Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters*

Aldo E. Calogero1,5, Adele De Palma1, Caterina Grazioso1,2, Nunziata Barone1, Rosa Romeo3, Giancarlo Rappazzo4 and Rosario D'Agata1

1 Division of Endocrinology and Master in Andrological Sciences: New Methodologies in Human Reproductive Medicine, University of Catania, 2 Master in Endocrinological and Metabolic Sciences, University of Naples, Naples, 3 Department of Human Anatomy and 4 Department of Animal Biology, University of Catania, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A large proportion of patients with oligoasthenoteratozoospermia (OAT) have an abnormal karyotype and hence they produce aneuploid gametes. However, a normal karyotype does not exclude the chance of having germ cell aneuploidy, since an altered intra-testicular environment not only damages spermatogenesis, but may also disrupt the mechanisms controlling chromosomal segregation during meiosis. Therefore, this study was undertaken to evaluate the rate of aneuploidy in the spermatozoa of selected patients with abnormal sperm parameters. For this purpose, sperm aneuploidy rate for chromosomes 8, 12, 18, X and Y was evaluated by multicolour fluorescence in-situ hybridization (FISH) in nine patients with teratozoospermia alone and 19 OAT patients of presumably testicular origin. Thirteen normozoospermic healthy men served as controls. Patients with teratozoospermia or OAT had significantly greater disomy and diploidy rates compared with controls, whereas the rate of nullisomy was similar. XY disomy was very low in all groups, suggesting that chromosomal non-disjunction occurs mainly during the second meiotic division. Autosome 12 disomy rate was low in both patients and controls. There was a marked variability of total sperm aneuploidy rate in both groups of patients. Sperm aneuploidy rate was negatively correlated with sperm concentration and particularly with the percentage of normal forms. In conclusion, patients with teratozoospermia or OAT have an increased rate of sperm aneuploidy. This increase is similar in both groups, suggesting that teratozoospermia may be the critical sperm parameter associated with aneuploidy.

Key words: chromosomes 8, 12 and 18/multicolour fluorescence in-situ hybridization/oligoasthenoteratozoospermia/sex chromosomes/sperm aneuploidy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Spermatogenetic impairment is responsible for diverse semen abnormalities, the most severe damage leading to azoospermia. The causes of spermatogenetic disorders in infertile men are multifactorial, including abnormal blood karyotype, leading to a variegated pathological impact on the germ cell line. A large number of studies has indeed shown that the prevalence of somatic chromosome abnormalities detectable with karyotype is 10-fold higher in infertile men with impaired spermatogenesis (Bourrouillou et al., 1985Go); however, a normal somatic karyotype does not exclude the possibility of producing aneuploid gametes in these patients (De Breekeleer and Dao, 1991Go; Huang et al., 1999Go; Vendrell et al., 1999Go). These data suggest a relationship between the incidence of chromosome abnormalities and the degree of spermatogenetic impairment. Indeed, an altered testicular environment, such as that found in patients with impaired spermatogenesis, may also cause meiotic errors (i.e. severe meiotic arrest, synaptic anomalies), resulting in cell instability and disorders during cell proliferation, as recently shown in the mouse (Mroz et al., 1998Go). Accordingly, using double- and triple-colour fluorescence in-situ hybridization (FISH), some authors have reported a higher frequency of sperm aneuploidy rate in patients with abnormal sperm parameters compared to normal controls, at least for some chromosomes (Moosani et al., 1995Go; Bernardini et al., 1997Go; Lahdetie et al., 1997Go; McInnes et al., 1998Go; Storeng et al., 1998Go; Pang et al., 1999Go; Rives et al., 1999Go; Nishikawa et al., 2000Go; Ushijima et al., 2000Go; Vegetti et al., 2000Go). Conversely, other studies have failed to show any significant difference in sperm aneuploidy rate between fertile and infertile patients (Miharu et al., 1994Go; Guttenbach et al., 1997Go). Although real population differences and different methodological approaches may account for this discrepancy, it is believed that the most important factor is the dissimilar criteria used in selecting the patients. Indeed, some authors have estimated sperm aneuploidy rate in patients with oligoasthenoteratozoospermia (OAT), whereas others have enrolled infertile patients with normal semen parameters. Even when only patients with OAT were included in the study, they did not always manifest a homogeneous alteration of the sperm output and no or little information was given about their andrological clinical history. On this account, the present study was undertaken to evaluate the sperm aneuploidy rate in selected patients with abnormal sperm parameters and its relationship with the sperm output. Since a preliminary study had suggested that teratozoospermia was associated with an increased aneuploidy rate (Colombero et al., 1997Go), patients were enrolled with teratozoospermia alone or with OAT, on the assumption that severe testicular damage might be associated with a higher sperm aneuploidy rate. Both groups of patients underwent accurate andrological diagnostic screening so that extra-testicular causes of teratozoospermia or OAT might be excluded. A group of healthy normozoospermic men served as control.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient selection
Twenty-eight patients, aged 30.1 ± 1.1 years (mean ± SEM), attending our Andrology and Reproductive Endocrinology Unit (AREU) for the work-up of their infertile status after experiencing at least 2 years of infertility, were recruited for this study. Nine patients had teratozoospermia (group 1) with normal sperm concentration, 19 had oligoteratozoospermia (group 2) in at least two semen samples. All but six (three in group 1 and three in group 2) also had asthenozoospermia. Patients were selected if they fulfilled the following criteria, chosen as an expression of semen abnormality mainly of testicular origin: normal or reduced testicular volume, normal or increased FSH concentrations and negative andrological history for causes or signs of pathology of the excretory genital tract. In the cases of normal testicular volume and serum FSH, patients had to have no clinical or ultrasound signs of epididymal abnormality (Vicari, 1999Go). Patients with abnormal forms thought to be of epididymal origin (cytoplasmic droplets, coiled tails, short tails) (Keel, 1990Go) were also excluded. To define normal sperm aneuploidy rate, 13 healthy men aged 26.5 ± 1.4 years (mean ± SEM) with normal sperm density, motility and morphology, served as controls (group 3).

Morphology assessment was performed on fresh seminal semen according to Kruger's strict criteria (Kruger et al., 1986Go), whereas the other parameters were evaluated according to the World Health Organization criteria (WHO, 1992). Semen was collected by masturbation after 4–5 days of abstinence. Semen samples with volume >=2 ml, sperm concentration >=20x106/ml, percentage of spermatozoa with total motility >=50% and with normal morphology >=14% were regarded as normal. The clinical, hormonal and seminal characteristics of patients (n = 28) and controls (n = 13) are shown in Table IGo.


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Table I. Clinical, hormonal and seminal characteristics of patients and controls enrolled in the study
 
Karyotype analyses
Blood (3 ml) was withdrawn into tubes containing heparin to prevent clotting. Metaphase spreads were made from phytohaemagglutinin-stimulated peripheral lymphocytes using standard cytogenetic techniques (Rooney and Czepulkowski, 1992Go).

Semen preparation for FISH analysis
Following liquefaction, sperm samples were washed three times in phosphate buffered saline (PBS), pH 7.2, centrifuged at 650 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.

Sperm head decondensation
Methanol/acetic acid-fixed spermatozoa were spread on slides and the slides were washed in 2xstandard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol (DTT) (Martin et al., 1995Go). This treatment did not disrupt the sperm structure, including the tail, making the differentiation between spermatozoa and other cells present in the ejaculate easier and unequivocal.

DNA probes
A double- and a triple-colour FISH were carried out on each patient and control, using {alpha}-centromeric probes for chromosomes 8, 12, 18, X and Y. The probe mixture for triple FISH consisted of a repetitive DNA sequence of centromeric probes for chromosome X (pDMX1), labelled with fluorescein isothiocyanate (FITC), for chromosome Y (pLAY5.5) labelled with Cy3 and for chromosome 12 (pBR12) labelled with FITC and Cy3. The probe mixture for double-colour FISH consisted also of a repetitive DNA sequence of centromeric probes for chromosome 8 (pZ8.4) and for chromosome 18 (2Xba), labelled with FITC and Cy3 respectively. The probes were provided by Prof. M. Rocchi, University of Bari (Bari, Italy).

Hybridization procedure
Each slide was denatured in a polymerase chain reaction (PCR) machine with a solution of 70% formamide (Merck Eurolab., Milan Italy) 2xSSC (pH 7.5) at 80°C for 150 s. The slides were immersed in a 70%, 90% and 100% ethanol series for 3 min each and dried by air. The probes, precipitated and denatured at 80°C for 8 min, were applied directly to the slides which were then covered with a coverslip and sealed with rubber cement. Hybridization occurred overnight in a dark humidified container at 37°C, after which one coverslip was removed and the slides were immersed in a post-hybridization wash of 50% formamide/2xSSC for three times at 37°C for 5 min, 2xSSC for three times at 42°C for 5 min and 2xSSC/0.1% Tween 20 at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-phenylindole (DAPI, DBA Srl, Milan, Italy) counterstain and antifade and stored in the dark at 4°C with a view of carrying out microscope observation.

Scoring criteria
The slides were observed using an Axiophot® fluorescent microscope (C. Zeiss, Oberkochen, Germany) with the appropriate set of filters: single band DAPI, FITC, and Cy3. Only slides with a hybridization rate >98% were analysed and, with few exceptions, about 2000 sperm nuclei per slide (about 4000 per patient) were scored. 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. Only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head were considered. Divided (split) signals were not scored as disomies. Spermatozoa were scored as nullisomic if they showed no signal for a given chromosome, whereas the signal of the other chromosome tested was present. Finally, a spermatozoon was considered diploid if it manifested two signals for each tested chromosome and if the tail as well as the normal oval shape of a sperm head were evident. No FISH signals in a spermatozoon head showing DAPI stain were considered a case of no hybridization.

Statistical analysis
Results are shown as median and range throughout the study, unless otherwise indicated. The data were analysed with the Mann–Whitney test, the Wilcoxon test, and the one-way analysis of variance (ANOVA), followed by the HSD Tukey test, as appropriate. Correlations between sperm aneuploidy rate and sperm concentration, percentage of normal forms or total motility were calculated with the Spearman correlation coefficient. Statistics Package for Social Sciences (SPSS) 9.0 for Windows was used for statistical calculation. A significant statistical difference was accepted when the P value was lower than 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The average age of the two groups of patients was not significantly different from that of the controls (Table IGo). A total of 147 243 spermatozoa were scored: 34 720 in group 1, 61 846 in group 2 and 50 677 in group 3. An average of 3951 sperm nuclei per subject was counted.

Total abnormal chromosomal rate
The total sperm aneuploidy rate (disomy and nullisomy) was 2.08% (range: 1.18–4.56%) in group 1, 1.83% (range: 1–9.73%) in group 2 and 0.87% in the control group (range: 0.3–1.18%) (P < 0.001 versus control for both groups). Pooling the data of the two groups of patients (n = 28), a total of 1.94% (range: 1–9.73%) of spermatozoa was found to be aneuploid. Total individual aneuploidy rate showed a wide variation in groups 1 and 2. Although all group 1 patients had an aneuploidy rate above the upper range of controls, four of them had values close to this value, whereas the others had values substantially higher. An even larger variation was observed among group 2 patients; one of them had a value within the normal range, six had values close to the upper range of controls and the remaining 12 had markedly elevated values (Figure 1Go).



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Figure 1. Sperm aneuploidy rate for chromosomes 8, 12, 18, X and Y in normozoospermic healthy men (CTL) (n = 13) and patients with teratozoospermia and normal sperm concentration (T) (n = 9) or with oligoasthenoteratozoospermia (OAT) (n = 19).

 
The diploidy rate of group 1 (0.05%, range: 0–0.42%) did not differ significantly from that of the controls, whereas the diploidy rate of group 2 (0.16%, range: 0–0.52%) was significantly higher (P < 0.001) compared to the one in the controls (0.03%, range: 0–0.20%). Overall the median diploidy rate of all patients was 0.18% (range: 0–0.52%), which was significantly higher than that of the controls (P < 0.002).

Chromosome 8 and 18
The number of spermatozoa scored and the nullisomy, disomy and diploidy rates for each patient and control are reported in Table IIGo. The incidence of disomy for chromosome 8 was significantly higher in group 1 and 2 (P < 0.005) compared to the one in the controls. The disomy rate for chromosome 18 was also significantly higher in groups 1 and 2 (P < 0.001 and P < 0.05 respectively) compared to that of the controls. The median incidence of nullisomy was zero in all groups. After pooling data from groups 1 and 2, nullisomy rates for chromosomes 8 and 18 were similar to those in the controls, whereas the incidence of disomy for the two above mentioned chromosomes (0.48%; range: 0–2.89% and 0.53%; range: 0.05–1.91% respectively) was significantly higher compared to those in the controls (P < 0.001).


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Table II. Chromosome 8 and 18 double colour fluorescence in-situ hybridization (FISH)
 
Chromosome 12 and sex chromosomes
The number of spermatozoa scored and the nullisomy, disomy and diploidy rates for each patient and control are reported in Table IIIGo. The incidence of disomy for chromosome 12 was low and was the same in both patients and controls. There were no nullisomic spermatozoa for chromosome 12 in the three groups. XY disomy was the same in the three groups, whereas XX and YY disomy rates were significantly higher in group 1 (P = 0.009 and P = 0.002 respectively) and 2 (P < 0.001 and P = 0.001 respectively). The total rate of disomy of the sex chromosomes was 0.73% (range: 0.5–2.18%) in group 1 and 0.91% (range: 0.55–6.58%) in group 2, significantly higher (P < 0.001 for both groups) than in the controls (0.38%, range: 0–0.62%). The median rate of the sex chromosome nullisomy was zero in all groups. The incidence of total disomy for the sex chromosomes in groups 1 and 2 was 0.89% (range: 0.5–6.58%), significantly higher than the one in the controls (0.38%, range: 0–062%) (P < 0.001).


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Table III. Chromosome 12, X and Y triple-colour FISH
 
Interchromosomal variations
The disomy rates of the different chromosomes were analysed to evaluate if they were differently prone to meiotic non-disjunction. The sex chromosome disomy rate was significantly higher than that of the three autosomes analysed in patients and controls (P < 0.02, ANOVA, followed by the HSD Tukey test). The chromosome 12 disomy rate was significantly lower than the disomy rate of chromosomes 8 and 18 (P < 0.05, ANOVA, followed by the HSD Tukey test). No statistically significant difference was observed between the disomy rates of chromosomes 8 and 18.

Correlation between aneuploidy rates and sperm parameters
Sperm aneuploidy rate was negatively correlated with sperm density (r = -0.49, P = 0.001) and normal forms (r = -0.63, P < 0.001), but not with total motility (r = -0.27, P = 0.08) (Figure 2Go).



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Figure 2. Scatterplot of sperm concentration (upper panel), percentage of normal forms (middle panel) and total motility (lower panel) versus the total sperm aneuploidy rate for chromosomes 8, 12, 18, X and Y in patients with abnormal sperm parameters (n = 28) and normozoospermic healthy men (n = 13).

 
X- and Y-bearing spermatozoa
The median rates of X- and Y-bearing spermatozoa were 49.7% and 49.4% in group 1, 49.2% and 49.2% in group 2, and 49.6% and 49.6% in group 3. These rates were not significantly different from the expected 1:1 ratio.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of this study was to investigate the aneuploidy rate in the spermatozoa of men with abnormal semen parameters. The patients were selected according to criteria established with a view to including only patients with an impaired spermatogenesis as the aetiopathogenetic factor of their semen abnormality. Certainty of spermatogenetic impairment, however, arises only from testicular histological evaluation, which was not performed on these patients for ethical reasons. Therefore, the intervention of excretory factors responsible for sperm abnormality in this and other similar studies might not be completely ruled out.

Primary testiculopathy is characterized by diverse histological phenotypes which as a common feature have a more or less severe spermatogenetic impairment, resulting in abnormal semen parameters. An impaired spermatogenetic process is commonly associated with a compromised testicular environment, which might make the germ cells particularly susceptible to meiotic abnormalities, as shown in mice (Mroz et al., 1998Go). Isolated teratozoospermia or teratozoospermia associated with oligoasthenozoospermia is taken as evidence of disorder in cell division, proliferation and differentiation in the germ cell line of infertile males (Egozcue et al., 1983Go; Vendrell et al., 1999Go). Consistent with this, the results of the present study provide evidence of increased chromosomal numerical abnormalities in the spermatozoa from men with teratozoospermia but with normal sperm concentration as well as in patients with OAT. The effect was largely attributable to an increase in sex chromosome and autosome disomies, both in patients with teratozoospermia and in patients manifesting a severer semen alteration such as OAT.

Pooling the data of the two groups of patients, a total of 1.94% of the spermatozoa was found to be aneuploid; the highest aneuploidy rate found was 9.73% in a patient with OAT. Chromosome 12 showed the lowest aneuploidy rate and there was a very low frequency of nullisomic spermatozoa in the controls and patients. The rate of chromosomally abnormal spermatozoa was quite comparable in patients with teratozoospermia and OAT, suggesting that the hallmark for such abnormal genetic constitution was represented by teratozoospermia, which to some extent reflected some degree of spermatogenetic impairment (Bernadini et al., 1998). Therefore, the data presented here pointed to an association between abnormal genetic constitution and sperm abnormal morphology, regardless of the sperm number; in spite of the disparate individual sperm aneuploidy rate, this finding was quite consistent in each patient. This association has been proved to be quite strong in the extreme case of an infertile patient, with 100% macrocephalic sperm heads, and in whom chromosomal abnormalities were virtually detected in all sperm cells (In't Veld et al., 1997Go; Viville et al., 2000Go). More recently, Bernardini et al. have found high frequencies of aneuploidy and diploidy rates in spermatozoa with enlarged heads and with two tails, one head (40% and 30% respectively) (Bernardini et al., 1998Go). Moreover, semen with severe teratozoospermia has been studied in a few cases but, after multicolour FISH, these types of abnormal sperm cells were constantly found to carry enormously high rates of aneuploidy and diploidy (Bergère et al., 1997Go; Weissenberg et al., 1998Go).

Previous studies using FISH for the detection of disomy rates in infertile patients with varying degrees of sperm abnormalities support the current findings regarding patients with OAT despite some variability within the studies (Moosani et al., 1995Go; McInnes et al., 1998Go; Pang et al., 1999Go; Pfeffer et al., 1999Go; Rives et al., 1999Go; Nishikawa et al., 2000Go; Vegetti et al., 2000Go). Consequently, the results of the current study, as well as previous studies, provide evidence of an increase in numerical abnormality of sperm chromosomes in infertile patients. The current study has shown that the presence of teratozoospermia requires a sperm chromosomal evaluation in order to estimate the genetic risk in the era of intracytoplasmic sperm injection (ICSI).

The population herein studied might meet the criteria for it to be included in moderate-to-severe non-obstructive teratozoospermia and OAT syndrome, which is apparently sustained by more or less severe conditions of impaired spermatogenesis. In such a population of patients, all of the few studies performed report a high incidence of meiotic abnormalities (37.9%) and synaptic anomalies on testicular tissue samples (Egozcue et al., 1983Go; Vendrell et al., 1999Go), which seems to suggest a strict interrelationship between these abnormalities and the derangement of the well-orchestrated process of human spermatogenesis. In any case, meiotic arrest due to synaptic anomalies or to unknown causes results in an arrest of spermatogenesis and is usually associated with incomplete meiotic arrest and oligoasthenozoospermia or, in the case of complete meiotic arrest, to azoospermia (Vendrell et al., 1999Go). A consequence of such disorders might be the subsequent production of chromosomally abnormal spermatozoa. This as well as other studies indicate a large inter-individual variability in this abnormal chromosomal pattern, therefore it would seem logical to envisage a different pathological involvement of the germ line in these infertile patients with impaired spermatogenesis; these patients are prone to the risk of mitotic and meiotic errors during cell division and proliferation (Ushijima et al., 2000Go). In these patients an inverse correlation between total aneuploidy rate and sperm concentration and/or normal forms has been found, confirming findings by others (Colombero et al., 1997Go; Rives et al., 1999Go; Ushijima et al., 2000Go; Vegetti et al., 2000Go). This seems to justify considering patients with oligozoospermia or teratozoospermia at high risk of carrying chromosomal abnormality, mainly if they choose to undergo ICSI programmes. In fact, although patients with increased sperm aneuploidy rate seem to have an overall impaired reproductive function in vitro, some of them are still able to father a child (Pfeffer et al., 1999Go; Colombero et al., 1999Go; Calogero et al., 2001Go).

In conclusion, patients with abnormal sperm parameters have an increased aneuploidy rate which was negatively correlated with sperm concentration and the percentage of normal forms. These findings suggest the necessity of proper counselling before carrying out ICSI on infertile patients with a severe male factor and that it would be prudent to screen these men for increased sperm aneuploidy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank David Farrugia, Lecturer, Faculty of Economics, University of Catania, for the English text editing. This paper was supported in part by a grant from MURST (Cofinanziamento 1999), Rome, Italy.


    Notes
 
5 To whom correspondence should be addressed at: Istituto di Medicina Interna e Specialità Internistiche, Cattedra di Endocrinologia, Ospedale Garibaldi, Piazza S. Maria di Gesù, 951213 Catania, Italy. E-mail: acaloger{at}unict.it Back

* Presented at the 16th Meeting of the European Society of Human Reproduction and Embryology, Bologna, Italy, June 25–28, 2000. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 11, 2000; accepted on February 23, 2001.