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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Key words: aneuploidy/azoospermia/epididymal sperm/fluorescence in-situ hybridization/testicular sperm
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Introduction |
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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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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 (4595%) 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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Discussion |
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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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Notes |
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References |
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accepted on October 19, 2001.