Segregation of chromosomes in spermatozoa of four Hungarian translocation carriers

Transcription

1 Segregation of chromosomes in spermatozoa of four Hungarian translocation carriers Anna Kékesi, a Edit Erdei, M.D., Ph.D., b Miklós Török, M.D., Ph.D., a Sándor Drávucz, M.D., Ph.D., a and András Tóth, Ph.D. a a Department of Obstetrics and Gynecology, and b Department of Andrology and Urology, Medical Health Center, Budapest, Hungary Objective: To determine the segregation pattern of the translocated chromosomes in spermatozoa of human males with translocations. Design: Retrospective case control study. Setting: Hospital-based genetic laboratory for reproductive biology. Patient(s): A carrier with Y autosome reciprocal translocation, two with autosome autosome reciprocal translocations, and one with Robertsonian translocation. Intervention(s): Blood sample and sperm sample collection from each translocation carrier. Main Outcome Measure(s): Fluorescence in situ hybridization on lymphocyte slides to characterize each translocation case. Fluorescence in situ hybridization with specific DNA probes for each of the sperm samples to characterize the chromosomes involved in the rearrangement and to evaluate the possible interchromosomal effect for chromosomes 18, X, and Y. Result(s): Each translocation carrier showed a specific mode of segregation pattern of the translocated chromosomes, confirming the dependence on chromosomes involved in the translocation. The highest frequency from alternate segregation was with the carrier of Robertsonian translocation (90.9%), and the lowest was with the carrier of Y autosome translocation (29.7%). No evidence of an interchromosomal effect for chromosomes 18, X, and Y were detected. Conclusion(s): Depending on the rate of the genetically normal and abnormal segregation modes, we can evaluate the chance of having a healthy proband. These results ensure more accurate genetic counseling for patients in assisted reproduction centers. (Fertil Steril 2007;88:212.e by American Society for Reproductive Medicine.) Key Words: Genetics, infertility, FISH, chromosome translocation, sperm Chromosomal abnormalities often lead to infertility. Structural chromosomal disorders are detectable from peripheral blood by karyotyping and metaphase fluorescence in situ hybridization (FISH) and account for 21% of all chromosome abnormalities (1). Robertsonian translocations and balanced reciprocal translocations are the most frequent structural chromosomal abnormalities, with an incidence of 1.23/1,000 (2) and 1/625 (3) newborns, respectively. The translocation carriers exhibit no particular phenotype, but they produce a large number of chromosomally unbalanced gametes. The widespread use of intracytoplasmic sperm injection technology may increase the possibility of transmitting the genetic defects to the offspring by avoiding severe steps of natural selection (4). By using FISH with specific DNA probes, we can determine the chromosomal segregation pattern of the translocated chromosomes in spermatozoa. The segregation pattern Received April 27, 2006; revised October 10, 2006; accepted November 15, Supported by the Hungarian Scientific Research Fund (OTKA, Budapest, Hungary; grant T ). Reprint requests: András Tóth, Ph.D., Department of Obstetrics and Gynecology, Medical Health Center, Budapest, Hungary, H-1135 Szabolcs u. 35, Budapest, Hungary (FAX: ; dr.atoth@gmail.com). is variable, depending on the translocated segments involved. Robertsonian translocation occurs when two acrocentric chromosomes fuse together, resulting in an abnormal, generally dicentric chromosome that has the long arms of the original two. Balanced Robertsonian translocation carriers have only 45 chromosomes. The most common translocations to be found are usually between two autosomes with only one breakpoint in each. One specific and rare category is the Y autosome translocation with the incidence of 1/2,000 (5), which falls into the following three groups. [1] Translocation of the long arm of the Y chromosome to an acrocentric chromosome. There is no loss of euchromatin. This type is frequently familial without clinical significance. [2] Translocation of the Y-euchromatin to an autosome, resulting in 45 chromosomes, including the Y autosome fusion product. These males are mostly infertile. [3] A balanced Y autosome translocation in which the autosome is not an acrocentric. Carriers are frequently mentally retarded and/or infertile. The infertility may be a consequence of abnormal sex vesicle (X Y association) formation disrupting meiosis, which causes spermatogenetic arrest (6). In some cases, infertility can be related to partial or complete loss of the AZF regions within the translocation-derived acentric fragment /07/$32.00 Fertility and Sterility Vol. 88, No. 1, July 2007 doi: /j.fertnstert Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. 212.e5

2 The use of FISH allows the investigation of interchromosomal effects with an analysis of thousands of spermatozoa. Some studies suggest that interchromosomal effects do exist (7, 8), but others have been unable to demonstrate them (9, 10). We analyze the segregation pattern of the translocated chromosomes in spermatozoa of four different translocation carriers and evalute the possible interchromosomal effects for chromosomes 18, X, and Y in three of the four patients were also investigated. MATERIALS AND METHODS This study was approved by the Hungarian Scientific Research Fund. Patient History Seminal parameters from translocation carriers are given in Table 1. Patient 1 The first patient was a 38-year-old male with cryptozoospermia ( per microliter). Ten percent of the spermatozoa had decreased mobility, and 90% were immotile. Only 3% of the spermatozoa had normal morphology. The patient was normal at the physical examination, including his reproductive system. Concentrations of FSH, LH, PRL, and T were within normal ranges. Chromosome investigation of peripheral blood revealed a balanced 46,X,t(Y;3)(q12;p21) karyotype. Deoxyribonucleic acid analysis showed no deletions of the AZFa, b, and c regions. Patient 2 The second patient was a 41-year-old male with normal reproductive system. Semen analysis showed normozoospermia (sperm concentration: per microliter). Chromosome investigation revealed a balanced 46,XY,t(1; 17)(p11;q11) karyotype. The patient s wife had bilateral tubular occlusion. After four unsuccessful intracytoplasmic sperm injection treatments, they were referred to our center. Patient 3 The third patient was a 34-year-old male with varicocele. Levels of FSH, LH, PRL, and P levels were within normal ranges. Semen analysis showed oligozoospermia ( per microliter). Chromosome investigation revealed a balanced 45,XY,t(13;14)(q10;q10) karyotype. His wife had left-side tubular occlusion and polycystic ovarian syndrome. Patient 4 The fourth patient was a 25-year-old male. Semen analysis showed normozoospermia (sperm concentration: per microliter). Chromosome investigation revealed a balanced 46,XY,t(4;6)(q31.1;p25) karyotype. He and his wife were referred to genetic counseling because of one spontaneous and two missed abortions at 9 weeks of gestation. Deoxyribonucleic Acid Probes A dual-color FISH was used to characterize the translocations from metaphase spreads of peripheral lymphocytes and to determine the meiotic segregation of the chromosomes involved in the rearrangement in sperm cells. For patient 1, DNA probes were purchased from Oncor (Gaithersburg, MD). Alpha-satellite (DYZ3) classicalsatellite (DYZ1) probe complex specific for chromosome Y (biotin labeled) and alpha-satellite DNA probe (DXZ1, DIG labeled) specific for chromosome X were used. The probes were detected by fluorescein isothiocyanate conjugate (FITC) (chromosome Y) and rhodamine (chromosome X) fluorochromes. For patient 2, alpha-satellite, direct-labeled probe specific for the heterochromatic part of chromosome 1 (1qh, green; Q-Biogene) and telomere-specific, direct-labeled probe for chromosome 17 (Tel17q, red) were used. For patients 3 and 4, DNA probes were purchased from Vysis Inc. For patient 3, a locus-specific probe for chromosome 13q14 region (LSI13, Spectrum Green) and a subtelomeric probe specific for the 14q region (TEL Vysion 14q, Spectrum Orange) were used; for patient 4, telomere-specific probes for chromosome 4 (Tel Vysion 4q, Spectrum Orange) and for chromosome 6 (Tel Vysion 6p, Spectrum Green) were used to identify the different segregation modes. In three patients (patients 2, 3, and 4), the occurrence of interchromosomal effect (ICE) for chromosomes 18, X, and Y also was evaluated. The DNA probes for the chromosomes studied were purchased from Vysis Inc. (CEP18, TABLE 1 Characteristics of the four translocation carriers analyzed. Name Age (y) Density ( 10 6 ml) Volume (ml) Normal motility (%) Normal morphology (%) Clinical diagnosis Karyotype Patient OATS 46,X, t(y;3)(q12;p21) Patient NS 46,XY, t(1;17)(p11;q11) Patient OS 45,XY, t(13;14)(q10;q10) Patient NS 46,XY, t(4;6)(q31.1;p25) 212.e6 Kékesi et al. Chromosomal analysis of spermatozoa Vol. 88, No. 1, July 2007

3 TABLE 2 Segregation analysis results from spermatozoa in the three translocation carriers analyzed. Segregation patter Patient No. sp. Alternate Adjacent II Adjacent I 3:1 Diploidy Other 2 t(1;17)(p11;q11) 1, t(13;14)(q10;q10) 1, t(4;6)(q31.1;p25) 1, Note: Dashes indicate none. Spectrum Aqua/CEPX, Spectrum Green/CEPY, and Spectrum Orange). Data were statistically analyzed by using a heteroscedastic t-probe. Fluorescence In Situ Hybridization Studies on Lymphocyte Metaphases and on Sperm Cells Fluorescence in situ hybridization analysis on lymphocyte metaphases was performed according to Oncor protocol for patient 1, according to Q-Biogene protocol for patient 2, and according to Vysis protocol for patient 3 and 4. The fresh ejaculate was washed twice with phosphate-buffered saline, and after centrifugation, it was fixed three times with methanol glacial acetic acid (3:1). Before hybridization, the spermatozoa were treated for 5 minutes with a solution of 25 mm dithiothreitol in 1 M Tris, ph 9.5 (11) to decondense the sperm heads. The protocol for probes and sample denaturation, incubation, and detection were performed according to manufacturer s instructions (Oncor, Q-Biogene, and Vysis). Briefly, after sperm-head decondensation, the slides were washed in 2 SSC at 37 C for 8 minutes, then the sperm spreads were denatured in 70% formamide: 2 SSC at 72 C for 4 minutes, whereas the DNA probes were denatured at 73 C for 5 minutes (Oncor, Vysis). According to Q-Biogene protocol, samples were denatured together with DNA probes at 75 C for 10 minutes and were hybridized in a humidified chamber in 37 C for hours. Slides were washed in 0.4 SSC at 73 C for 2 minutes (Vysis), in 0.5 SSC at 65 C for 4 minutes (Q-Biogene), and then in phosphate-buffered detergent (BPD) for 2 minutes and were counterstained with 6-diamino-2-phenylindole (DAPI) solution. According to Oncor protocol, after the washing step, DNA probes were immunodetected by fluorescein isothiocyanate conjugate and rhodamine fluorochromes. Microscopic Analysis and Scoring Criteria Analyses were performed with an epifluorescence microscope equipped with filter sets for DAPI, Spectrum Aqua, Spectrum Green, and Spectrum Orange. Sperm nuclei without fluorescent signal and sperm cells without intact tail were not evaluated. To differentiate split signals, sperm cells were scored as having two signals for a particular probe if the signals were separated from each other by at least one spot diameter. RESULTS Dual-color FISH was performed in all of our patients with hybridization rates of 95%. Segregation analysis results are shown in Table 2. Patient 1 The balanced Y autosome translocation was confirmed by FISH analysis from lymphocytes metaphases. Digoxigeninlabeled probe complex specific for the centromeric and heterochromatic part of chromosome Y revealed the translocation of the heterochromatic region to an autosome (chromosome 3). Because this patient had a very low sperm concentration ( per microliter), only 450 spermatozoa could be analyzed by FISH. The rate of genetically normal and balanced spermatozoa in ejaculate was only 20.2% (3/X): 9.5% of spermatozoa [der(3)/der(y)] resulted from 2:2 alternate segregation during meiosis, 67.3% resulted from adjacent I and II, and 3% resulted from one type of 3:1 segregation [der(3)/der(y)/x] or were diploid [3/X/der(3)/der(Y)]. The methodological approach used did not allow differentiation of the other types of 3:1 segregation from the other segregation modes and differentiation between adjacent I and II segregations. Because this patient has very few sperm, his data are not described in Table 2. Patient 2 Fluorescence in situ hybridization technique on lymphocyte metaphase spreads demonstrated the reciprocal translocation between chromosome 1 and chromosome 17. One thousand five hundred seventy-five sperm heads were scored by using double FISH. The segregation pattern showed that 53.7% of the spermatozoa analyzed originated from 2:2 alternate segregation [1/17 and der(1)/der(17)], 38% resulted from adjacent I [17/der(1) and 1/der(17)], 7% resulted from adjacent II segregation [1/der(1) and 17/der(17)], and 1.3% had diploidy Fertility and Sterility 212.e7

4 [1/17/der(1)/der(17)]. The proportion of alternate and adjacent segregation was close to the theoretical 1:1 (53.7% and 45%, respectively). DNA probes. Other spermatozoa were unbalanced and resulted from adjacent I segregation [4/der(6) and 6/der(4); 33.6%] or from 3:1 segregation (12.2%) or were diploid (0.2%; Fig. 2). Patient 3 In lymphocyte FISH, we could demonstrate the Robertsonian translocation t(13;14)(q10;q10). Segregation analysis was ascertained in 1,629 spermatozoa. Most spermatozoa (90.9%) resulted from 2:1 alternate segregation [13/14 and der(13;14)] during meiosis. The proportion of unbalanced spermatozoa resulting from 2:1 adjacent segregations [13, 14, 13/der(13;14) and 14/der(13;14)] were 8.2% of the cells analyzed. Diploidy or 3:0 segregation mode proved to result in 0.7% [13/14/der(13;14)]. Spermatozoa with an unexpected combination of signals were 0.2% (classified as other). To evaluate the possible ICE, a total of 4,531 spermatozoa were analyzed from three translocation carriers (patients 2, 3, and 4). These results were compared with those of five normozoospermic control donors. From these donors, we analyzed 9,885 spermatozoa. Table 3 shows the details of the evaluation of chromosomes 18, X, and Y. The frequencies of disomy for chromosome 18 (0.2%, 0.2%, and 0.0, respectively), X (0.06% each), Y (0.2%, 0.4%, and 0.06%), and XY (0.1%, 0.06%, and 0.0) and the diploidy rate (0.06%, 0.3%, and 0.06%) were similar to the rates of spermatozoa of control donors (0.08%, 0.06%, 0.07%, 0.08%, and 0.19%, respectively). Patient 4 Double-FISH analysis with DNA probes specific for chromosome 4q and for chromosome 6p confirmed the result of standard cytogenetic investigation, which showed reciprocal autosomal translocation between chromosomes 4 and 6 (Fig. 1). One thousand fifty spermatozoa of this translocation carrier were scored. Fifty-four percent of the sperm cells resulted from alternate or adjacent II segregations [4/6, der(4)/der(6), 4/der(4), 6/der(6)]. Differentiation between alternate and adjacent II segregation modes was not possible by using these The numbers of sperm studied were not high enough to support a statement, but it appears that there were no statistical differences between the patients and control males regarding the aneuploidy and diploidy rates of chromosomes 18, X, and Y (P.1) in sperm. DISCUSSION The aim of this study was to determine the segregation pattern of the translocated chromosomes in sperm cells of four translocation carriers. Segregation studies have been FIGURE 1 Fluorescence in situ hybridization on a lymphocyte metaphase of a translocation carrier with 46,XY,t(4;6)(q31.1;p25). The chromosomes were counterstained with 6-diamino-2-phenylindole. 212.e8 Kékesi et al. Chromosomal analysis of spermatozoa Vol. 88, No. 1, July 2007

5 FIGURE 2 Fluorescence in situ hybridization analysis on spermatozoa of patient 4. (A) 2:2 Alternate [4/6 or der(4)/der(6)] or adjacent II [4/der(4) and 6/der(6)] segregations. (B) 3:1 segregation [6 or der(4)]. (C) Adjacent I segregation [6/der(4)]. (D) Adjacent I segregation [4/der(6)]. (E) 3:1 segregation [4 or der(6)]. performed by FISH for the last decade (12 15) on spermatozoa from those who carry structural chromosomal abnormalities as Robertsonian translocations and reciprocal translocations. The four different types of segregation were found in the translocation carriers we analyzed, where the methodological approach used allowed differentiation between the different types of segregation. In the patient who carried Robertsonian translocation (patient 3), during meiosis (prophase I), the rearranged chromosomes form a trivalent structure (16, 17). When TABLE 3 Results of ICE for chromosomes 18, X, and Y in balanced reciprocal translocation carriers and in control, normozoospermic males analyzed. Patient 18/X, 18/Y, Disomy 18, Disomy X, Disomy Y, Disomy XY, Diploidy, Total Patient (51.3) 721 (48.0) 3 (0.20) 1 (0.06) 3 (0.20) 2 (0.10) 1 (0.06) 1,502 Patient (48.8) 758 (50.3) 3 (0.20) 1 (0.06) 6 (0.40) 1 (0.06) 5 (0.3) 1,509 Patient (49.5) 763 (50.2) 0 1 (0.06) 1 (0.06) 0 1 (0.06) 1,520 Totals for patients 2,260 (49.9) 2,242 (49.5) 6 (0.13) 3 (0.06) 10 (0.22) 3 (0.05) 7 (0.14) 4,531 Control (47.2) 908 (52.0) 4 (0.20) 2 (0.10) 2 (0.10) 4 (0.20) 2 (0.10) 1,747 Control 2 1,030 (49.4) 1,045 (50.0) 0 2 (0.10) 0 1 (0.05) 11 (0.50) 2,081 Control 3 1,004 (49.4) 1,024 (50.3) 1 (0.05) 0 2 (0.10) 0 1 (0.05) 2,032 Control (47.8) 1,043 (51.6) 3 (0.14) 1 (0.05) 1 (0.05) 3 (0.14) 2 (0.14) 2,021 Control 5 1,018 (50.8) 980 (48.9) 0 1 (0.05) 2 (0.10) 0 3 (0.15) 2,004 Totals for controls 4,844 (49) 5,000 (50.6) 8 (0.08) 6 (0.06) 7 (0.07) 8 (0.08) 19 (0.19) 9,885 Fertility and Sterility 212.e9

6 this trivalent form is in cis-configuration, alternate segregation is promoted during meiosis I, resulting in the production with equal frequencies of a normal gamete, including the acrocentric chromosomes and the balanced gamete with only the translocated chromosomes. The other segregation modes, adjacent I and the rare 3:0, produce unbalanced gametes with abnormal embryos that cause miscarriage or aneuploid offspring. Spermatozoa in seven male carriers of Robertsonian translocation were analyzed by Anton et al. (15). Their results were correlated with ours (alternate segregations, 83% 88.23%; adjacent segregations, 11.11% 14.53%; and 3:0 mode, % compared with our investigations 90.9%, 8.2%, and 0.7%, respectively). Most carriers of Robertsonian translocations have oligozoospermia. This may be a result of the intervention of meiotic checkpoints during meiosis (erratic chromosomes, lack of tension), when the cell may be unable to complete the meiotic process that leads the cell into apoptosis. If the cell is capable of completing the division process, the result may be the production of aneuploid or diploid spermatozoa. Usually, carriers of reciprocal translocations have an increased risk of producing unbalanced gametes as compared with carriers of Robertsonian translocations. The meiotic behavior of reciprocal translocations depends on the chromosomes involved in the rearrangement, the position of the breakpoints, crossovers in the translocated chromosomes, and the morphological characteristics of the rearranged chromosomes. In the first metaphase of meiosis, a close configuration, such as a ring, produces mainly 2:2 segregations, whereas an open configuration, such as a chain, produces 3:1 segregations (18). During meiosis I, the segregation of the quadrivalent that is formed by the translocated chromosomes and their normal homologues produces a variety of balanced and unbalanced gametes. The alternate phenotype is the only segregation mode that yields normal offspring. Unbalanced gametes are produced by adjacent I, adjacent II, and 3:1 segregation. In adjacent I segregation, homologous centromeres move to opposite poles, whereas they move to the same pole in adjacent II and 3:1 segregations. In the two autosomal-translocation carriers (patient 2 and 4), almost equal proportions of normal (alternate segregation) and abnormal (adjacent and 3:1 segregation) sperm cells were analyzed. An excess of unbalanced gametes usually is higher in sex chromosome autosome translocations than in the autosome autosome translocations. This may be a result of the involvement of the sex chromosomes because the translocation may prevent the normal formation of the sex vesicle, which is essential for a normal meiotic process. Pseudoautosomal regions of the Y chromosome (PAR1 and PAR2) are responsible for a correct pairing between the two sex chromosomes. Pseudoautosomal region 1 is at the telomere of Xp/Yp, and PAR2 is at the telomere of Xq/Yq. Our patient 1 had t(y;3)(q12;p21) translocation with breakpoints at Yq12 and 3p21, which means that PAR2 was translocated to der(3). This phenomenon may cause asynapsis within the quadrivalent at the pachytene stage in some cells, which leads to severe oligozoospermia. Pinho et al. (19) analyzed a male with t(y;1)(q12;q12) translocation with an otherwise normal phenotype. In this male, the PAR2 region in Yq was translocated to der(1), causing meiotic I arrest, which led to azoospermia. Hsu (20) described 25 cases, among which 80% of males had azoospermia. Sperm analysis was performed in a male with a reciprocal t(y;16)(q11.21;q24) translocation by Giltay et al. (13). This patient had severe oligoasthenoteratozoospermia. Segregation analysis of morphologically normal spermatozoa showed that 51% originated from alternate segregation. If morphologically abnormal sperm cells were also included, nearly 90% of all the spermatozoa were unbalanced. Thus, selecting morphologically normal sperm cells for intracytoplasmic sperm injection treatments reduces but does not preclude the likelihood of unbalanced spermatozoa. These literature data propose that males with Y autosome translocations produce little or no sperm cells or that they produce a high percentage of unbalanced spermatozoa. In the ICE evaluation, the numbers of investigated sperm were not high, but the data obtained did not appear to provide any evidence of an ICE for chromosomes 18, X, and Y. According to Pellestor et al. (21), ICE in translocation carriers could be restricted to those carriers who have abnormal semenograms. Oliver-Bonet et al. (22) analyzed two reciprocal translocation carriers with normozoospermia and also did not find any evidence for an ICE (chromosomes 6, 18, 21, X, and Y were analyzed). In our study, the two reciprocal translocation carriers analyzed (patient 2 and 4) had normal semen parameters, and patient 3 with Robertsonian translocation had oligozoospermia (other semen parameters, such as sperm cell morphology and motility, were within normal ranges). Interchromosomal effect analysis of spermatozoa with patient 1 was not possible because of the extremely low sperm concentration ( per microliter). Our results confirm the investigations mentioned in the previous paragraph (21, 22), although the Robertsonian translocation carrier had a low sperm concentration that could have been related to aneuploidy and diploidy. We found only a slight increase in the rate of disomy for chromosome Y (0.4%) and in the rate of diploidy (0.3%) for this patient, although we analyzed only three chromosomes (18, X, and Y). Fluorescence in situ hybridization analysis of spermatozoa can be a very useful technique for evaluating the risk of chromosomally abnormal embryos in translocation carriers, although the rate of unbalanced spermatozoa observed is much higher than that detected in studies of human fetuses from translocation carriers (23). This difference is explained by the low viability of most unbalanced conceptions. These 212.e10 Kékesi et al. Chromosomal analysis of spermatozoa Vol. 88, No. 1, July 2007

7 results are very informative in applying assisted reproductive techniques. For those translocation carriers whose partners become pregnant, prenatal or preimplantation diagnosis are offered. REFERENCES 1. Pandyan N, Jequier AM. Mitotic chromosomal anomalies among 1210 infertile men. Hum Reprod 1996;11: Nielsen J, Wohlert M. Chromosome abnormalities found among newborn children: results from 13-year incidence study in Arhus, Denmark. Hum Genet 1991;87: Van Dyke DL, Weiss L, Roberson JR, Babu VR. The frequency and mutation rate of balanced autosomal rearrangements in man estimated from prenatal genetic studies for advanced maternal age. Am J Med Genet 1983;35: Buonadonna AL, Cariola F, Caroppo E, Di Carlo A, Fiorente P, Valenzano MC, et al. Molecular and cytogenetic characterization of an azoospermic male with a de-novo Y;14 translocation and alternate centromere inactivation. Hum Reprod 2002;17: Nielsen J, Rasmussen K. Y/autosomal trasnlocations. Clin Genet 1976; 9: Gardner RJM, Sutherland GR. Chromosome abnormalities and genetic counseling. New York: Oxford University Press, 1989: Burns J, Koduru P, Alonso M, Chaganti R. Analysis of meiotic segregation in a man heterozygous for two reciprocal translocations using the hamster in vitro penetration system. Am J Hum Genet 1986;38: Templado C, Navarro J, Benet J, Genesca A, Perez MM, Egozcue J. Human sperm chromosome studies in a reciprocal translocation, t(2;5). Hum Genet 1988;79: Pellestor F, Sele B, Jalbert H, Jalbert P. Direct segregation analysis of reciprocal translocations: a study of 283 sperm karyotypes from four carriers. Am J Hum Genet 1989;44: Estop A, van Kirk V, Cieply K. Segregation analysis of four translocations, t(2,8), t(3;15), t(5;7) and t(10;12), by sperm chromosome studies and a review of the literature. Cytogenet Cell Genet 1995;70: Martini E, Speel EJM, Geraedts JPM, Ramaekers FCS, Hopman AHN. Application of different in-situ hybridization detection methods for human sperm analysis. Hum Reprod 1995;10: Mercier S, Morel F, Fellman F, Roux C, Bresson JL. Molecular analysis of the chromosomal equipment in spermatozoa of a 46,XY,t(7;8) (q11.21;cen) carrier by using fluorescence in situ hybridization. Hum Genet 1998;102: Giltay JC, Kastrop PMM, Tiemessen CHJ, van Inzen WG, Scheres JMJC, Pearson PL. Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH. Cytogenet Cell Genet 1999;84: Oliver-Bonet M, Navarro J, Carrera M, Egozcue J, Benet J. Aneuploid and unbalanced sperm in two translocation carriers: evaluation of the genetic risk. Mol Hum Rep 2002;8: Anton E, Blanco J, Egozcue J, Vidal F. Sperm FISH studies in seven male carriers of Robertsonian translocation t(13;14)(q10;q10). Hum Reprod 2004;11: Vidal F, Templado C, Navarro J, Marina S, Egozcue J. Meiotic and synaptonemal complexes studies in a 14/21 translocation carrier. Int J Androl 1982;5: Luciani JM, Guichaoua MR, Mattei A, Morazzani MR. Pachytene analysis of a man with a 13q;14q translocation and infertility. Cytogenet Cell Genet 1984;38: Escudero T, Abdelhadi I, Sandalinas M. Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations. Fertil Steril 2003;79: Pinho MJ, Neves R, Costa P, Ferras C, Sousa M, Alves C, et al. Unique t(y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report. Hum Reprod 2005;20: Hsu LYF. Phenotype/karyotype correlations of Y chromosome aneuploidy with emphasis on structural aberrations in postnatally diagnosed cases. Am J Med Genet 1994;53: Pellestor F, Imbert I, Andreo B, Lefort G. 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 2001;16: Oliver-Bonet M, Navarro J, Codina-Pascual M, Guitart AM, Egozcue J, Benet J. From spermatocytes to sperm: meiotic behaviour of human male reciprocal translocations. Hum Reprod 2004;19: Boue A, Gallano P. Collaborative study of the segregation of inherited chromosome structural rearrangements in 1356 prenatal diagnosis. Prenat Diagn 1984;4: Fertility and Sterility 212.e11