli et al., 1997Go; Munné et al., 1998Go). However, in our opinion, such an increase in aneuploidy rate in spermatozoa, inversely correlated with sperm concentration and motility, should not be underestimated. If each chromosome shows this slight increase in aneuploidy rate, it is possible to speculate a total rate of aneuploidy of ~33–74% in spermatozoa from OAT men (Pang et al., 1999Go). It would also be of great interest to analyse the chromosome complements of epididymal and testicular spermatozoa, since many ICSI procedures are now performed in patients who do not have spermatozoa in the ejaculate.

As a consequence we believe that prenatal diagnosis after an ICSI pregnancy should be offered to all couples, especially to those with a severe male factor and certainly when a structural chromosomal rearrangement in the paternal karyotype is present.

Another point of interest is to study the segregation of rearranged chromosomes in carriers of a reciprocal translocation. These patients show a different risk of conceiving a child with an unbalanced karyotype and also different semen parameters, ranging from a normal semen analysis to severe OAT, possibly depending on the chromosomes involved in the rearrangement and on the breakpoints. So far these segregation studies have been performed in <5000 spermatozoa from 36 carriers using the zona-free hamster egg–sperm fusion technique (reviewed by Guttenbach et al., 1997a) and in few patients using the FISH technique (Lu et al., 1994Go; Spriggs and Martin, 1994Go; Rousseaux et al., 1995a,b; Colls et al., 1997Go; Estop et al., 1997bGo; Martini et al., 1998Go; Mercier et al., 1998Go), mainly due to the difficulties in obtaining probes to study the chromosomes involved in the translocation. A segregation study on our patients carrying a reciprocal translocation (10 patients in group 2) is in progress and data from one of these patients (patient no. 25) have been published elsewhere (Van Assche et al., 1999Go).

In conclusion, our data suggest a correlation between poor semen parameters and an increase in chromosomal aneuploidy rate of spermatozoa. Nevertheless, the majority of spermatozoa in these patients seems to be normal for chromosomes 13, 18, 21, X and Y. The risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated with sperm concentration and total progressive motility. Patients with abnormal karyotype showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls, strongly suggesting the presence of an interchromosomal effect of the cytogenetic rearrangement. These observations suggest the necessity for proper counselling before ICSI in couples with a severe male factor or with a male partner carrying a chromosomal rearrangement.

Finally, men who fathered a child with an abnormal karyotype through an ICSI procedure did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to similar male factor. These data, as well as other publications, support the hypothesis that the increase in sex chromosomal anomalies after ICSI is possibly the consequence of sperm aneuploidies rather than a consequence of micromanipulation.


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Table IA. Semen parameters of three groups (nos. 1–3) of patients and a control group (no. 4)
 

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Table IB. Clinical characteristics of three groups (nos. 1–3) of patients and a control group (no. 4)
 

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Table IVA. Studies of sperm disomy in infertile men for chromosomes 13, 18, 21, X and Y using in-situ hybridization (ISH) and fluorescent in-situ hybridization (FISH)
 

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Table IVB. Studies of sperm disomy in infertile men for chromosomes 13, 18, 21, X and Y using in-situ hybridization (ISH) and fluorescent in-situ hybridization (FISH)
 

    Acknowledgments
 
Dr W.Vegetti would like to thank Prof. Pier Giorgio Crosignani, Chief of the First Department of Obstetrics and Gynaecology of the University of Milan, who offered him the opportunity to spend 11 months in the Medical Genetics Centre in the Free University of Brussels. The authors are grateful to Yvonne Pomposo for correcting the manuscript. The authors would also like to thank the technical and clinical staff of the Centre for Medical Genetics and Centre for Reproductive Medicine of the Free University of Brussels. Grants from the Flemish foundation for Medical Research are gratefully acknowledged.


    Notes
 
4 To whom correspondence should be addressed

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    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 13, 1999; accepted on September 7, 1999.