1 Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Biology, Siena University, Regional Referral Center for Male Infertility, Azienda Ospedaliera Universitaria Senese, Siena and 2 Department of Pathological Anatomy and Genetics D.A.P.E.G., University of Bari, via Amendola 165/A, 70126, Bari, Italy
3 To whom correspondence should be addressed at: Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, Via Tommaso Pendola 62, 53100 Siena, Italy. Email: baccetti{at}unisi.it
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Abstract |
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Key words: FISH/ICE/Robertsonian translocation/sperm/TEM
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Introduction |
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Different opinions concerning chromosomal influence on testicular function have been reported. In 1976, Plymate and colleagues described the effect of translocated D group chromosomes on spermatogenesis (Plymate et al., 1976). Up to that time it had been accepted that defective testicular function may only be associated with sex chromosome abnormalities, as in the classic and variant forms of Klinefelter's syndrome (Paulsen et al., 1968
).
Impairment of spermatogenesis in carriers of chromosome anomalies was demonstrated by Chandley et al. (1976) and seems to be due particularly to translocations. On the contrary, Marmor et al. (1980)
did not find any differences in sperm parameters of balanced translocation carriers compared with individuals with normal karyotypes. Nevertheless, Guichaoua et al. (1990)
reported that a sterile male t(14;22) carrier had oligoasthenospermia with normal sperm morphology evaluated by light microscopy. In't Veld et al. (1997)
and Ogawa et al. (2000)
observed oligospermia in carriers of different Robertsonian translocations (13;14 and 13;13). More recently, Baccetti et al. (2002)
analysed spermatozoa from a sterile male carrier of t(14;22) by transmission electron microscope (TEM) and found unusual ultrastructural sperm defects related to immaturity that could be due to the effect of the translocation on sperm differentiation and development.
The spermatogenetic derangement in translocation carriers is not only related to sperm concentration, motility and morphology, but also concerns sperm chromosomal constitution. Using the hamster oocyte test, Martin (1995) demonstrated that 327% of sperm are chromosomally unbalanced, depending on the specific translocation. Fluorescence in-situ hybridization (FISH) studies showed that the meiotic segregation of Robertsonian translocation t(14q;21q) (Rousseaux et al., 1995
; Honda et al., 2000
) and t(13q;14q) had similar frequencies of unbalanced gametes, producing a majority of normal or balanced spermatozoa (Escudero et al., 2000
; Morel et al., 2001
; Anton et al., 2004
). The recent study of Liu and Zhu (2004)
gave conflicting results, showing a high percentage of unbalanced spermatozoa.
The possibility that chromosomal rearrangements may interfere with meiotic behaviour of chromosomes not involved in translocation led to the introduction of the concept of interchromosomal effect (ICE), first postulated in humans by Lejeune (1965). Using the humanhamster system to highlight human sperm karyotype, no significant ICE was detected in Robertsonian translocation carriers (Guttenbach et al., 1997
). However, ICE on meiotic segregation of sex chromosomes and autosomes has also been investigated directly in sperm nuclei by FISH (Rousseaux et al., 1995
; Vegetti et al., 2000
; Blanco et al., 2000
; Morel et al., 2001
; Anton et al., 2004
), and the results generally suggested that ICE was restricted to translocation carriers with abnormal semen parameters (Vegetti et al., 2000
; Pellestor et al., 2001
) at the light microscopic level.
In order to clarify the relationship between chromosomal rearrangements, sperm morphology and presence of ICE, we selected seven infertile males with different Robertsonian translocations. Semen analysis was performed by light and electron microscopy to evaluate morphological quality of spermatozoa in detail, and a FISH study was carried out in six out of seven patients to evaluate the possibility of ICE in these Robertsonian translocation carriers.
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Materials and methods |
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Sexual development, medical history and physical examination were normal and serum hormone concentrations (FSH, LH, PRL, androstenedione, DEAS, estradiol, testosterone, free-testosterone -inhibin) were in the standard range. Microbiological investigations did not reveal any urogenital infection. Patients had never received hormone therapy.
Karyotype
Conventional cytogenetic analysis of 2448 h cultures of blood lymphocytes of the patients was performed using standard techniques and evaluated by Giemsa-Trypsin-Giemsa (GTG) banding at about the 400 band level according to the 1995 International System for Human Cytogenetic Nomenclature (Mitelman, 1995).
Karyotype FISH analysis was performed with alphoid probes pX14 and pZ21a. The probe pX14 labelled both chromosomes 14 and 22 and the probe pZ21a the chromosomes 13 and 21.
Plasmidic DNA from bacterial cultures was directly used as probe for FISH experiments on chromosome metaphases of phytohaemagglutinin-stimulated peripheral blood lymphocytes of the patient. Chromosome preparations were hybridized in situ essentially as described previously (Lichter et al., 1990), albeit with minor modifications (Marzella et al., 1997
). In FISH experiments, chromosomes were identified by diamino-phenylindole (DAPI) counterstaining, and digital images obtained using Leica epifluorescence microscope equipped with cooled charge-coupled device camera. Cy3 (Amersham and FL-X) and DAPI fluorescence signals were recorded separately as greyscale images. Pseudocolouring and merging were performed using commercial Adobe Photoshop software.
Light and electron microscopy
Semen samples of selected patients were collected by masturbation after 4 days of sexual abstinence and examined after liquefaction for 30 min at 37°C. Volume, pH, concentration and motility were evaluated according to WHO guidelines (World Health Organization, 1999). The eosin Y test was performed to detect viable spermatozoa.
For electron microscopy, sperm samples were fixed in cold Karnovsky fixative and maintained at 4°C for 2 h. Fixed semen was washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, post-fixed in 1% buffered osmium tetroxide for 1 h at 4°C, dehydrated and embedded in Epon Araldite. Ultra-thin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted on copper grids, stained with uranyl acetate and lead citrate and observed and photographed with a Philips CM10 TEM (Philips Scientifics, Eindhoven, The Netherlands).
For each patient 300 sperm were analysed in ultra-thin sections. Major submicroscopic characteristics were recorded by highly trained examiners who were blind to the experiment. TEM data was evaluated using the mathematical statistical formula of Baccetti et al. (1995), which calculates the number of spermatozoa free of structural defects (healthy) and the percentages of three main phenotypic sperm pathologies: immaturity, necrosis and apoptosis.
Controls. Semen samples from 10 fertile men (aged 2634 years) with normal karyotype were used as controls.
FISH analysis of sperm
In order to evaluate aneuploidy frequency, FISH was performed according to Baccetti et al. (2003) in sperm nuclei of six selected patients. A mix of
-satellite DNA probes (CEP, Chromosome Enumeration Probes, Vysis, IL, USA) for chromosomes 18, X and Y, directly labelled with different fluorochromes, was used.
Scoring criteria. The overall hybridization efficiency was >99%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995), namely, they were only scored if they were intact, non-overlapped and had a clearly defined border. In the case of aneuploidy, the presence of a sperm tail was confirmed. A sperm was considered disomic if the two fluorescent spots were of the same colour, similar in size, shape and intensity, and disposed inside the edge of the sperm head, at least one domain apart. Diploidy was recognized by the presence of two double fluorescent spots, according to the above criteria. Observation and scoring were performed using a Leitz Aristoplan Optical Microscope equipped with fluorescence apparatus, with a triple bandpass filter for aqua, orange and green fluorochromes (Vysis) and a monochrome filter for DAPI.
Controls. Semen samples from seven fertile men (aged 2639 years) were analysed and used as controls (Baccetti et al., 2003).
Statistical analysis
Differences in aneuplody rates between the two groups, translocation carriers and fertile males, used as controls, were analysed by Wilcoxon scores (rank sums); P<0.05 was considered significant.
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Results |
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Discussion |
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In previous studies morphological sperm quality has always been evaluated by light microscopy, sometimes applying Kruger's criteria. Subcellular sperm anomalies can only be detected by electron microscopy. The mathematical formula developed by Baccetti et al. (1995) calculates the percentage of sperm devoid of structural defects and the probability of sperm pathologies, suggesting the best assisted reproduction technique (ART) for each ejaculate. We observed that the lowest number of spermatozoa free of defects that would assure a normal fertility was a little higher than two million. Other investigations carried out by our group on the relationship between sperm quality and IVF (Piomboni et al., 1996
) and ICSI (Strehler et al., 1995
) allowed us to set the range of healthy sperm used to select the best ART in each patient.
Sperm ultrastructural examination in this group of patients showed a higher percentage of immaturity, apoptosis and necrosis than in controls. Diffuse impairment of spermatogenesis was indicated by incompletely mature sperm, often in the process of apoptotis and late stages of necrosis. Many binucleate sperm were found in every ejaculate, indicating impairment of germ cell division during spermiogenesis. The percentage of healthy sperm was very low and the mathematical formula indicated ICSI as the preferential treatment.
In Robertsonian translocations, when the chromosomes organize into pairs during meiosis, the translocated chromosome and its homologous chromosome do so as a trivalent. The resulting gametes may be chromosomally normal or aneuploid with an extra chromosome or a missing chromosome q arm. This particular meiotic configuration could interfere with the correct segregation of chromosomes not involved in translocation, leading to ICE.
The presence of ICE in translocation carriers is a subject still under debate.
Although it is true that some results support ICE of gonosomes and/or autosomes (Rousseaux et al., 1995; Morel et al., 2001
; Anton et al., 2004
), other authors have reported that the presence of ICE is restricted to those carriers with poor sperm quality evaluated at the light microscopic level (Vegetti et al., 2000
; Pellestor et al., 2001
).
In addition, infertile patients with normal somatic karyotypes seem to have an increase in the frequency of aneuploidy (Bernardini et al., 1997; Rives et al., 1999
; Martin et al., 2003
), and the presence of compromised testicular environments could favour meiotic errors (Mroz et al., 1999
).
In the present study aneuploidies of gonosomes were detected in sperm of five out of six carriers of Robertsonian translocation, whereas aneuploidy of chromosome 18 was evident in three carriers out of six. A high frequency of chromosome 18 disomy was observed in cases in which all FISH values were altered.
The increment of aneuploidies in sperm from the six patients examined could be linked to ICE; however, as none of the carriers was classified as normozoospermic, an effect of poor testicular environment on the meiotic process should not be excluded.
Moreover, it is commonly thought that the occurrence of ICE depends on the type of chromosomes involved in translocation and on the rearranged chromosomal region. Our results confirm that Robertsonian translocations have variable effects on spermatogenesis and that the same rearrangement may be associated with spermatogenetic impairment in some cases but not in others. The sperm of the four t(13;14) carriers showed different meiotic segregation patterns (detected by FISH), revealing that the same translocation may or may not lead to aneuploidies.
The frequencies of diploidy were particularly interesting, being significantly higher in patients than controls. Diploidy of sperm cells may be generated by a binucleate sperm head or by diploid nuclei: the first case was demonstrated by the membranous sperm septum between the two nuclei, the second was deduced from the presence of double sets of chromosomes 18, X and Y.
FISH analysis performed directly on sperm provides a better understanding of the meiotic process and could improve the risk assessment of chromosomally abnormal embryos of carriers of Robertsonian translocation, who constitute a group of candidates for ICSI (Munnè et al., 2000; Scriven et al., 2001
). Gianaroli et al. (2002)
reported that infertile patients may produce embryos with a high incidence of aneuploidies and that ICE in particular seems to play a role in Robertsonian translocation carriers. The substantial contribution of aneuploidies exposes these couples to an additional risk of abnormal pregnancy.
In our experience (unpublished observations), a couple, the male partner of which was a carrier of t(13;21), underwent ICSI in 1997 and PGD of the embryos. PGD was performed by FISH in four embryos: one embryo was apparently euploid, but balanced for the translocation, whereas the other three embryos were monosomic, haploid and tetraploid. The baby was therefore a carrier of 13;21 translocation.
Until now genetic counselling of Robertsonian translocation carriers has been based on outcomes observed during prenatal diagnosis or at birth (Bouè and Gallano, 1984). Since the infertile patients analysed showed severe spermatogenetic impairment from the morphological and meiotic points of view, detailed ultrastructural and chromosomal analysis of sperm of Robertsonian translocation carriers is advisable before undertaking ICSI cycles, especially in countries like Italy, where PGD is not allowed.
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Acknowledgements |
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References |
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Submitted on January 14, 2005; resubmitted on March 16, 2005; accepted on April 1, 2005.
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