Ultrastructural studies of spermatozoa from infertile males with Robertsonian translocations and 18, X, Y aneuploidies

B. Baccetti1,3, G. Collodel1, R. Marzella2, E. Moretti1, P. Piomboni1, G. Scapigliati1 and F. Serafini1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In order to clarify the relationship between chromosomal rearrangements, sperm morphology and interchromosomal effects (ICE), we studied the spermatogenetic defects in seven infertile Robertsonian translocation carriers. METHODS: Lymphocyte karyotypes were evaluated using Giemsa-Trypsin-Giemsa banding and fluorescence in-situ hybridization (FISH). Semen analysis was performed by light and transmission electron microscopy. FISH of sperm nuclei was carried out to detect possible ICE. RESULTS: Lymphocyte karyotype analysis revealed five t(13;14), one t(13;21) and one t(14;22) carriers. Sperm ultrastructural examination highlighted a higher percentage of immaturity, apoptosis and necrosis than in controls. Aneuploidies of gonosomes were detected in sperm from five out of six carriers of Robertsonian translocation, whereas aneuploidy of chromosome 18 was evident in three out of six carriers. The frequencies of diploidy were altered in all cases. CONCLUSIONS: Since these infertile patients showed severe spermatogenetic impairment from the morphological and meiotic points of view, we recommend detailed sperm ultrastructural and chromosomal analysis before undertaking ICSI cycles in Robertsonian translocation carriers.

Key words: FISH/ICE/Robertsonian translocation/sperm/TEM


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Structural chromosomal anomalies are more frequent in infertile men than in the general population (Van Assche et al., 1996Go; Kalantari et al., 2001Go). Robertsonian translocations and numerical sex chromosome aberrations are the most common structural anomalies in these subjects. Robertsonian translocations are characterized by centric fusion of two acrocentric chromosomes resulting in a 45 chromosome karyotype. Among them, t(13q;14q) and t(14q;21q) have an estimated frequency of 0.97 and 0.20, respectively (Frydman et al., 2001Go). Abnormal meiotic segregation of these reorganizations lead to almost complete trisomy 14 (not viable), almost complete trisomy 13 (probability of survival to birth 2.8%) and almost complete trisomy 21 (probability of survival to birth 22.1%) (Egozcue et al., 2000Go).

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., 1976Go). 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., 1968Go).

Impairment of spermatogenesis in carriers of chromosome anomalies was demonstrated by Chandley et al. (1976)Go and seems to be due particularly to translocations. On the contrary, Marmor et al. (1980)Go did not find any differences in sperm parameters of balanced translocation carriers compared with individuals with normal karyotypes. Nevertheless, Guichaoua et al. (1990)Go 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)Go and Ogawa et al. (2000)Go observed oligospermia in carriers of different Robertsonian translocations (13;14 and 13;13). More recently, Baccetti et al. (2002)Go 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)Go demonstrated that 3–27% 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., 1995Go; Honda et al., 2000Go) and t(13q;14q) had similar frequencies of unbalanced gametes, producing a majority of normal or balanced spermatozoa (Escudero et al., 2000Go; Morel et al., 2001Go; Anton et al., 2004Go). The recent study of Liu and Zhu (2004)Go 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)Go. Using the human–hamster system to highlight human sperm karyotype, no significant ICE was detected in Robertsonian translocation carriers (Guttenbach et al., 1997Go). However, ICE on meiotic segregation of sex chromosomes and autosomes has also been investigated directly in sperm nuclei by FISH (Rousseaux et al., 1995Go; Vegetti et al., 2000Go; Blanco et al., 2000Go; Morel et al., 2001Go; Anton et al., 2004Go), and the results generally suggested that ICE was restricted to translocation carriers with abnormal semen parameters (Vegetti et al., 2000Go; Pellestor et al., 2001Go) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We selected seven infertile male Robertsonian translocation carriers (age range 36–46 years) referred to our centre for semen analysis, after several years of unprotected sexual intercourse without conception. Five patients, including two brothers, were carriers of t(13;14), another of t(13;21) and the last of t(14;22). The patient with t(13;21) fathered a child with the same translocation following ICSI and prenatal genetic diagnosis (PGD).

Sexual development, medical history and physical examination were normal and serum hormone concentrations (FSH, LH, PRL, androstenedione, DEAS, estradiol, testosterone, free-testosterone {beta}-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 24–48 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, 1995Go).

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., 1990Go), albeit with minor modifications (Marzella et al., 1997Go). 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, 1999Go). 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)Go, 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 26–34 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)Go in sperm nuclei of six selected patients. A mix of {alpha}-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, 1995Go), 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 26–39 years) were analysed and used as controls (Baccetti et al., 2003Go).

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Lymphocyte karyotyping performed with conventional GTG banding and FISH analysis on chromosomal metaphases revealed that five patients, including two brothers, had a Robertsonian translocation (13;14), one patient was carrier of t(13;21) and the other of t(14;22) (Table I). With regard to semen parameters, three carriers (patients 4, 6 and 7) had a normal number of sperm/ml according to WHO guidelines (World Health Organization, 1999Go). Two individuals (patients 2 and 5) were severely oligozoospermic. Total progressive motility was reduced in all cases (Table I). The eosin test revealed 70–80% of viable spermatozoa.


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Table I. Spermatological characteristics of seven infertile Robertsonian translocation carriers

 
TEM analysis was carried out in only six out of seven patients (Table II) because one of them (patient 2) was severely oligozoospermic and had insufficient sperm cells. The percentage of sperm devoid of ultrastructural defects (‘healthy’) and sperm pathologies according to the formula of Baccetti et al. (1995)Go are reported in Table II. These figures revealed severe spermatogenetic impairment in the samples of all six patients. The sperm pathologies evaluated by Baccetti's formula, namely apoptosis, immaturity and necrosis, were more frequent than in spermatozoa from control subjects (Table II).


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Table II. TEM spermatological characteristics of six infertile Robertsonian translocation carriers

 
As far as immaturity is concerned, the nuclei were irregularly shaped, generally elliptical or spherical, with uncondensed chromatin (Figure 1) and cytoplasmic residues embedding the head or midpiece regions. Spermatids and binucleated or multinucleated spermatozoa were generally observed. Axonemal structure was often altered, lacking central microtubules, and some doublets had incomplete or absent dynein arms. The tail was often rolled up into cytoplasmic droplets with a disorganized fibrous sheath and accessory fibres, mitochondrial helix was badly assembled.



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Figure 1. TEM micrograph of longitudinal and cross sections of immature sperm characterized by irregular nuclei with uncondensed chromatin (uCh). A large cytoplasmic residue (CR) embeds the coiled axoneme (AX) and disorganized, swollen mitochondria (M). A=acrosome. Original magnification x10 000.

 
Apoptotic sperm (Figure 2) were present and characterized by misshapen acrosome and nucleus with marginated chromatin and swollen mitochondria, irregularly organized into large cytoplasmic residues with translucent vacuoles. Broken plasma membrane, absent or reacted acrosomes, disrupted chromatin texture and disassembled axonemes were typical features of necrosis (Figure 3).



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Figure 2. TEM micrograph of longitudinal and cross sections of apoptotic sperm characterized by altered nuclei with marginated chromatin (mCh), coiled axoneme (AX) irregularly organized into large cytoplasmic residues (CR) with translucent vacuole (V). The plasma membrane (PM) is intact. aA=altered acrosome. Original magnification x6000.

 


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Figure 3. TEM micrograph of longitudinal and cross sections of necrotic binucleated sperm characterized by the absence of acrosome (aA) and misshapen nuclei (N) with necrotic chromatin (nCh). Axonemal structure (AX) is altered, lacking microtubules and dynein arms. Mitochondria (M) are swollen and dispersed. The plasma membrane is broken (arrow). Original magnification x12 000.

 
FISH sperm analysis was performed in six out of seven patients with Robertsonian translocation because one of them (patient 5) was severely oligozoospermic. A total of 22 367 sperm nuclei were scored evaluating the aneuploidy and diploidy frequencies of chromosomes 18, X and Y (Table III). FISH data from the seven fertile males used as controls are shown in Table IV.


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Table III. Frequencies of disomies and diploidies of chromosomes 18, X and Y in six male carriers of Robertsonian translocations

 

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Table IV. Frequencies of disomies and diploidies of chromosomes 18, X and Y in seven fertile male

 
In the group of translocation carriers, the diploidy rate was significantly higher compared with controls (P<0.0027). In the t(13;21) and t(14;22) carriers (patients 6 and 7), disomy values were also out of the normal range. Among carriers of t(13;14), gonosome disomy was more frequent in three cases (patients 2, 3 and 4), whereas disomy 18 was substantially more frequent in only one case (patient 2). The total rate of disomy of gonosomes was significantly higher than controls in the group of translocation carriers (P<0.05). No statistically significant difference was found between the two groups with respect to the disomy rate of chromosome 18 (P<0.15).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, spermatogenetic derangement was analysed in seven carriers of Robertsonian translocation. The spermatozoa of these sterile individuals were examined by light and electron microscopy to determine semen parameters and details of fine morphology. FISH analysis was performed in order to investigate the meiotic behaviour of chromosomes not involved in translocation, namely gonosomes and chromosome 18. Four out of seven Robertsonian translocation carriers were oligospermic, probably because any factor that delays anaphase, such as erratic separation of chromosomes, may arrest division, leading the cells into apoptosis (Anton et al., 2004Go).

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)Go 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., 1996Go) and ICSI (Strehler et al., 1995Go) 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., 1995Go; Morel et al., 2001Go; Anton et al., 2004Go), 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., 2000Go; Pellestor et al., 2001Go).

In addition, infertile patients with normal somatic karyotypes seem to have an increase in the frequency of aneuploidy (Bernardini et al., 1997Go; Rives et al., 1999Go; Martin et al., 2003Go), and the presence of compromised testicular environments could favour meiotic errors (Mroz et al., 1999Go).

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., 2000Go; Scriven et al., 2001Go). Gianaroli et al. (2002)Go 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, 1984Go). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was financed by University of Siena P.A.R. grant 2003 and Azienda Ospedaliera Senese, Siena, Italy.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Anton E, Blanco J, Egozcue J and Vidal F (2004) Sperm FISH studies in seven male carriers of Robertsonian translocation t(13;14)(q10;q10). Hum Reprod 19, 1345–1351.[Abstract/Free Full Text]

Baccetti B, Bernieri G, Burrini AG, Collodel G, Crisà N, Mirolli M, Moretti E and Piomboni P (1995) Notulae seminologicae 5. Mathematical evaluation of interdependent submicroscopic sperm alterations. J Androl 16, 365–371.

Baccetti B, Capitani S, Collodel G, Estenoz M, Gambera L and Piomboni P (2002) Infertile spermatozoa in a human carrier of Robertsonian translocation 14;22. Fertil Steril 78, 1127–1130.[CrossRef][ISI][Medline]

Baccetti B, Bruni E, Collodel G, Gambera L, Moretti E, Marzella R and Piomboni P (2003) 10;15 reciprocal translocation in an infertile man: ultrastructural and fluorescence in-situ hybridization sperm study: case report. Hum Reprod 18, 2302–2308.[Abstract/Free Full Text]

Bernardini L, Martini E, Geraedts JP, Hopman AH, Lanteri S, Conte N and Capitanio GL (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol Hum Reprod 3, 431–488.[Abstract]

Blanco J, Egozcue J and Vidal F (2000) Interchromosomal effects for chromosome 21 in carriers of structural chromosome reorganizations determined by fluorescence in situ hybridization on sperm nuclei. Hum Genet 106, 500–505.[CrossRef][ISI][Medline]

Bouè A and Gallano P (1984) A collaborative study of the segregation of inherited chromosome structural rearrangements in 1356 prenatal diagnoses. Prenat Diagn 4, 45–67.[ISI][Medline]

Chandley AC, Maclean N, Edmond P, Fletcher J and Watson GS (1976) Cytogenetics and fertility in man II. Testicular histology and meiosis. Ann Hum Genet 40, 165–176.[ISI][Medline]

Egozcue S, Blanco J, Vendrell JM, Garcia F, Veiga A, Aran B, Barri PN, Vidal F and Egozcue J (2000) Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum Reprod Update 6, 93–105.[Abstract/Free Full Text]

Escudero T, Lee M, Carrel D, Blanco J and Munné S (2000) Analysis of chromosome abnormalities in sperm and embryos from two 45, XY t(13; 14) (q10; q10) carriers. Prenat Diagn 20, 599–602.[CrossRef][ISI][Medline]

Frydman N, Romana S, Le Lorc'h M, Vekemans M, Frydman R and Tachdjian G (2001) Assisting reproduction of infertile men carrying a Robertsonian translocation. Hum Reprod 16, 2274–2277.[Abstract/Free Full Text]

Gianaroli L, Magli MC, Ferraretti AP, Munnè S, Balicchia B, Escudero T and Crippa A (2002) Possibile interchromosomal effect in embryos generated by gametes from translocation carriers. Hum Reprod 17, 3201–3207.[Abstract/Free Full Text]

Guichaoua MR, Quack B, Speed RM, Noel B, Chandley AC and Luciani JM (1990) Infertility in human males with autosomal translocations: meiotic study of a 14;22 Robertsonian translocation. Hum Genet 86, 162–166.[ISI][Medline]

Guttenbach M, Michelmann HW, Hinney B, Engel W and Schmid M (1997) Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations: a review. Hum Genet 100, 1–21.[CrossRef][ISI][Medline]

Honda H, Miharu N, Samura O, He H and Ohama K (2000) Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization. Hum Genet 106, 188–193.[CrossRef][ISI][Medline]

In't Veld PA, Weber RF, Los FJ, den Hollander N, Dhont M, Pieters MH and Van Hemel JO (1997) Two cases of Robertsonian translocations in oligozoospermic males and their consequences for pregnancies induced by intracytoplasmic sperm injection. Hum Reprod 12, 1642–1644.[Abstract]

Kalantari P, Sepehri H, Behjati F, Ashtiani ZO and Akbari MT (2001) Chromosomal studies in infertile men. Tsitol Genet 35, 50–54.

Leujeune J (1965) Les concéquences méiotiques des remaniements chromosomiques. Ann Genet 8, 9–10.[Medline]

Lichter P, Tang Chang CJ, Call K, Hermanson G, Evans GA, Housman D and Ward DC (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69.[ISI][Medline]

Liu Y and Zhu H (2004) Detection of sperm chromosomes in Robertsonian translocation carriers by dual-color fluorescence in situ hybridization. Zhonghua Nan Ke Xue 10, 90–93.[Medline]

Marmor D, Taillemite JL, Van Den Akker J, Portnoi MF, le Porrier N, Delafontaine D and Roux C (1980) Semen analysis in subfertile balanced-translocation carriers. Fertil Steril 34, 496–502.[ISI][Medline]

Martin RH (1995) Sperm cell-genetics aspects. (ed. Grudzinskas JG, Yovich JL, Simpson JL, Chard T), Cambridge Reviews in Human Reproduction. Cambridge University Press, Cambridge, UK, pp. 104–121.

Martin RH and Rademaker A (1995) Reliability of aneuploidy estimates in human sperm: results of fluorescence in situ hybridization studies using two different scoring criteria. Mol Reprod Dev 42, 89–93.[CrossRef][ISI][Medline]

Martin RH, Rademaker AW, Greene C, Ko E, Hoang T, Barclay L and Chernos J (2003) A comparison of the frequency of sperm chromosome abnormalities in men with mild, moderate, and severe oligozoospermia. Biol Reprod 69, 535–539.[Abstract/Free Full Text]

Marzella R, Viggiano L, Ricco A, Tanzariello A, Fratello A, Archidiacono N and Rocchi M (1997) A panel of radiation hybrids and yac clones specific for chromosome 5. Cytogenet Cell Genet 77, 232–237.[ISI][Medline]

Mitelman F (ed.) (1995) An International System for Human Cytogenetic Nomenclature. ISCN, Karger, Basel.

Morel F, Roux C and Bresson JL (2001) FISH analysis of the chromosomal status of spermatozoa from three men with 45,XY,der(13;14)(q10;q10) karyotype. Mol Hum Reprod 7, 483–488.[Abstract/Free Full Text]

Mroz K, Hassold TJ and Hunt PA (1999) Meiotic aneuploidy in the XXY mouse: evidence that a compromised testicular environment increases the incidence of meiotic errors. Hum Reprod 14, 1151–1156.[Abstract/Free Full Text]

Munnè S, Escudero T, Saudalinas M, Sable D and Cohen J (2000) Gamete segregation in female carriers of Robertsonian translocation. Cytogenet Cell Genet 90, 303–308.[CrossRef][ISI][Medline]

Ogawa S, Araki S, Ohno M and Sato I (2000) Chromosome analysis of human spermatozoa from an oligoasthenozoospermic carrier for a 13;14 Robertsonian translocation by their injection into mouse oocytes. Hum Reprod 15, 1136–1139.[Abstract/Free Full Text]

Paulsen CA, Gordon DL, Carpenter RW, Gandy HM and Drucker WD (1968) Klinefelter's syndrome and its variants: a hormonal and chromosomal study. Recent Prog Horm Res 24, 321–363.[Medline]

Pellestor F, Imbert I, Andreo B and Lefort G (2001) Study of the occurrence of interchromosomal effect in spermatozoa of chromosomal rearrangement carriers by fluorescence in-situ hybridization and primed in situ labelling techniques. Hum Reprod 16, 1155–1164.[Abstract/Free Full Text]

Piomboni P, Strehler E, Capitani S, Collodel G, De Santo M, Gambera L, Moretti E, Baccetti B and Sterzik K (1996) Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction 2. In vitro fertilization (Notulae seminologicae 7). J Assist Reprod Genet 13, 635–646.[ISI][Medline]

Plymate SR, Bremner WJ and Paulsen CA (1976) The association of D-group chromosomal translocations and defective spermatogenesis. Fertil Steril 27, 139–144.[ISI][Medline]

Rives N, Saint Clair A, Mazurier S, Sibert L, Simeon N, Joly G and Mace B (1999) Relationship between clinical phenotype, semen parameters and aneuploidy frequency in sperm nuclei of 50 infertile males. Hum Genet 105, 266–272.[CrossRef][ISI][Medline]

Rousseaux S, Chevret E, Monteil M, Cozzi J, Pelletier R, Delafontaine D and Sele B (1995) Sperm nuclei analysis of a Robertsonian t(14q;21q) carrier by FISH, using three plasmids and two YAC probes. Hum Genet 96, 655–660.[CrossRef][ISI][Medline]

Scriven PN, Flinter FA, Brande PR and Ogilvie CM (2001) Robertsonian translocations—reproductive risks and indications for preimplantation genetic diagnosis. Hum Reprod 16, 2267–2273.[Abstract/Free Full Text]

Strehler E, Capitani S, Collodel G, De Santo M, Moretti E, Piomboni P, Sterzik S and Baccetti B (1995) Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. I. Intracytoplasmic sperm injection (Notulae seminologicae 6). J Submicrosc Cytol Pathol 27, 573–586.[ISI][Medline]

Van Assche E, Bonduelle M, Tournaye H, Joris H, Verheyen G, Devroey P, Van Steirteghem A and Liebaers I (1996) Cytogenetics of infertile men. Hum Reprod 11, 1–24.

Vegetti W, Van Assche E, Frias A, Verheyen G, Bianchi MM, Bonduelle M, Liebaers I and Van Steirteghem A (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum Reprod 15, 351–365.[Abstract/Free Full Text]

World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, UK.

Submitted on January 14, 2005; resubmitted on March 16, 2005; accepted on April 1, 2005.





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Articles by Baccetti, B.
Articles by Serafini, F.