10, 15 reciprocal translocation in an infertile man: ultrastructural and uorescence in-situ hybridization sperm study: Case report

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1 Human Reproduction Vol.18, No.11 pp. 2302±2308, 2003 DOI: /humrep/deg460 10, 15 reciprocal translocation in an infertile man: ultrastructural and uorescence in-situ hybridization sperm study: Case report B.Baccetti 1,2,4, E.Bruni 1, G.Collodel 1, L.Gambera 1,2, E.Moretti 1,2, R.Marzella 3 and P.Piomboni 1,2 1 Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, 2 Regional Referral Center for Male Infertility, Azienda Ospedaliera Senese, Siena and 3 Department of Pathological Anatomy and Genetics D.A.P.E.G, University of Bari, Bari, Italy 4 To whom correspondence should be addressed at: Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, via T. Pendola 62, Siena, Italy. baccetti@unisi.it BACKGROUND: Peculiar sperm defects are described in a sterile man heterozygous for a balanced translocation t(10;15) (q26;q12). As this structural reorganization was absent in the parents, the translocation must have appeared de novo in the present patient. METHODS: Spermatozoa were analysed under light and transmission electron microscopy (TEM). Fluorescence in-situ hybridization (FISH) was performed on the lymphocyte karyotype. Aneuploidy frequencies of chromosomes 18, X and Y in sperm nuclei, not involved in the translocation, were investigated using three-colour FISH. Dual- colour FISH was used to evaluate segregation of chromosomes 10, 15 in decondensed sperm nuclei. Moreover, three-colour FISH, using telomeric probes for chromosomes 10, 15 was performed in order to distinguish balanced and unbalanced gametes. RESULTS AND CONCLUSIONS: Overall, structural characteristics indicate general immaturity of the germinal cells. FISH sperm analysis detected an increase in chromosome 18 disomy (0.81%) suggesting an interchromosomal effect. A high frequency of diploidies, particularly 18,18,X,X and 18,18,X,Y, was also found. FISH segregation analysis for chromosomes 10, 15 indicated that 32.8% were balanced gametes, whereas 68.2% were unbalanced. Taken together, these data demonstrate in a male carrier of a reciprocal translocation t(10;15) the presence of diffuse ultrastructural sperm alterations and a high frequency of sperm aneuploidies. The existence of a correlation among these factors is proposed. Key words: electron microscopy/ uorescence in-situ hybridization/male infertility/reciprocal translocation/spermatozoa Introduction Human male infertility and chromosomal anomalies are often closely related. In particular, reciprocal translocations are the most frequent (1 in 600) structural chromosomal anomalies in humans (Estop et al., 1997), and among infertile males chromosomal reorganizations are about 10 times more frequent than in the general population (Van Assche et al., 1996). The frequency of chromosomally unbalanced spermatozoa in cases of reciprocal translocations is 50% on average, and depends heavily on the chromosomes involved in the translocation (Shi and Martin, 2001). The risk of miscarriage or birth defects resulting from unbalanced gamete formation in balanced translocation carriers is very high. The possible correlation between human male infertility due to impaired spermatogenesis and chromosome anomalies (mainly translocations) was postulated almost 30 years ago (Chandley, 1975; 1976). Likewise, others (Plymate et al., 1976; Leonard et al., 1979) found a correlation between derangement of spermatogenesis and autosomal translocations. Recently, the correlation between chromosomal anomalies and human male infertility has become more evident. One group (Pellestor et al., 2001) reported oligoteratozoospermia in carriers of reciprocal or Robertsonian chromosomal translocation; nevertheless, they found normal sperm number and motility in males with different structural chromosomal anomalies. Other authors (Marmor et al., 1980; Matsuda et al., 1991; Testart et al., 1996), when examining the morphology and motility of human spermatozoa with light microscopy, did not nd any correlation between sperm quality and chromosome translocations or inversions. Electron microscopy enables more detailed evaluation of sperm alterations (Baccetti, 1984; Zamboni, 1987; Bartoov et al., 1994; Chemes et al., 1998), and permits a distinction to be made between phenotypic and genotypic defects (Baccetti, 2000; Baccetti et al., 2001). Hereditary male sterility due to genotypic sperm defects is a possibility. The original idea of 2302 Human Reproduction 18(11) ã European Society of Human Reproduction and Embryology 2003; all rights reserved

2 Infertile male 10, 15 reciprocal translocation carrier generic human male infertility correlated to chromosome anomalies could give way to the concept that male infertility is due to particular defects, caused by chromosome anomalies, heralding research into the genes responsible for hereditary sperm characteristics and their mutations. Recently, brous sheath dysplasia of `stump' spermatozoa was associated with pericentric inversion of chromosome 9 (Baccetti et al., 1997). However, the clearest connection between a sterilizing human sperm defect and a speci c chromosome alteration was the discovery of mutations in the DNAI1 gene, mapping to 9 p13- p21, in three Kartagener's syndrome patients (Blouin et al., 2000; Guichard et al., 2001). In studying the association between chromosome mutations and sperm defects in sterile men, immature sperm were found in a human carrier of Robertsonian translocation 14;22 (Baccetti et al., 2002). This anomaly was most likely in a chromosome region involved in spermatogenesis. Herein, peculiar ultrastructural sperm defects are described in a sterile male carrier of a balanced translocation t(10;15) (q26;q12). Fluorescence in-situ hybridization (FISH) was used in decondensed sperm nuclei to evaluate chromosomes 10, 15 segregation and possible interchromosomal effects, detected through an increase in aneuploidy frequencies of chromosomes 18, X, Y that were not involved in the translocation. Materials and methods A 32-year-old man, an only child, was referred to the authors' laboratory for semen analysis after 3 years of sexual intercourse without conception. His wife, who was aged 30 years, had no apparent fertility problem. A testicular echo-colour-doppler examination did not reveal the presence of any functional alterations such as varicocele or hydrocele; testicle size was small bilaterally. The subject had never smoked, drunk alcohol or been addicted to drugs, and was not exposed to chemical contaminants or radiation. Baseline plasma concentrations of FSH, LH, estradiol, cortisol, prolactin, thyroid-stimulating hormone (TSH) and free thyroxine were normal, but the testosterone level was low. The patient had never received hormone therapy. Karyotype Conventional cytogenetic analysis of 24±48 h cultures of blood lymphocytes of the patient and his parents 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 (ISCN, 1995). Karyotype FISH analysis was performed with a painting probe for chromosome 15, obtained from human±hamster hybrid (GM11418) retaining chromosome 15 as the only human contribution (a kind gift from the Coriell Institute for Medical Research, NJ, USA). DNA from the hybrid was dual Alu-PCR ampli ed according to published methods (Liu et al., 1993). The PCR products were biotinlabelled by nick translation and used as probe for FISH experiments on chromosome metaphases of phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes of the patient. Chromosome preparations were hybridized in situ essentially as described previously (Lichter et al., 1990), albeit with minor modi cations (Marzella et al., 1997). In FISH experiments, chromosomes were identi ed by diamino-phenylindole (DAPI) counterstaining, and digital images obtained using a Leika epi uorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Cy3 (Amersham) and DAPI uorescence signals were recorded separately as grey-scale images. Pseudocolouring and merging were performed using commercial Adobe Photoshop software. Light and electron microscopy Semen samples were obtained 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 following published guidelines (World Health Organization, 1999). Semen analysis was performed three times at 6-month intervals. For electron microscopy, a single sperm sample was xed in cold Karnovsky xative 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 xed for 1 hat4 C in 1% buffered osmium tetroxide, dehydrated, and embedded in Epon Araldite. Ultra-thin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted onto copper grids, stained with uranyl acetate and lead citrate, observed and photographed with a Philips CM 10 transmission electron microscope (TEM; Philips Scienti cs, Eindhoven, The Netherlands). A total of 300 ultra-thin sperm sections was examined for major submicroscopic characteristics. TEM data were evaluated using a mathematical formula (Baccetti et al., 1995). An aliquot from the same sperm sample was also processed for scanning electron microscopy (SEM), xing the spermatozoa as described above and smearing them onto poly-lysine (1%) -coated coverslips. After dehydration, specimens were dried using the critical point technique, coated in gold and examined using a Philips CM 515 scanning electron microscope (Philips Scienti cs). FISH analysis of spermatozoa An aliquot from the same sperm sample as used for TEM and SEM analyses was washed with 150 mmol/l NaCl and 10 mmol/l Tris±HCl (ph 8), smeared onto glass slides and air-dried. Slides were then xed in methanol:acetic acid (3:1) for 10 min, dehydrated in 70, 80 and 100% cold ethanol, and air-dried. Samples were swell-treated with 0.01 mol/l dithiothreitol (Biorad) in 0.1 mol/l Tris±HCl (ph 8), followed by 20 mmol/l 3,5-diiodosalicylic acid, lithium salt (Sigma) in the same buffer, checking sperm head swelling. The slides, rinsed in 23 standard saline citrate (SSC), ph 7, air-dried and then dehydrated and denatured in 70% formamide (Aldrich) 23 SSC at 73 for 4 min. Slides were then quickly dehydrated in a graded ethanol series at 0 C and air-dried. During this last step, chromosome enumeration probes (Vysis, IL, USA), a-satellite DNA probes for chromosomes X, Y, 10, 15 and 18 directly labelled with different uorochromes, were used. Moreover, self-made telomeric probes RP11-10D13 for 10p, RP11-108K14 for 10q and RP11-9B21 for 15q, were used to distinguish alternate and adjacent-1 segregations. The probe mix was denatured for 5 min at 73 C in a water bath. Hybridization was carried out at 37 C in a moist chamber for 12 h. The slides were then washed with 0.43 SSC±0.3% Nonidet P40 (NP40) for 2 min at 73 C, quickly in 23 SSC±0.1% NP40 at room temperature, and nally mounted with DAPI 125 ng/ml in antifade solution (Vysis). In total, 3023 spermatozoa were analysed using triple-colour FISH (18, X, Y), 2606 were scored by dual-colour FISH (10, 15), and 2100 sperm cells were examined with telomeric probes. Scoring criteria The overall hybridization ef ciency was >99%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995). According to these criteria, sperm nuclei are scored only if they are intact, non-overlapping and have a clearly de ned border. In the case of aneuploidy, the presence of a sperm tail was con rmed. A spermatozoon was considered disomic if the two uorescent spots 2303

3 B.Baccetti et al. Figure 1. Fluorescence in-situ hybridization (FISH) analysis of lymphocyte karyotype performed with speci c painting probes for chromosome 15, showing reciprocal translocation t(10;15) (10q26;15q12). Chromosome 15 shows a breakpoint at 15q12 and chromosome 10 at 10q26, giving rise to der(10)t(10;15) and der(15)t(10;15). Note normal chromosome 10, normal chromosome 15, derivative chromosome 10, fused with a fragment of chromosome 15 devoid of centromere, and fragment of derivative chromosome 15 including centromere, fused with telomeric region of chromosome 10. were of the same colour, comparable in size, shape and intensity and positioned inside the edge of the sperm head at least one domain apart. Diploidy was recognized by the presence of two double uorescent spots, following the above criteria. Observation and scoring was performed on a Leitz Aristoplan Optic Microscope equipped with uorescence apparatus, with a triple bandpass lter for Aqua, Orange, Green Fluorochromes (Vysis) and a monochrome lter for DAPI. Controls Semen samples from seven healthy normal men (age range 26±39 years) were collected to constitute a control group. All subjects were of proven fertility and showed normal seminal parameters, according to published guidelines (World Health Organization, 1999). Ultrastructural analysis evaluated by the mathematical formula (Baccetti et al., 1995) con rmed that sperm quality was in the range of natural fertility. Results Lymphocyte karyotyping performed with conventional GTGbanding revealed balanced reciprocal translocation 10;15 (10q26;15q12). The karyotypes of the patient's parents were normal. FISH analysis on chromosomal metaphases from blood lymphocytes using speci c painting probes for chromosome 15 Table I. Semen analysis a Parameter Value b Volume (ml) ph 7.6 Sperm concentration (310 6 /ml) Motility (%; 30 min) Absent In situ 0 Slow progressive Rapid progressive a Baseline seminal parameters evaluated 30 min after ejaculation. b Values are mean 6 SEM of three examinations. showed a reciprocal translocation involving chromosomes 10 and 15 (Figure 1). Chromosome 15 showed the breakpoint at 15q12 and chromosome 10 at 10q26, giving rise to der(10)t(10;15) and der(15)t(10;15). Semen analysis data are reported in Table I. The characteristic feature was a very poor progressive motility of only 6% in the three samples analysed. Sperm ultrastructure was evaluated by transmission electron microscopy as suggested previously (Baccetti et al., 1995). The percentage of sperm probably devoid of ultrastructural defects was 0%. The main defects were in the head region, where the 2304

4 Infertile male 10, 15 reciprocal translocation carrier Figure 2. TEM micrograph of an immature sperm: a large cytoplasmic residue (CR) is present around the head showing deformed acrosome (A) and altered nucleus (N), with uncondensed chromatin and intranuclear vacuole (iv). Coiled tail shows disorganized axoneme (AX). Mitochondria (M) are swollen and not assembled in regular helix. See also translucent vesicle (V) in cytoplasmic residue. Scale bar = 0.87 mm. Figure 4. TEM micrograph of a binucleated germinal cell showing altered acrosome (A) and nuclei (N), with intranuclear vacuoles (iv), all embedded in a large cytoplasmic residue (CR). Scale bar = 0.87 mm. Figure 3. TEM micrograph of sperm: the nucleus (N) has regular shape, but the acrosome (A) is altered and an intranuclear vacuole (iv) is present. A large cytoplasmic residue (CR) embeds the neck region and the middle piece with displaced mitochondria (M). Scale bar = 0.85 mm. acrosome was often absent and never normal, having reduced dimensions, altered position, sparse content, abnormal shape and often being far from the nucleus (Figures 2 and 3). The nucleus showed similar defects (Figures 2 and 3); shape was generally abnormal, spherical or irregular, with uncondensed chromatin in half of the sections examined, and 10% of sperm showed double nuclei (Figure 4). Taken together, these characteristics indicate immaturity of most head structures, with apoptosis (marginated chromatin) or necrosis (disrupted chromatin) affecting 20±25% of the total sperm population, including immature sperm, which represented 70% of the sperm population. Cytoplasmic residuesðanother index of Figure 5. TEM micrograph of sperm tail cross-section. A cytoplasmic residue (CR) embeds a coiled tail. The mitochondria (M) are swollen and not assembled in a regular helix. The axonemal pattern (AX) and accessory bres (AF) are disorganized; the brous sheath (FS) is scanty and disrupted. Scale bar = 0.19 mm. immaturityðwere observed in 25% of sperm, embedding the head or the midpiece regions (Figures 2±5). Mitochondria were often swollen and assembled in a very irregular helix (Figure 5). The tail had normal shape (50%), but the axoneme was generally anomalous due to missing or abnormal doublets, dynein arms (99%), accessory bres (90%, usually reduced in size) or brous sheath (80%, generally scanty and disrupted) (Figures 5 and 6). Some of these characteristics may be due to necrosis, which evidently also occurred in immature spermatozoa. 2305

5 B.Baccetti et al. Table II. FISH analysis for chromosomes 18, X and Y Normal spermatozoa (%) Disomy frequency (%) Diploidy frequency (%) X-bearing Y-bearing 18,18 X,X Y,Y X,Y 18,18,X,X 18,18,Y,Y 18,18,X,Y Patient Controls a In the patient, 3023 cells were scored by triple-colour FISH analysis. Frequencies of nullisomies were: 18/0 = 0.47%; X/0 = 0.21%; Y/0 = 0.05%. Multiple uorescent signals (>4) were detected in 0.14% of cells. a Mean percentages (6 SD) of disomies and diploidies for the examined chromosomes in seven healthy men of proven fertility (controls). The mean number of sperm scored by triple-fish analysis was Table III. FISH analysis for chromosomes 10, 15 Disomy frequency Diploidy frequency 10,10 10,10,15 15,15 15,15,10 10,10,15, % 0.90% 0.30% 0.30% 1.15% In the patient, 2602 sperm were scored by dual-colour FISH analysis. Frequencies of nullisomies were: 10/0 = 0.70%; 15/0 = Triple sets of chromosomes were detected in 0.20% of cells. Multiple uorescent signals (>4) were detected in 0.10% of cells. Figure 6. TEM micrograph of a 9+2 axonemal pattern, but with some microtubular doublets displaced. Dynein arms (arrows) are present only in some doublets. Scale bar = 0.03 mm. SEM analysis of a large cell sample con rmed the presence of spermatozoa with immature features as the presence of spheroid heads, surrounded by large cytoplasmic residues, detected also in the tail region. Meiotic segregation in the t(10;15) (10q26;15q12) translocation carrier was investigated by FISH. The aneuploidy frequencies of chromosomes 18, X and Y, not involved in the translocation, are summarized in Table II. A very high value of chromosome 18 disomy and diploidies (particularly 1818XX and 1818XY) was found in comparison with values of the control group (Table II). FISH analysis of chromosomes involved in translocation (10;15) showed a very high percentage of chromosome 10 disomy and diploidy (Table III). The diploidy of sperm cells could be generated by a binucleate sperm head or by diploid nuclei. The rst case was demonstrated by the presence of a membranous septum between the two nuclei; the second was deduced by the presence of double sets of 10, 15, and 18, X, Y chromosomes. Almost the same frequency of diploid cells (mean ) was detected in the scoring of double- and triple-colour FISH. With regard to the meiotic process, by using dual-colour FISH 93.4% of sperm were found with both 10, 15 chromosome signals, indicating an alternate or adjacent-1 segregation pattern. The products of adjacent-2 segregation were 2.8%, and those of 3:1 segregation 2.4%, of total products. By using FISH with telomeric probes for chromosomes 10, 15 it was possible to distinguish alternate segregation (producing balanced gametes) in 32.8% of sperm from other segregation patterns, all of which generated unbalanced gametes in 68.2% of sperm. Discussion In this case of male sterility, the patient was heterozygous for a balanced reciprocal translocation t(10;15) (10q26;15q12) which was absent in his parents. The translocation therefore appeared de novo in the present patient. It has been suggested previously (Olson and Magenis, 1988) that chromosome rearrangements are of preferential paternal origin. In infertile men, structural chromosomal anomalies occur more frequently than in the general population (Van Assche et al., 1996; Kalantari et al., 2001). Semen analysis carried out in a large population of infertile males (Bourrouillou et al., 1997; Pandiyan and Jequier, 1996) revealed a strong correlation between the rate of chromosomal anomalies and the severity of oligozoospermia. Other authors (Pellestor et al., 2001) observed oligoteratozoospermia in carriers of structural chromosomal alterations. Nevertheless, morphological sperm quality is always evaluated with optical microscopy, sometimes applying Kruger's strict criteria. Structural sperm anomalies can be highlighted only by the use of electron microscopy, which allows detailed evaluation of inner sperm organelles. By using this method, one group (Quintana de la Rosa et al., 2001) reported a generalized agellar abnormality in an infertile man carrier of t (3;22), while others (Baccetti et al., 2002) observed 2306

6 Infertile male 10, 15 reciprocal translocation carrier an unusual structural sperm immaturity in a sterile carrier of t(14;22). In the present report, the electron microscopic analysis of ejaculated spermatozoa from an infertile male carrier of reciprocal translocation 10;15, demonstrated severe ultrastructural sperm alterations, indicating diffuse sperm immaturity. Nevertheless, no particular monomorphic sperm defect was observed which affected the entire sperm population, as reviewed previously (Baccetti et al., 2001). Derangement of meiotic segregation is indicated by the abnormal chromosomal constitution of spermatozoa detected using FISH analysis. The high frequency of chromosome 18 disomy could be considered as a positive interchromosomal effect. This effect depends on the type of structural reorganization, the chromosomes involved and the chromosome breakpoints (Blanco et al., 2000; Anton et al., 2002), and results in a particular meiotic behaviour, namely unsynapsed regions or preferential meiotic con gurations that could lead to the observed increase in chromosome 18 disomies. Moreover, the high frequency of sperm diploidies detected by dual- and triple-colour FISH, using centromeric probes, indicates incomplete meiosis process leading to immature sperm cells with double nuclei, as observed by TEM, or with a double chromosome set. Dual-colour FISH performed in sperm nuclei highlighted the segregation pattern of chromosomes 10 and 15 involved in the translocation. The detection of both signals in 93.4% of sperm indicated alternate or adjacent-1 segregation patterns, producing normal, balanced or unbalanced gametes. The products of adjacent-2 segregation (2.8%) and 3:1 segregation (2.4%) were certainly unbalanced gametes. By using triple-colour FISH and telomeric probes it was shown that 32.8% of sperm were balanced, but 68.2% were unbalanced. These data were in agreement with previous reports of between 19 and 77% of the spermatozoa of reciprocal translocation carriers being chromosomally unbalanced, while about 50% were chromosomally abnormal (Martin and Spriggs, 1995; Shi and Martin, 2001). In conclusion, in this male carrier of a reciprocal translocation t(10;15), the existence of a correlation is proposed between these diffuse ultrastructural sperm alterations and the high frequency of sperm aneuploidies. Ultrastructural ndings indicated very poor sperm quality incompatible with natural fertility and, as in all cases of immature sperm, the only possibility for fertilization is ICSI. 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