Human Reproduction, Vol. 15, No. 10, 2165-2172,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients*
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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The hypothesis that sperm aneuploidy and diploidy increase as a function of spermatogenesis impairment was addressed. Ejaculated semen samples from a series of men (n = 22) with very low total normal motile count (1 x 106) was analysed in terms of sperm aneuploidy and diploidy by in-situ hybridization and compared with controls (n = 10). Germ cell aneuploidy was also analysed in an additional series of infertile patients presenting unexplained infertility (n = 3), congenital absence of the vas deferens (CAVD) (n = 6) and non-obstructive azoospermia (n = 3) undergoing IVF, microsurgical epididymal sperm aspiration (MESA)/ICSI and testicular sperm extraction (TESE)/ICSI cycles respectively. In-situ hybridization for chromosomes 1, 17, X and Y was performed on ejaculate, epididymal and testicular spermatozoa. Significantly higher sperm aneuploidy and diploidy rates where found (for the four chromosomes analysed) in spermatozoa from oligoasthenoteratozoospermia (OAT) over controls (18 versus 2.28% and 2.8 versus 0.13% respectively; P < 0.001). Testicular germ cells had even higher rates of sperm aneuploidy and diploidy. However, in this group it was difficult to determine whether the cells analysed were dysmorphic spermatozoa or spermatids. The data warrant further investigation on the cytogenetic abnormalities found in most germ cells identified in testicular tissue biopsies of azoospermic patients.
Key words: FISH/germ cells/male infertility/sperm aneuploidy
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Introduction |
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From the inception of intracytoplasmic sperm injection (ICSI) and its successful application in cases of severe oligoasthenoteratozoospermia (OAT) and azoospermia patients, the risk of potential genetic abnormalities in ICSI liveborns has been of general concern. To date, the overall evidence deriving from different lines of investigation suggests that in ICSI newborns the risk of paternal transmission of sex chromosome aneuploidy is highly realistic. Longitudinal and prospective surveillance studies have been addressed in different ways including traditional epidemiological studies on prenatal data (Bonduelle et al., 1998
), analyses on the pattern of parental chromosome segregation by DNA polymorphisms (Van Opstal et al., 1997) and indirectly by looking at the cytogenetic constitution of spermatozoa by using fluorescence in-situ hybridization (FISH) (Pang et al., 1999). Specifically, most FISH studies on sperm aneuploidy in infertile men have shown a generalized increased rate of disjunctional errors involving most human chromosomes. Nevertheless, variability from one author to another exists in terms of baseline aneuploidy rates reported in such patients. This is due to many factors and above all the subjectivity of the selection criteria adopted for male factor infertility definition and study group allocation (Aran et al., 1999). Despite this, our personal experience suggests that whenever the count of normal motile spermatozoa falls below 5x106 (compared with 7–15x106 in normal controls), an almost two-fold increase in sperm disomy and diploidy should be expected for chromosomes X, Y, 1 and 17 (total disomy rate of 2.34 versus 1.38% in controls) (Bernardini et al., 1997, 1998). Similar results have also been reported by other studies adopting more strict criteria for male factor infertility definition. However, few studies have so far evaluated the frequency of sperm aneuploidy and diploidy as a function of the ejaculate semen quality and almost no data have been reported on the frequency of aneuploidy in spermatozoa retrieved from the epididymides and testes in cases of azoospermia. In this study, we decided to investigate the sperm aneuploidy for chromosomes X, Y, 1 and 17 in ejaculated semen from a population of men presenting with very poor semen quality (total normal motile count of, on average, 1x106). Results were then compared with those previously obtained in similar groups of patients who were carrying minor degrees of spermatogenesis impairment (total normal motile count of, on average, 5x106). Also and for the first time, sperm aneuploidy was analysed in sperm samples surgically collected from a small series of azoospermic patients undergoing microsurgical epididymal sperm aspiration (MESA) because of congenital absence of vasa deferentia (CAVD) or testicular sperm extraction (TESE) due to late spermatogenetic arrest. |
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Semen samples
Semen samples were obtained from 10 healthy donors, used as controls (C), and 34 patients including three patients with unexplained infertility (U), 22 patients with severe OAT, six men with obstructive azoospermia [in five due to CAVD] and a group of three patients with non-obstructive azoospermia undergoing TESE due to incomplete spermatogenetic arrest. All patients studied had normal blood karyotypes (46, XY). In addition, patients with non-obstructive azoospermia or severe OAT were offered genetic screening with molecular analysis for Y chromosome microdeletions (Human Genetic Laboratory, Galliera Hospital, Genoa, Italy). Men with CAVD and their female partners were screened for cystic fibrosis gene mutations. Protocols for epididymal and testicular sperm retrieval and cryopreservation have been previously published (Gianaroli et al., 1996
, 1999). Semen samples were routinely analysed at the Andrology Laboratory of our hospital. Sperm cell count, motility and morphology were determined for each sample and total normal motile count (TNMC) in the pre-processed sample estimated (total countxpercentage of spermatozoa with linear progressive motilityxpercentage of spermatozoa with normal morphology) (Balmaceda et al., 1993). A total normal count of <5x106 was selected as the cut-off value for male factor definition. Patients with unexplained infertility (n = 3) had a TNMC between 7.3 and 9x106, and their female partners were apparently healthy. In two, past medical records showed histories of repeated IVF failures (absence of fertilization in 10 cycles) and recurrent abortions (n = 5; 3/5 with molar degeneration) respectively. Semen parameters and clinical profiles for the five groups are shown in Table I. In cases of epididymal and testicular sperm samples, information on cell morphology was not available. All patients underwent assisted reproduction treatment with IVF and ICSI cycles. Informed consent was obtained from all of them.
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Sperm nuclei preparation
Sperm preparation prior to in-situ hybridization (ISH) was performed following a previously described protocol (Martini et al., 1995
). Samples were washed 3 times in phosphate buffered saline (PBS, 0.15 mol/l NaCl, 10 mmol/l sodium phosphate, pH 7.2) and centrifuged at 280 g for 10 min. The pellet was then resuspended carefully in 1 ml of fresh, cold fixative (methanol:acetic acid, 3:1) and stored at –20°C. Sperm cells were cytospun on poly-L-lysine coated slides, washed in 2xSSC (saline sodium citrate) (0.3 mol/l NaCl, 30 mmol/l Na citrate) to remove the excess of fixative and incubated for 4–8 min in 1 mol/l Tris/HCl buffer, pH 9.5, containing 25 mmol/l dithiothreitol (DTT). After decondensation, the slides were washed once in 2xSSC, once in PBS, dehydrated through an ethanol series (70–96–96–100–100%) and air dried.
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 (MFITC, Dako A/S) and PO-conjugated rabbit anti-mouse IgG (RM-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 20–30 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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Table II
lists the frequencies of morphologically normal, abnormal spermatozoa and IGC found in FISH slides of the patient groups studied. With respect to controls and unexplained infertility patients, ejaculate semen from OAT patients was found to contain fewer morphologically normal spermatozoa (96.3% and 96.6% versus 77.9%) and higher percentages of morphologically abnormal spermatozoa and immature germ cells (2.47% and 2.35% versus 12.7%; 1.08% and 0.99% versus 9.4%). In TESE samples the percentage of IGC and abnormal spermatozoa were 71.28 and 24.9% respectively.
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The hybridization efficiency was high (98%) for ejaculate semen of controls, unexplained infertility patients, OAT patients as well as in epididymal spermatozoa from MESA samples while it significantly decreased in cases of TESE (80%; P < 0.0001). This was largely due to the prevalence in this group of abnormal and micro-headed spermatozoa showing signs of incomplete head decondensation and probe hybridization. Figure 1a
, b, c and d shows the quality of FISH results achieved in ejaculate of controls, OAT patients and TESE biopsies. In this last group a heterogeneous population of germ cells was found, including mostly immature germ cells of different size and shape, abnormal spermatozoa and spermatids and very rarely, a few normal spermatozoa. For each image, specific details are given in the legend. Of note, artefacts related to the effect of decondensation must be taken into consideration during the morphological evaluation of round and immature germ cells. In fact, the average size of the immature germ cells is here enlarged with respect to that normally reported by conventional classification of these cells (Sousa et al., 1999).
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Results of double target FISH are reported separately in Table IIIa and b
. The data were always based on the cytogenetic analysis of morphologically normal spermatozoa. However, in TESE samples the majority of sperm cells analysed were morphologically abnormal spermatozoa or spermatids.
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As for ejaculate semen, the overall rate of autosomal hyper- (1+1/17; 17+17/1) and hypohaploidy (1/0; 1+1/0; 17/0; 17+17/0) and diploidy (1+1/17+17) were significantly different in OAT patients compared with controls (4.47%, 7.27%, 2.8% versus 0.63%, 0.84%, 0.13% respectively; P < 0.0006, ANOVA) (Table IIIa
). Similar differences between OAT and controls were noted for X and Y chromosomes DNA-ploidy (6.3 versus 0.81% respectively; P < 0.009, ANOVA) (Table IIIb). Statistically significant differences were also scored for the sperm nullisomy rates found in OAT patients versus controls. Conversely, no differences in sperm cytogenetic constitution were noted between controls and unexplained infertility patients.
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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Frequencies of epididymal sperm disomy found in MESA patients were significantly higher than controls but much lower than in ejaculated spermatozoa of OAT patients. On the contrary, much higher values of sperm hyper- and hypohaploidy as well as diploidy were recorded in sperm cells found in TESE samples. In two out of three azoospermic patients it was possible to score only 22 morphologically normal spermatozoa. Nine per cent of these spermatozoa were disomic (disomy 1 and 17) (2/22) and 18% were monosomic (1/0 and 17/0) (4/22). In these few morphologically normal spermatozoa from TESE samples sex chromosomes abnormalities were not found.
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 (8–17%) 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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The ICSI performance of the cytogenetically analysed spermatozoa was very poor. Pregnancy rates per cycle obtained in OAT, MESA and TESE patients were very low [9% (2/34 cycles), 5% (1/20 cycles) and 0% (0/7 cycles) respectively]. Nevertheless, a pregnancy rate per patient of 16.6% was achieved in MESA group (1/6). In the three unexplained infertility patients, no pregnancy ensued (0/13 cycles) despite the number of repeated IVF cycles and the normality of sperm aneuploidy.
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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In this study, we have applied a standardized method of double target ISH to study the frequency of autosomal and gonosomal aneuploidy in germ cells found in ejaculate and testicular semen samples of infertile men. In order to distinguish accurately between sex chromosome diploidy and disomy a multi-probe FISH including a probe for autosomes would be the best approach (Martin et al., 1996
). Despite this, we believe that our protocol of ISH, based on a proper staining of the germ cell cytoplasm (including sperm tail) combined with brightfield microscope evaluation of no fading signals, guarantees, as compared with FISH, a more correct recognition of the morphologically normal spermatozoa among all the other germ cells.
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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The authors would like to thank Professor Josep Egozcue, Unitat de Biologia Cellular, Universitat Autonoma de Barcelona, Spain, for the suggestions received in preparing the manuscript.
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Notes |
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* Presented in part at the 15th Annual Meeting of the European Society of Human Reproduction (ESHRE) Conference, June 30 to July 3, 1999, Tours, France.
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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Submitted on March 10, 2000; accepted on June 26, 2000.
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