1 Department of Obstetrics/Gynaecology, S. Martino's Hospital, University of Genoa, Lgo R. Benzi 10, 16132 Genoa, 2 SISMER, Reproductive Medicine Unit, Bologna and 3 Human Genetic Laboratory, Galliera Hospital, Genoa, Italy
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Abstract |
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Key words: FISH/germ cells/male infertility/sperm aneuploidy
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Introduction |
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Materials and methods |
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Double target ISH procedure
The DNA probes used in this study included an alphoid probe specific for the centromeric region of the human X chromosome (Oncor, Milan, Italy), a satellite probe specific for the long arm of the human Y chromosome (Boeringher, Milan, Italy) and another two probes recognizing the (peri) centromeric regions of human chromosome 1 and 17 (Oncor and Boeringher). A final volume of 5 µl (corresponding to a concentration of 0.4 ng/µl for each probe) was added to each slide under a coverslip (18x18 mm). Denaturation was achieved at 73°C for 3 min and hybridization was performed for 4 h at 37°C. The slides were washed twice for 5 min at 42°C with 60% formamide, 2xSSC, pH 7.0, containing 0.05% Tween 20, followed by two 5 min washes with 2xSSC, pH 7.0 at 42°C and one 5 min wash with 4xSSC, pH 7.0, containing 0.05% Tween 20 at room temperature. The detection of signals was performed as described previously (Bernardini et al., 1997). Briefly, the chromosome X- or 1-specific probes were detected with horseradish peroxidase-conjugated avidin (AV-PO, Dako A/S, Glostrup, Denmark), amplified with biotinylated goat anti-avidin (BioG
A, Vector, Burlingame, CA, USA), followed by a second AV-PO layer and the diaminobenzidine (DAB) reaction. The slides were then incubated for 10 min at room temperature in 0.01 N HCl to inactivate peroxidase activity. Subsequently, the fluoresceinated chromosome Y- or 17-specific probes were detected with mouse anti-FITC (M
FITC, Dako A/S) and PO-conjugated rabbit anti-mouse IgG (R
M-PO, Dako A/S). After this last incubation step, the peroxidase-tetramethylbenzidine (TMB, Sigma) reaction was performed. Avidin conjugates were diluted in 4xSSC, pH 7.0, containing 5% non-fat dry milk, and the antibody conjugates were diluted in PBS containing 0.05% Tween 20 and 2% normal goat serum. After each incubation step of 2030 min at 37°C, the slides were rinsed twice in 4xSSC, pH 7.0, 0.05% Tween 20 (avidin conjugates) or PBS, 0.05% Tween 20 (antibody conjugates). Counterstaining was achieved by the combined use of haematoxylin and Diff-Quik (DADE s.p.a., Milan, Italy) as previously published (Martini et al., 1995
). The cytoplasmic staining step of the Diff-Quik consisting of Eosin G in phosphate buffer, pH 6.6 (1.22 g/l; solution 1) was utilized. Slides were embedded in Entellan (Merk, Bracco s.p.a., Milan, Italy), an organic mounting medium and stored at +4°C until evaluation. The evaluation of the FISH signals was performed with a standard Zeiss bright-field microscope.
Scoring criteria and statistics
Sperm-slides were scored by two independent observers according to previous recommendations (Wyrobek et al., 1994; Martin and Rademaker, 1995
). Both the investigators were blinded as to the origin of the slides being analysed. For controls, unexplained infertility patients and male factor patients, an average number of 3000 spermatozoa was scored. For MESA and TESE a lower number of 2000 and 200 sperm cells respectively could be analysed. Most sperm cells analysed in TESE samples were represented by abnormal spermatozoa or elongated spermatids. In this group an additional number of 600 immature germ cells was scored per patient. For spermatozoa to be recorded as disomic, two signals of similar size had to be located at a distance of approximately one diameter of the signal domain. These criteria were not taken into account for sperm cells with XY signals. Sperm cells showing normal decondensation but no signals at all were scored and considered valid for calculations of nullisomy. Other forms were observed and classified as hypohaploid spermatozoa (1/0; 1+1/0; 17/0; 17+17/0). However, here the possibility of hybridization failure could not be excluded. For each individual, two separate experiments of double target FISH were performed. By employing autosomal DNA probes, a realistic estimation of diploidy could be determined. Sperm nuclei were scored when they were morphologically preserved, i.e. not clumping or overlapping, with a well-defined outline and the tail and sperm-head decondensed to no more than twice the size of normal non-decondensed spermatozoa. The presence of the tail was considered essential for a reliable evaluation. The differentiation of the morphologically abnormal sperm cells (sperm with two heads, two midpieces but one tail, two tails but one head, micro-heads) from diploid somatic cells or aneuploid spermatozoa was made possible by Diff-Quik staining and three sperm cells were not included in the count of the aneuploid cells. The big, brightly stained, round cells showing efficient ISH signals were recorded as immature germ cells (IGC). The cytogenetic evaluation on this type of cells found in ejaculate semen has formed a part of previous studies (Bernardini et al., 1998
). In this study the cytogenetic analysis on immature germ cells was attempted only for testicular semen derived from TESE samples. Specifically, the cells were separately scored on the bases of their size and morphology. All haploid or aneuploid round cells <8 µm in diameter were recorded as round spermatids. Other similarly stained cells of larger size were observed including some showing abortive tail-parts and recorded as abnormally large spermatids as well as other bigger and round cells considered as generally more immature forms (spermatocytes or spermatogonia) (IGC). Among the five patient groups, comparison of the sample means was performed by non-parametric rank sum test for independent samples and analysis of variance (ANOVA).
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Results |
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Frequencies of sperm aneuploidy found in ejaculate semen of OAT patients were significantly higher than those previously found in cases of less severe impairment of spermatogenesis (TNMC 5x106) (Table IV
). Since the type of investigations, protocol of ISH and scoring criteria have remained constant throughout these studies, data were re-plotted altogether and analysed by ANOVA.
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The analysis of immature germ cells found in TESE samples was accomplished according to the criteria previously cited and is reported in Table V. The rate of autosomal aneuploidy found in round spermatids and larger cells (IGC) did not differ (817%) while the diploidy rate was significantly different (62.5 versus 4.34% in IGC versus RS). Most of these large diploid cells were shown to carry an XY or XX or XXYY chromosomal constitution and were probably primary spermatocytes.
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Analysis on chromosome Y deletions was available for 11 out of 22 men with OAT. The Yq-interval 6-STS of peripheral leukocytes DNA were amplified by multiplex PCR. No microdeletions in this DNA interval on the long arm of Y chromosome were found. On the contrary, all men presenting with congenital absence of the vas deferens (n = 5) were heterozygous for the cystic fibrosis gene mutations more commonly observed among the Northern Italian population.
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Discussion |
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This study provides an additional demonstration that in men with normal peripheral karyotype, the poorer the ejaculate semen quality is, the higher the chances are of finding increased rates of sperm chromosome numerical abnormalities (aneuploidy and diploidy). In fact, the rate of hyper-, hypohaploid and diploid spermatozoa found in men with severe male factor infertility (OAT) was very high and different from that observed in previous studies on men with milder forms of male infertility (Bernardini et al., 1997, 1998
). Mean values of sperm chromosome disomy found in these patients with very low semen quality strongly resembled that reported previously (Pang et al., 1999
; Pfeffer et al., 1999
; Vegetti et al., 2000
) for a similarly compromised group of infertile men (TNMC of 1x106) and should not be compared with disomy rates reported by most studies where FISH was applied on spermatozoa from men with a minor level of compromised spermatogenesis (Moosani et al., 1995
; Bernardini et al., 1997
, 1998
; Lahdetie et al., 1997
; Finkelstein et al., 1998
; McInnes et al., 1998
; Storeng et al., 1998
; Aran et al., 1999
; Colombero et al., 1999
; Rives et al., 1999
; Nishikawa et al., 2000; Ushijima et al., 2000
). A cumulative review of the literature data clearly indicates higher sperm aneuploidy rates in male factor patients (n = 301) over controls (n = 170) and supports the contention for a generalized disruption of chromosomal segregation in meiosis of infertile men. Nevertheless, in most of these reports the patients studied presented with mild forms of male factor infertility (TNMC of 5x106) and often, with the exception of one study (Aran et al., 1999
), most other papers included low numbers of patients with poorly defined seminal characteristics. This has led many authors to conclude that the difference in the level of sperm aneuploidy between infertile men and normal donors was only slightly significant. Conversely, we would like to emphasize that studies in poorly defined groups are worthless and more strict criteria for male factor definition should be adopted before attempting to make conclusions on the true frequencies of sperm aneuploidy in infertile males. We have already published the theoretical clinical implications related to the utilization of ICSI for these men and the importance of proper reproductive counselling depending on the type of sperm chromosome disomy involved (Bernardini et al., 1998
; Egozcue et al., 2000
).
No one has yet demonstrated convincingly that there is a relationship between aneuploidy or diploidy and sperm morphology (Templado et al., 2000). So far, aneuploidy and diploidy have only been related to sperm numbers and high FSH concentrations (Egozcue et al., 2000
). Nevertheless, the data obtained in the present study show that the frequency of disomy in morphologically normal spermatozoa are consistently higher whenever the number of morphologically abnormal spermatozoa and immature germ cells of the ejaculate is also higher. In fact, the percentage of abnormal spermatozoa was significantly increased in the OAT group (12.7 versus ~2.5% in controls; P < 0.0001) and it was even higher in the TESE group (24.9 versus ~2.5% in controls; P < 0.00001). Most disomic spermatozoa had an XY arrangement suggesting that the majority of non-disjunction errors occur at meiosis I. Overall these data suggest, as already reported, that abnormalities in chromosome segregation and sperm morphology differentiation and maturation are probably associated phenomena. Most probably this alteration may originate during the earliest stages of the spermatogenesis process (Bernardini et al., 1998
), and agrees with the notion that high cellular exfoliation is indicative of a possible meiotic anomaly (Egozcue et al., 1983
).
This concept is reinforced by the data obtained in cases of particularly severe male infertility such as that of TESE patients. Though the results achieved on testicular spermatozoa should not be compared with those found in ejaculated semen, we found that in men with incomplete spermatogenetic arrest, the numerical ratio of normal spermatozoa to abnormal and immature germ cells was basically reversed with respect to normal ejaculated semen. In parallel the rate of sperm hyper-, hypohaploidy and diploidy was the highest ever noted. Our data show that the cytogenetic constitution found in germ cells of TESE samples markedly varies according to the type of cells analysed with an inverse relationship between stage of maturation and aneuploidy rate (IGC >round spermatids> elongated spermatids and abnormal spermatozoa>normal spermatozoa). The presence of higher numbers of IGC in complete or incomplete arrest is to be expected. It is very likely that most of these cells analysed were primary spermatocytes (diploid, XY). Almost no morphologically normal spermatozoa were found in this group of patients and only a limited number of abnormal spermatozoa or spermatids could be scored making it difficult to attempt fair comparisons with ISH results obtained in normal spermatozoa of ejaculated sperm samples. Consequently, any conclusion based on this small number of cells analysed is obviously far from being final and other studies are requested to validate these preliminary data. Nevertheless, recent studies on meiotic anomalies in men with severe oligoasthenozoospermia and azoospermia have been reported which suggest a direct relationship, at the testicular level, between incidence of meiotic abnormalities and spermatogenic parameters (Huang et al., 1999; Vendrell et al., 1999
). Studying meiosis on testicular biopsies, it has been shown (Vendrell et al., 1999
) that sperm concentration
1x106/ml of ejaculated semen and blood FSH concentrations >10 IU/l are strong predictors of testicular meiotic abnormalities (synaptic anomalies). Huang et al. (Huang et al., 1999
) performed three-colour FISH on testis tissue biopsy specimens to evaluate sex chromosome non-disjunction at the mitotic stage and distinguish it from errors in meiosis. Their results provide direct evidence of an increased aneuploidy rate in both mitotic and meiotic spermatogenetic cells of candidates for TESE-ICSI. The finding of a high incidence of aneuploidy in diploid testicular germ cells led the authors to conclude that chromosome instability may also be the result of altered genetic control occurring not only during meiosis but also during mitosis and proliferation of spermatogonia. Both these studies confirm the results originally reported (Egozcue et al., 1983
).
Novel data on the baseline frequency of aneuploidy in epididymal spermatozoa from patients presenting CAVD are here reported for the first time. Despite the small series of cases analysed it is concluded that in comparison with ejaculated spermatozoa of controls a moderate increase of aneuploidy is present in these sperm cells. With the exception for chromosome 1 disomy, an almost two-fold increased rate of aneuploidy on average was noted, a difference reaching statistical significance. In contrast, the finding of the rate of aneuploidy scored in epididymal spermatozoa was lower than that present in ejaculated spermatozoa of severe male factor patients suggested that gene mutations for cystic fibrosis have no important effect on chromosome disjunction processes during spermatogenesis. In agreement with previous data, no significant changes in sperm disomy and diploidy were found in semen samples from men presenting with unexplained infertility versus controls.
Very low pregnancy and implantation rates were observed for all the groups studied. While for the OAT and TESE patients a detrimental influence on clinical results might be directly ascribed to the levels of sperm hyper- and hypohaploidy present in these patients, other considerations are implied to explain results achieved in the group of unexplained infertility and MESA patients. In these last groups, sperm aneuploidy rates were found to be basically normal and the mean maternal age was <35 years, thus making it improbable that the occurrence of chromosomal disorders is due to oocyte quality. Perhaps these couples belong to special categories whose poor morphological embryo quality may derive from unknown genes or biochemical anomalies present in either male or female gametes.
In conclusion, substantially increased rates of 1, 17, X and Y chromosome non-disjunction in ejaculate sperm of men with severe OAT as well as testicular cells of men with non-obstructive azoospermia are shown in this study. These results continue to support the possibility of a paternal origin of sex chromosome anomalies in the karyotype of ICSI offspring. In particular our data have shown a strong inverse association between rate of disjunctional errors present in germ cells and their degree of maturation and morphology differentiation. Since men with extremely severe infertility may be offered the option of using different types of sperm cells during ICSI, the information here specifically provided in regard to the cytogenetic constitution of variable types of germ cells may become clinically useful and worthy to be taken into account during pre-conceptional genetic counselling. Additional work is required to confirm the real frequency of hyperhaploidy in the heterogeneous population of germ cells retrieved at testicular level.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Department of Obstetrics/Gynaecology, S. Martino's Hospital, University of Genoa, Lgo R. Benzi 10, 16132 Genoa, Italy.E-mail: bernar01{at}aleph.it
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References |
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Submitted on March 10, 2000; accepted on June 26, 2000.