Human Reproduction, Vol. 17, No. 3, 570-575,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI
1 The Center for Reproductive Medicine and Infertility and 2 The James Buchanan Brady Foundation, Department of Urology, The New York Presbyterian-Weill Medical College of Cornell University, New York, NY, USA
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Abstract |
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BACKGROUND: Although ICSI provides a way of treating azoospermic men, concern has been raised about the potential risk for transmission of genetic abnormalities to the offspring. We quantified the incidence of chromosomal abnormalities in epididymal and testicular sperm retrieved from azoospermic patients undergoing ICSI. METHODS: Individual testicular sperm were collected from testicular biopsies with an ICSI pipette, and epididymal sperm were retrieved by microsurgical epididymal sperm aspiration. Samples were processed by fluorescent in-situ hybridization (FISH) for chromosomes 18, 21, X and Y and the results compared with those from normal ejaculated samples. RESULTS: The overall aneuploidy rate of 11.4% in men with non-obstructive azoospermia was significantly higher (P = 0.0001) than the 1.8% detected in epididymal sperm from men with obstructive azoospermia and also the 1.5% found in ejaculated sperm. No significant difference was found between the epididymal and ejaculated samples. When the chromosomal abnormalities were analysed, gonosomal disomy was the most recurrent abnormality in both obstructive and non-obstructive azoospermic patients, while autosomal disomy was the most frequent in ejaculated sperm. CONCLUSIONS: Sperm of non-obstructive azoospermic men had a higher incidence of chromosomal abnormalities, of which sex chromosome aneuploidy was the most predominant. Genetic counselling should be offered to all couples considering infertility treatment by ICSI with testicular sperm.
Key words: aneuploidy/azoospermia/epididymal sperm/fluorescence in-situ hybridization/testicular sperm
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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ICSI (Palermo et al., 1992
) is now widely acknowledged as the most effective treatment for male infertility due to fertilization failure, yielding fertilization and pregnancy rates comparable with those obtained in couples with normal semen parameters undergoing IVF. ICSI has been used successfully also with immature sperm, such as epididymal (Tournaye et al., 1994) and testicular sperm (Schoysman et al., 1993), with results comparable with those achieved with freshly ejaculated semen (Palermo et al., 1996a, 1999).
Since the introduction of ICSI, a major concern has been the safety of this technique (Cummins and Jequier, 1994; In't Veld et al., 1995), since it entails arbitrary selection and injection of a single spermatozoon into the oocyte cytoplasm, thus bypassing many anatomical and physiological steps in the fertilization process. Additional worries were raised when ICSI was employed with immature sperm, because it was assumed that the risk of chromosomal aberrations might be higher in the male population with obstructive or non-obstructive azoospermia. So far, follow-up studies of children born after ICSI have not revealed more congenital malformations as compared with the general population, but there seems to be a slightly increased risk for transmission of chromosomal aberrations, mainly sex chromosomal abnormalities (Palermo et al., 1996b; Tarlatzis and Bili, 1998; Bonduelle et al., 1998, 1999).
For a long time, it has been recognized that constitutional chromosomal abnormalities are much more frequent in infertile men than in the general male population (Chandley, 1979). Furthermore, men with a normal peripheral karyotype may have chromosomal abnormalities limited exclusively to the germ cells (Egozcue et al., 1983), probably arising from non-disjunction during spermatogenesis (Moosani et al., 1995). Recently, an increased incidence was found of numerical chromosome abnormalities in fertilized human oocytes after ICSI (Macas et al., 2001), and it was shown using polymorphic microsatellite markers that most chromosomal abnormalities in ICSI pregnancies are of paternal origin (Van Opstal et al., 1997). Utilizing fluorescence in-situ hybridization (FISH), we and others have demonstrated that men with suboptimal semen quality have a higher incidence of chromosomal abnormalities in their sperm (Bernardini et al., 1997; Aran et al., 1999; Colombero et al., 1999; Pang et al., 1999; Shi and Martin, 2001). Nevertheless, data on the incidence of aneuploidy in immature sperm are limited and controversial (Bernardini et al., 2000; Martin et al., 2000; Morel et al., 2001). In this study, we have compared the frequency of aneuploidy in epididymal and testicular sperm versus ejaculated cells.
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Materials and methods |
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Patients
Germ cells were obtained from 27 patients at our infertility centre. Epididymal sperm were retrieved from eight patients undergoing microsurgical epididymal sperm aspiration (MESA) due to congenital absence of the vas deferens, and testicular sperm were obtained from testicular biopsy in five patients with non-obstructive azoospermia. Ejaculated sperm (control) were provided by 14 men with normal semen parameters undergoing IVF due to female factor infertility. All patients had a peripheral karyotype done (Palermo et al., 1999
). The Internal Review Board of the New York Presbyterian Hospital-Weill Medical College of Cornell University approved this study. All patients gave informed consent to participate in this study.
Semen collection and preparation
Ejaculates were provided by masturbation and evaluated according to the standards of the World Health Organization (1999) and Kruger strict criteria for assessment of morphology (Kruger et al., 1986). Normal semen parameters were considered as follows: concentration 
20x106 sperm/ml, progressively motile 40 and 4% of normal morphology. Ejaculates were centrifuged after 1:1 dilution in human tubal fluid medium buffered with HEPES (H-HTF; Irvine Scientific, Santa Ana, CA, USA) at 300 g for 20 min on a three layer (90/70/50%) discontinuous density gradient Isolate® to select progressive motile sperm. Sperm retrieved in the 90% fraction were rinsed in H-HTF, centrifuged for 5 min at 500 g, and resuspended preferably to a concentration of approximately 10x106 sperm/ml. Ten µl of washed semen were smeared on a pre-cleaned glass slide and allowed to dry (Colombero et al., 1999). Epididymal sperm were obtained by MESA as previously described (Palermo et al., 1996a, 1999). Epididymal fluid was diluted, analysed and processed by one (95%) or two (45–95%) layers discontinuous density gradient centrifugation, following which a 10 µl aliquot was smeared on a glass slide. Testicular sperm were retrieved by testicular biopsy, as described previously (Palermo et al., 1999). After mechanical dissection and centrifugation, the specimen was placed into a drop under oil (Palermo et al., 1993, 1995), and individual sperm collected with an ICSI pipette and expelled directly onto a microslide. When possible, several slides from the same patient were made in all three groups to allow the analysis of a larger number of sperm.
Preparation of sperm for FISH analysis
For sperm decondensation, slides were placed in a Coplin jar containing 10 mmol/l dithiothreitol (DTT; Sigma Chemical Co., St Louis, MO, USA) in 100 mmol/l tris(hydroxymetyl)aminomethane (Trizma HCl; Sigma Chemical Co.), pH 8 for 30 min at room temperature (Colombero et al., 1999). Subsequently, they were transferred to another jar containing 10 mmol/l 3,5-diiodosalicylic acid (Sigma Chemical Co.) and 1 mmol/l DTT in 100 mmol/l Tris, pH 8 for 30 min. Slides were washed for 2 min in 2xstandard saline citrate (SSC; Vysis, Downers Grove, IL, USA), pH 7 and observed under phase contrast microscopy for evidence of sperm nucleus decondensation. Partial decondensation was considered to be adequate when the majority were decondensed but the shape of the nuclei was maintained and the tails still evident. Excessive agitation of the slides was avoided in all decondensation and washing steps in an effort to limit sperm loss, especially from smears performed with testicular sperm. Slides with decondensed sperm were hybridized with probes specific to chromosomes 18, 21, X, and Y (Colombero et al., 1999). For each patient, half of the slides were assessed for chromosomes 18, X, and Y and the remainder for chromosomes 21, X, and Y.
Denaturation and hybridization were carried out as described previously (Colombero et al., 1999). Sperm nuclei were counterstained with 15 µl of 4',6-diamino-2-phenylindole (DAPI) in antifade solution (0.5mg/ml, Vysis), cover-slipped and assessed at x1000 with an epifluorescence microscope (Olympus B Max 60®; New York/New Jersey Scientific, NJ, USA). The incidence of disomy, nullisomy and diploidy was recorded, with only unequivocal fluorochrome signals related to each chromosome being recorded using the criteria described previously (Colombero et al., 1999). Briefly, normal haploid sperm nuclei were those carrying one signal for an autosome and one signal for a gonosome. Sperm missing one of the two signals were nullisomic for the corresponding chromosome, and sperm with an extra signal were disomic. The simultaneous scoring of one autosome and two sex chromosomes allowed the distinction between nullisomy and hybridization failure (no signals), and between disomy and diploidy (two signals for autosomes and two signals for gonosomes). A spermatozoon was considered disomic for a specific chromosome when two fluorescent domains of the same colour were clearly positioned within the sperm head, comparable in brightness and size, and at least one domain apart. One domain was considered to be the diameter of the signal.
Statistical analysis
The analysis was performed using SAS software (Statistical Analysis System, Cary, NC, USA). Statistical procedures were carried out by two-tailed 
2 tests using a 5% level of significance (P < 0.05) to evaluate all hypotheses. Where appropriate, Fisher's exact tests were used to ensure no violations owing to small cell counts in 2 procedures.
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Results |
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The mean paternal age (±SD) was 37.1 ± 6 years for controls, 36.5 ± 6 years for men with obstructive azoospermia, and 41.6 ± 6 years for non-obstructive azoospermic patients with characteristics of the samples presented in Table I
. From the infertile men, 490 testicular sperm and 6675 epididymal sperm were analysed and 25 150 ejaculated sperm were scored in the control group. All patients studied had normal blood karyotypes. The efficiency of FISH for the three groups was estimated to be 98.4%. Overall, the sperm aneuploidy rate was 11.4% in non-obstructive azoospermic men, 1.8% in patients with obstructive azoospermia, and 1.5 % in the ejaculate controls (Figure 1a,b). Thus, the incidence of chromosomal abnormalities in the non-obstructive azoospermia group was significantly higher than in both the obstructive azoospermic and the control groups (P = 0.0001). However, the aneuploidy rate for epididymal sperm (Figure 2a,b) did not differ significantly from that in ejaculated sperm.
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The frequency of nullisomy, autosomal disomy, sex chromosome disomy and diploidy for the three groups is presented in Table II
. All types of chromosomal abnormalities were more common in testicular sperm (Figure 3a,b) as compared with controls (P = 0.0001), the most predominant abnormality in this group was sex chromosome disomy (37.5% of 56 abnormal sperm), followed by nullisomy (32.1%). When sperm cells displaying X or Y disomy (n = 21) were considered together with those bearing a nullisomy for sex chromosomes (n = 11), such sex chromosome abnormalities accounted for 57.1% of the overall aneuploidy rate. The specific autosomal and gonosomal disomies are listed in Table III. The obstructive azoospermic group displayed a modest increase of nullisomy, however significant (0.43 versus 0.27% in the control group, P < 0.05), with the remaining sex chromosome disomy as the most common abnormality.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In this study, rates of aneuploidy detected by tricolour FISH in epididymal sperm from obstructive azoospermic men were not significantly different from those in ejaculated spermatozoa from putatively normal men. These observations suggest that the gene mutations responsible for congenital absence of the vas deferens do not have a significant impact on chromosome disjunction during spermatogenesis (Bernardini et al., 2000
). However, Bernardini et al. (2000) reported a significantly higher incidence of sex chromosome disomy/diploidy (2.7 versus 0.8% in the control group). In our study, although gonosomal disomy was the predominant abnormality among these men, the incidence was not significantly different from that in ejaculated sperm controls. This discrepancy may be partially explained by the fact that Bernardini et al. (2000) utilized bicolour target in-situ hybridization—an approach which prevents the accurate distinction between diploidy and sex chromosome disomy (Martin and Rademaker, 1995).
Since the underlying pathology of non-obstructive azoospermia is mainly genetic in nature, a higher frequency of aneuploidy might be expected in testicular sperm from such men. Indeed, it has been suggested that the defects in meiotic pairing and recombination observed in infertile men may lead to both meiotic arrest (azoospermia) and chromosome non-disjunction (aneuploidy) (Martin, 1996). Consonant with this hypothesis, testicular sperm in our study revealed an almost eight-fold increase in the incidence of chromosomal aneuploidy when compared with normal ejaculated sperm, more than half being sex chromosomal abnormalities. Previous reports on the incidence of chromosome anomalies in testicular sperm have been contradictory. Martin et al. (Martin et al., 2000) found no increase among sperm extracted from the testis of azoospermic men when compared with ejaculated sperm from normal healthy donors. Although they did not specify semen characteristics, Martin et al. (2000) were able to retrieve more than 3000 sperm from the biopsies in three patients, suggesting that these individuals may not have had severe spermatogenic defects. Due to the very limited number of cells recoverable from testicular tissue in five patients in our study, we were able to analyse only 490 sperm. Typically, only a small number of sperm can be recovered from the biopsies of men with severe testicular failure and usually only after an extensive, meticulous, and time-consuming search.
In agreement with our observations, an increased incidence of chromosomal abnormalities in testicular sperm of azoospermic men was reported (Bernardini et al., 2000), but their overall aneuploidy rate of 24.9% was considerably higher than the 11.4% observed here. While the basis of this difference is not readily clear, the different techniques utilized for sperm retrieval, DNA hybridization as well as the different chromosomes assessed, may be responsible for the discrepancy. It is possible that the higher rate of aneuploidy reported by Bernardini et al. (2000), and also here, may be something of an overestimate because of the lower number of sperm counted and the effect of paternal age. Nevertheless, recent meiotic studies performed in germ cells of men with impaired spermatogenesis provide further, if indirect, evidence to support our findings (Huang et al., 1999).
In the study by Verheyen et al. (2001), the incidence of sperm aneuploidy in non-obstructive azoospermic men was 10.3%, similar to the one found in our study of 11.4%. The incidence of aneuploidy in obstructive azoospermia of 7.2% (testicular biopsy) was considerably higher than our 1.8% (epididymal aspiration). A possible selective effect carried out in the epididymis by either apoptosis or phagocytosis may explain the difference.
In summary, although a relatively small number of testicular cells were evaluated, these findings point to a higher incidence of sperm chromosomal abnormalities in sperm of non-obstructive azoospermic men, where sex chromosome aneuploidy appears to be the most common. These observations support the possibility of an increased incidence of paternally derived sex chromosomal abnormalities in pregnancies achieved with testicular sperm. Therefore, appropriate genetic counselling as well as screening of the resulting pregnancies should be strongly recommended in these patients.
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Acknowledgements |
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The authors thank the clinical and scientific staff of The Center for Reproductive Medicine and Infertility, Prof. J.Michael Bedford for his critical review of the manuscript, and Ms Queenie V.Neri for editorial assistance.
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Notes |
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3 To whom correspondence should be addressed: The Center for Reproductive Medicine and Infertility, New York Presbyterian-Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA.E-mail: gdpalerm{at}med.cornell.edu.
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