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
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Abstract |
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Key words: chromosomes 8, 12 and 18/multicolour fluorescence in-situ hybridization/oligoasthenoteratozoospermia/sex chromosomes/sperm aneuploidy
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Introduction |
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Materials and methods |
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Morphology assessment was performed on fresh seminal semen according to Kruger's strict criteria (Kruger et al., 1986), whereas the other parameters were evaluated according to the World Health Organization criteria (WHO, 1992). Semen was collected by masturbation after 45 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 I
.
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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., 1995). 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 -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 MannWhitney 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.
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Results |
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Total abnormal chromosomal rate
The total sperm aneuploidy rate (disomy and nullisomy) was 2.08% (range: 1.184.56%) in group 1, 1.83% (range: 19.73%) in group 2 and 0.87% in the control group (range: 0.31.18%) (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: 19.73%) 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 1).
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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 II. 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: 02.89% and 0.53%; range: 0.051.91% respectively) was significantly higher compared to those in the controls (P < 0.001).
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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 2).
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Discussion |
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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., 1998). 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., 1983
; Vendrell et al., 1999
). 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., 1997; Viville et al., 2000
). 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., 1998
). 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., 1997
; Weissenberg et al., 1998
).
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., 1995; McInnes et al., 1998
; Pang et al., 1999
; Pfeffer et al., 1999
; Rives et al., 1999
; Nishikawa et al., 2000
; Vegetti et al., 2000
). 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., 1983; Vendrell et al., 1999
), 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., 1999
). 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., 2000
). 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., 1997
; Rives et al., 1999
; Ushijima et al., 2000
; Vegetti et al., 2000
). 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., 1999
; Colombero et al., 1999
; Calogero et al., 2001
).
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.
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Acknowledgements |
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Notes |
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* Presented at the 16th Meeting of the European Society of Human Reproduction and Embryology, Bologna, Italy, June 2528, 2000.
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References |
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Submitted on October 11, 2000; accepted on February 23, 2001.