1 S.I.S.Me.R. Reproductive Medicine Unit, via Mazzini 12, 40138 Bologna, 2 Dept Ob/Gyn, S. Martino Hospital, University of Genoa, Largo Rosanna Benzi 10, 16132 Genoa and 3 Dept Ob/Gyn, University of Pisa, via Roma 67, 56126 Pisa, Italy
4 To whom correspondence should be addressed. Email: sismer{at}sismer.it
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Abstract |
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Key words: aneuploidy/disomy/fluorescence in situ hybridization/male factor infertility/preimplantation genetic diagnosis
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Introduction |
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During the last decade, the application of multicolor fluorescence in situ hybridization (FISH) was proposed and enabled comprehensive studies on numerical chromosomal abnormalities in human sperm (reviewed by Egozcue et al., 1997, 2003
; Shi and Martin, 2000
, 2001
). FISH is a highly sensitive and specific technique that permits the analysis of large numbers of sperm by using chromosome-specific probes labelled with fluorochromes. The packaging of DNA arrays by disulphide bonds between adjacent protamines makes sperm nuclei very condensed and inaccessible to DNA probes. Therefore, a decondensation treatment with a reducing agent is necessary and is strictly related to the success of the technique (for reviews see Downie et al., 1997
; Egozcue et al., 1997
). The quality of the procedure affects the efficiency of hybridization and the definition of the fluorescent signals, and the possibility of distinguishing sperm heads from non-sperm cells.
This technique, through a combination of differently labelled fluorescent probes, opens up the possibility of evaluating the frequency of aneuploidy for two or three chromosomes in each round of FISH. The advantage of using multi-probes simultaneously resides in a more accurate estimation of aneuploidy, allowing differentiation between nullisomy, disomy and diploidy. However, due to the small size of the decondensed sperm head, the number of probes that can be used is restricted (generally no more than three). This limitation can be bypassed if sperm spreads are prepared on different slides and hybridization is performed with different combinations of probe. In this way, sperm samples can theoretically be diagnosed for any chromosome in the human complement. These characteristics allowed FISH to be included as a test for the study of infertile couples.
Unfortunately, severe oligozoospermic samples do not have sufficient numbers of sperm to be scored following this approach. In the most severe cases, including testicular samples, the low count numbers do not even permit the preparation of one slide, based on the assumption that the size of the sample affects the validity of the results. Consequently, a substantial proportion of infertile men are excluded from the possibility of having a FISH test done on their sperm due to the scarcity of cells available for analysis. The reports of higher frequency of chromosomal abnormalities in infertile males' sperm (Pang et al., 1995; Egozcue et al., 1997
; Int't Veld et al., 1997
; Bernardini et al., 2000
) and the increased incidence of gonosome abnormalities in children conceived after ICSI (Bonduelle et al., 2002
) suggest that this proportion of male infertility could be the best candidates to benefit from FISH analysis on sperm. The frequency of high levels of aneuploidy is inversely correlated with sperm count and progressive motility in infertile men (Vendrell et al., 1999
; Vegetti et al., 2000
). It has been reported that up to 18% of men with severe oligoasthenoteratospermia (OAT) carry synaptic abnormalities originated at prophase I (Egozcue et al., 2003
). This predisposes to the production of aneuploid and diploid sperm and to the consequent risk of chromosomally abnormal conceptuses (Gianaroli et al., 2000
). The end result could be an increase in failed implantation and/or repeated abortions (Egozcue et al., 2000
; Bernardini et al., 2004
).
The aim of this study was to define a protocol for the chromosomal analysis of samples with a severely reduced number of sperm and to assess its reliability. Reliability was defined as the studied sperm being representative of the original population.
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Materials and methods |
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Semen sample collection
The study was conducted between February 2002 and December 2003. Ejaculated sperm samples were analysed to evaluate concentration and motility according to World Health Organization (WHO) parameters (WHO, 1999), and sperm morphology was assessed, when possible, following strict criteria (Kruger et al., 1988
). OAT samples were fixed immediately after the collection with the exception of four samples, which had been previously stored in liquid nitrogen and were thawed and fixed. Sperm retrieved by TESE had been cryopreserved after the surgical intervention for subsequent use in assisted reproduction cycles (Gianaroli et al., 1999
); according to the patient's consent, two or three straws were thawed for FISH analysis.
Sperm nuclei preparation
Sperm samples were prepared as already described (Bernardini et al., 2000). Briefly, the samples were washed three times in phosphate-buffered saline (PBS, pH 7.2) at 300g for 10 min. The pellet was resuspended in 1 ml of cold methanol:acetic acid (3:1) and eventually stored at 20°C until further processing. The fixed sperm were cytospun on poly-L-Lys-coated slides, washed in 2xsaline sodium citrate (SSC) with 0.3 mol/l NaCl, and decondensed in 1 mol/l TrisHCl (pH 9.5) with 25 mmol/l dithiothreitol for 5 min at room temperature. After rinsing in 2xSSC and PBS, the slides were dehydrated in increasing ethanol series (70, 96 and 100%) (Martini et al., 1995
).
Cytoplasmic staining
The cytoplasmic staining of Papanicolaou was used with some modifications (WHO, 1999). Slides were immersed in OG6 (Orange G solution, Merck, Germany) for 30 s and repeatedly washed in 95% ethanol; immersion in EA-50 (polychromatic solution EA-50, Merck, Germany) for 30 s followed by repeated washings in 95% and absolute ethanol were the final steps.
FISH on sperm
Multicolor FISH was used in a two-step protocol to diagnose each sperm cell for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The probe mixture used in the first round contained the probes specific for the chromosomes 13, 16, 18, 21 and 22 (Multivysion PB panel from Vysis; Vysis Inc., USA). A second round followed, with probes specific for chromosome X (CEP X alpha satellite, Xp11.1-q11.1), Y (CEP Y alpha satellite, Yp11.1-q11.1), 15 (CEP 15 satellite III, 15q11.2) and 17 (CEP 17 alpha satellite, 17p11.1-q11.1).
Sperm nuclei were denaturated at 75°C for 10 min in 70% formamide 2xSSC followed by dehydration in the ethanol series; 3 µl of probes were denaturated at 75°C for 5 min and added to the slide. Hybridization was performed overnight in a humid chamber at 37°C followed by washings at 46°C in 2xSSC/50% formamide for 10 min, in 2xSSC for 10 min, in 2 x SSC/0.1% Nonidet P 40 for 5 min. The slides were then counterstained in antifade solution (Antifade II; Vysis) when using the first panel probes or with 4',6-diamino-2-phenylindole (DAPI II; Vysis) for the second panel probes. Slides were observed using an Olympus BX40 fluorescence microscope at x600 magnification equipped with a Ludl filter wheel with the following filter sets: dual band pass filters (Red/Green and Aqua/Blue) and single band pass filters (Red, Green, Yellow, Aqua). Images were captured using a CCD PVCAM camera controlled by a MacIntosh computer and an image analysis software (Vysis Quips).
Sperm were scored according to previously described criteria (Blanco et al., 1996). Briefly, they were diagnosed as abnormal if they presented two or more fluorescent signals for the same chromosome whose size and intensity were similar to those detected in normal nuclei; diploidy was defined by the presence of two signals for each of the studied chromosomes in the presence of the sperm tail and an oval head shape; sperm were defined as nullisomic when no fluorescent signals appeared. All signals were separated from each other by at least a single domain.
During the analysis at the fluorescence microscope, the position of each sperm cell was assessed by the coordinates defined by the graduated scale on the microscope table, allowing diagnosis of each spermatozoon for the nine chromosomes studied by the two round FISH.
FISH on embryos
Day 3 embryos with regular morphology and development were selected for embryo biopsy which was performed at 6264 h after insemination according to a previously described protocol (Gianaroli et al., 2002). Briefly, one nucleated blastomere was removed by mechanical opening of the zona pellucida. Fluorescent probes were used for the simultaneous detection of different chromosomes in successive rounds of FISH, including those implicated in the most frequent aneuploidies detected in spontaneous abortions and trisomic pregnancies (X, Y, 13, 15, 16, 18, 21, 22) (Munné et al., 1998
).
Data management and statistical analysis
Evaluation of the incidence of aneuploidy followed a conservative approach by doubling the incidence of disomy (Blanco et al., 1996; Egozcue et al., 1997
).
The end-points of the current study were to ascertain whether: (1) a significant difference between the studied population and the theoretical sperm aneuploidy frequency exists (Williams et al., 1993; Spriggs et al., 1995
; Egozcue et al., 1997
; Pang et al., 1999
); (2) the studied number of total, haploid and aneuploid sperm, as well as the frequency of aneuploidy for each chromosome, are representative of the original population; (3) aneuploidy within the same sperm cell is randomly or not randomly generated; (4) significant differences in terms of aneuploidy exist between ejaculated and testicular sperm.
Items 1 and 4 were investigated by 2-test for rare frequencies; item 2 was analysed by
2-test for observed frequencies (
2c-k-1); and item 3 was evaluated by goodness-of-fit test (
n2) (Camussi et al., 1995
).
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Results |
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The incidence of aneuploidy was evaluated following the conservative approach of doubling the incidence of disomy and yielded an overall value of 12.0%. After 2-test for rare frequencies, 30 samples (79%) showed a significantly higher proportion of total aneuploidy compared to the normal population: 22 out of 27 OAT (81%) and eight out of 11 TESE (73%). The incidence of diploidy was found to be significantly increased in seven samples, of which six were also abnormal for aneuploidy (Tables III and IV).
Figure 2 represents the incidence of total aneuploidy, evaluated as double disomy, in relation to the number of chromosomes with a significant variation. The highest values were detected for samples having between two and six chromosomes with a significant degree of aneuploidy (2-test for rare distribution = 83.6, P<0.01).
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Discussion |
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There are a few landmarks to ascertain the reliability of a FISH test. The most accepted in the literature is represented by the higher frequency of aneuploidy in OAT patients compared to normozoospermic (Williams et al., 1993; Spriggs et al., 1995
; Egozcue et al., 1997
; Pang et al., 1999
). This external landmark (i.e. not pertinent to the test itself) has been chosen to check the reliability of the test presented here. Ejaculated sperm reach a higher maturation degree compared to those retrieved from the testis by TESE (Aboulghar et al., 1997
). Accordingly, they have a lower degree of aneuploidy; this constitutes the second external landmark of this study. Finally, internal landmarks were selected to assess the test reliability, by verifying: (i) whether an unselected population was studied; and (ii) whether the pathophysiological mechanism generating aneuploidy could be recognized in the studied sample.
The present data demonstrate that the designed FISH protocol for the analysis of severe OAT and testicular sperm is reliable, implying that the studied sample may be considered representative of the original population. The proportion of aneuploidy for each chromosome between the original and the examined population was determined, indicating that the preparation technique does not select aneuploidy, which is randomly represented in the studied sample.
According to the results reported in this study, significant differences between the observed and expected FISH frequencies exist, with testicular sperm being significantly more prone to aneuploidy than ejaculated sperm (Table III, IV). In addition, the simultaneous presence of aneuploidy events in the same gamete appeared to be not randomly distributed (Figure 2) confirming, by statistical analysis, that sperm aneuploidy derives from alterations of the meiotic spindle, in which each chromosome is allocated to a settled place (Egozcue et al., 2000). These findings support the hypothesis that FISH analysis should be regarded as a marker of spermatogenesis, which influences sperm functions more positively than sperm count (Silber, 2000
; Biagiotti et al., 2002
).
The introduction of the multi-probe FISH technique for sperm analysis has included the numerical study of chromosomes as a routine test for the screening of infertile couples. The results obtained in combination with epidemiological studies could assist the comprehension of paternally derived chromosome abnormalities. In view of the data regarding the karyotype of the children born after ICSI (Liebaers et al., 1995; Bonduelle et al., 2002
), the paternal contribution to aneuploidy could be especially notable in cases of severe male factor infertility.
The study of aneuploidy in sperm from normozoospermic patients has demonstrated a mean autosomal disomy rate of 0.13%; this value for the gonosomes is higher, corresponding to 0.37% (Egozcue et al., 2003). As a result, the total disomy frequency in a normal ejaculate should be
3%, with total aneuploidy corresponding to 6%. This theoretical estimation is based on the assumption that each sperm cell only presents one aneuploid event, and that all chromosomes have the same rate of variation. Nevertheless, some chromosomes, such as chromosome 21 and the gonosomes, have a higher tendency to non-disjunction compared to the others (Blanco et al., 1996
; Martin et al., 1996
; Vegetti et al., 2000
; Rubio et al., 2001
; Carrell et al., 2003
; Egozcue et al., 2003
). Although this finding could represent a factor of reproductive risk, its clinical implications are considered almost irrelevant when compared to the maternal contribution to aneuploidy, as trisomic pregnancies, in 90% of cases, are maternal in origin (Koehler et al., 1996
). In this respect, it has been postulated that trisomies of paternal origin could have a lower survival potential compared to those generated during oogenesis, yielding an underestimation of their frequency at implantation (Hall, 1990
). These data derive from the analysis of abortuses in the general population to which conceptuses generated by assisted reproduction give a small numerical contribution. Therefore, the situation could be different in cases of severe male factor infertility, including azoospermia, whose treatment by ICSI completely bypasses any mechanism of natural sperm selection.
There is increasing evidence that the outcome of ICSI is closely and positively related to the quality of spermatogenesis as well as to the degree of male gamete haploidy (Aboulghar et al., 1997; De Croo et al., 2000
; Escudero et al., 2003
). One of the major problems in sperm FISH studies is represented by the low amount of available DNA (Harkonen et al., 2001
), for which a solution could be represented by the protocol described in this study. It has been suggested that sperm aneuploidies are provoked by an alteration of the meiotic spindle (Egozcue et al., 2000
) caused by a modification of the intracellular redox potential (Zini et al., 2001
). Estimating the degree of sperm aneuploidy associated with severe male factor infertility could be particularly interesting in view of the availability of therapies able to improve sperm count and euploidy by restoring the physiological sperm redox potential in idiopathic infertile males (Wong et al., 2002
; Cavallini et al., 2004
). As the chromosomal defects in gametes can be transmitted to the resulting embryos, an attempt was made to relate the FISH results generated by sperm analysis to those obtained on preimplantation embryos (Table VI
). Although a higher number of cases will be necessary to draw valuable conclusions by controlling the dominant effect of female age, this approach seems to be promising in indicating correlations between sperm aneuploidy and the resulting embryos.
In conclusion, preimplantation genetic diagnosis for aneuploidy has been reported to have a prognostic role on subsequent attempts at assisted reproduction (Ferraretti et al., 2004). For sperm, a threshold in the aneuploidy frequency could be proposed which may enable estimation of the reproductive possibilities for each infertile couple. This strategy, in the framework of an adequate therapeutic programme, represents the most advantageous situation in patients' management.
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Submitted on January 11, 2005; resubmitted on March 14, 2005; accepted on March 22, 2005.