Sperm analysis by FISH in a case of t(17; 22) (q11; q12) balanced translocation

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1 Human Reproduction Vol.17, No.2 pp , 2002 CASE REPORT Sperm analysis by FISH in a case of t(17; 22) (q11; q12) balanced translocation Aimé Geneix 1,3, Benoît Schubert 2, André Force 2, Karen Rodet 1, Georges Briançon 1 and Daniel Boucher 2 1 Laboratoire de Cytogénétique, Faculté de Médecine, Place Henri Dunant, Clermont-Ferrand and 2 Laboratoire de Biologie de la Reproduction et du Développement et CECOS, Hôtel-Dieu, Boulevard Léon Malfreyt, Clermont-Ferrand, France 3 To whom correspondence should be addressed. ageneix@chu-clermontferrrand.fr Individual sperm from men with balanced translocations have different chromosomal contents. Thus, an estimation of the overall sperm chromosomal imbalance of such patients could help to give the couple an adapted genetic counselling. We report here the study of a balanced translocation carrier, t(17;22) (q11;q12) whose reproductive history reported four miscarriages. Moreover, he had an abnormal semen analysis with oligoteratozoospermia. The meiotic segregation pattern was examined in 700 sperm, using fluorescence in-situ hybridization (FISH). Nineteen percent of the sperm had balanced translocations or were normal. All other sperm were unbalanced (81%) and their distribution was observed as follows: the frequencies of adjacent 1, adjacent 2 and 3:1 segregations were 12.9, 5.8 and 46.8% respectively. Among the segregations scored, 13.7% were related to second meiotic division abnormalities. Less than 2% of the total sperm scored were not explained. The 3:1 segregation was present at a very high rate, which is very unusual. In cases of balanced translocations, we believe that no general features can be drawn. Thus, the FISH technique may be very helpful for genetic counselling, which remains an important step and must be done with care. Key words: balanced translocation/chromosome segregation/fish/sperm Introduction Sperm from men with balanced translocations can show several types of nuclear contents: normal, balanced or unbalanced karyotype. No usual sperm parameter can distinguish each of them in an ejaculate. Each of these sperm can fertilize an oocyte with or without ART and some of them can give rise to abnormal offspring or recurrent pregnancy loss (Egozcue et al., 2000). The ART currently used do not allow the selection of a normal spermatozoon with regard to its karyotype. Thus, it is of great interest to estimate the overall sperm chromosomal imbalance of such patients in order to give the couple a better genetic counselling. Until recently, the best method for sperm chromosomal analysis was to perform a zona-free hamster egg penetration test which is still now the only way to get a complete analysis of all chromosomes (Spriggs et al., 1992; Guttenbach et al., 1997). However, although effective, this technique is difficult, time-consuming and expensive. In addition, only a few cells can be studied. This limited number of analysed sperm is not generally enough to provide a statistically meaningful analysis of the entire sperm population. Furthermore, sperm from certain subgroups of infertile men show an impaired capacity to penetrate eggs. The relative ability of genetically abnormal sperm to penetrate hamster eggs has not been extensively studied. The fluorescence in-situ hybridization (FISH) technique has now been used in several studies for the detection of sperm aneuploidy (Robbins et al., 1993; Downie et al., 1997; Colls et al., 1998; Cifuentes et al., 1999; Giltay et al., 1999; Lim et al., 1999; Rives et al., 2000; Sloter et al., 2000; Ushijima et al., 2000). In contrast to the zonafree hamster egg penetration test, FISH allows the study of a high number of sperm and in a faster and more precise way. Thus, the statistical analysis is meaningful in these conditions. We report here the study of a couple with a history of recurrent miscarriage and infertility. The patient bore a t(17, 22) (q11; q22) balanced translocation in his somatic cells and we studied the sperm cell chromosomes using FISH with specific probes to the particular loci. The main purpose was to evaluate the distribution of this translocation, after meiosis had occurred, in the entire sperm population, in order to give the couple an adapted genetic counselling. European Society of Human Reproduction and Embryology 325

2 A.Geneix et al. Figure 1. Pedigree of the proband. Materials and methods Patients The couple were attending the centre of Developmental and Reproductive Biology, Hospital of Clermont-Ferrand, France, for a secondary infertility referral. They had had two miscarriages 7 years before their naturally conceived pregnancy, which resulted in the birth of a healthy boy. Five years ago, two other miscarriages occurred, each during the first trimester of pregnancy. The patients were a healthy 30 year old woman, with a normal physical examination and a healthy 35 year old man. His physical examination was normal and his history revealed a surgical operation for cryptorchidism at the age of 8 years. An elevated serum FSH concentration of 15.4 miu/ml (normal range ) suggested abnormal spermatogenesis. The patient had oligoteratozoospermia and carried a (17; 22) balanced translocation. These chromosomal abnormalities were inherited from his father, who carried the same balanced translocation and had naturally conceived two healthy children. The second child of his father was a girl who gave birth to two healthy babies and had no history of miscarriages (Figure 1). The constitutional karyotypes were established from cultured lymphocytes with reverse heating Giemsa (RHG) banding. The male formula was 46,XY, t(17; 22) (q11; q12) (Figure 2). This balanced translocation was verified by FISH to confirm the breakpoints (Figure 3). In this control, we used a kit for Bcr and Abl genes location. The Bcr locus was painted in green and located on chromosome 22 or der(22). The Abl locus was painted in red, located on the terminal part of chromosome 9. The centromere of chromosome 17 appeared in green and the Her2/neu locus in 17q11, in red. These colours are different from others used later for the sperm labelling. The female s constitutional karyotype was normal: 46,XX. Semen collection and preparation Semen was collected by masturbation after 3 days of sexual abstinence. Analyses were performed in our laboratory according to standard 326 World Health Organisation criteria (WHO, 1992) and showed a volume of 4.5 ml (reference range for normal 2 5 ml); n sperm/ml (reference range ); teratozoospermia was 74% according to David s criteria (David et al., 1975). Motility was 63% fast and progressive and 22% weakly or non-progressive. Sperm were studied from a fresh ejaculate, after processing by migration on a two layer discontinuous Percoll gradient ( %) (Sigma-Aldrich, Saint Quentin Fallavier, France). First, the entire semen was washed 4 times by centrifugation in Earle s solution (3 min, 700 g, 20 C). The final pellet was resuspended in 5 10 µl of Earle s solution and dropped onto clean glass slides. The slides were air-dried and sperm were fixed by alcohol/ether (1/1, V/V, 15 min). Sperm nuclear decondensation was obtained by plunging the slides into a solution of NaOH 1 N for 1 min at room temperature. The slides were rinsed in 2 sodium chloride/sodium citrate ( 2 SSC, ph 7). The sperm were then dehydrated in an ethanol dilution series from 70 to 100% and air dried. DNA probes used for FISH Three specific probes were used in this study and were purchased from Vysis (Vysis Inc., Voisins, Le Bretonneux, France). Her2/neu is located on 17q11.2-q12 and labelled with Spectrum Orange TM. Two pericentromeric probes of the chromosome 17 (CEP 17) were used: one is labelled with Spectrum Green TM and the other one with Spectrum Orange TM. The combination of these two labelling gave a yellow colour. Bcr is located on 22q11.2 and is labelled with Spectrum Green TM. Thus a three-colour FISH was performed. These probes can identify: (i) a locus from 17 pter to 17 q11: CEP 17 probe (yellow spot), on the centromere of the chromosome 17; (ii) a locus from 17 q11 to 17 qter on the long arm of the chromosome 17: Her2/neu probe (red spot), under the breakpoint of this chromosome; (iii) a locus from 22 pter to 22 q11 on the long arm of the chromosome 22: Bcr probe (green spot) and above the breakpoint of this chromosome.

3 Sperm analysis by FISH Figure 2. RHG banded proband s karyotype (from peripheral blood sample). A balanced translocation between chromosome 17 and chromosome 22 is observed. FISH The probes were denatured for 2 min at 73 C. The hybridization mixture (1 µl of each probe, 1 µl H 2 O, 7 µl of hybridization solution) was applied to each slide and covered with a coverslip mm (Labonord, Templemars, France). This hybridization mixture is a solution of dextran sulphate, formamide in SSC (ph 7). Each slide was then sealed with Rubber Cement (Kleertak; Mecanorama, France) before hybridization was carried out overnight in a moist chamber at 37 C. After hybridization, the slides were washed for 3 min in a solution of 0.4 SSC at 73 C and a second time for 30 s in a solution of 2 SSC/0.1% Nonidet P40. After the final wash, slides were air dried in the dark. The slides were counterstained with a solution of 4,6-diamidino-2-phenylindole.2HCl (DAPI II; Vysis Inc.) diluted in an antifade mounting medium (Vectashield; Valbiotech, France). Scoring criteria and data collection The slides were then examined with an epifluorescence microscope (DMRB, Leica, Germany) at a magnification of The nuclear decondensation resulted in a sperm morphology similar to that already published. (Martini et al., 1995), i.e. each cell seen under the microscope could be clearly identified as a mature spermatozoon because of its structure and could be distinguished from other cells. Only clearly identifiable sperm were scored and only the spots appearing on the sperm head were counted. Retained spots were clearly seen and of equivalent size. Two spots were separated by at least the size of one of them, and if not, they were not taken into account. Results FISH Four hundred sperm were scored for the control and 700 for the patient (Figure 4). Control The hybridization rates of the probes used here were determined by screening 200 nuclei from diploid cells: 197/200 for CEP 17; 200/200 for Bcr and 191/200 for Her2/neu. Control sperm came from a man with a normal karyotype who had two naturally conceived healthy children. The same experimental conditions and the same probes were applied to both control and patient samples. These sperm revealed a very low percentage of aneuploidy (3.3%), which could be considered as normal. For 1.2% of these cells, no spots were present, and could probably be explained by artefacts. Patient t(17; 22) (q11; q12) Nineteen percent of the analysed sperm were balanced (alternate segregation): der(22), der(17) or 17, 22 bearing sperm. Due to the probes we used, the FISH technique did not allow the distinction between these two patterns. All other sperm (81%) were unbalanced and their distribution was unequal among each of all the theoretical possibilities (see Table I). The 3:1 segregation was the most represented in our study, with a rate of 47%. All the results are presented in Table I. Fewer than 2% of all the spots scored were unexplained. Discussion The FISH technique could not distinguish whether the studied sperm bore a balanced translocation or a normal karyotype. All that could be observed was coloured spots, from which we estimated the presence of the involved loci. Of course, other abnormalities corresponding to these two chromosomes, or to other chromosomes, could be present without being visible. We considered that these events were unlikely and insignificant. Thus, even if the FISH technique is far from giving a result as complete as a full karyotype, a statistical analysis can easily be performed. No other cases with similar breakpoints were found in other databases, such as HC FORUM (Dr O.Cohen, Grenoble, France). In our study, 20% of the sperm bore a normal or balanced genetic complement. This is in agreement with the patient s history considering that he naturally conceived a healthy boy. In addition, his father, bearing the same balanced translocation, also had two naturally conceived healthy children. His sister had no history of miscarriages and also naturally conceived two healthy children. Unfortunately, her karyotype was unknown. The t(17; 22) (q11; q12) patient s severe oligozoospermia could 327

4 A.Geneix et al. Figures 3 and

5 Sperm analysis by FISH possibly be a consequence of his karyotype abnormality. However, his cryptorchidism was the most likely cause of this infertility. His weak testicular function can be related to either or both abnormalities. The history of four miscarriages could clearly be explained by unbalanced translocation bearing sperm: 80% of the sperm scored had an unbalanced karyotype. However, it seems surprising that so many abnormal sperm can reach complete maturation. One could expect that the major part of the sperm population would be represented by normal sperm regarding their karyotypes, but in our study, they only represented 20% of the entire population. As a hypothesis, we propose that all mis-segregations were much more common than the normal segregations; thus, even if some did not reach complete maturation because of their abnormal karyotype, a large part of them could finally pass through the spermatogenesis control check points. The unequal rates between unbalanced translocation and its complement (for example: 17, der(22): 3.2% and der(17), 22: 9.7%) could possibly be explained by the quality and the quantity of the genes involved in this translocation: in some cases missing genes could be more important to reach complete maturation than others. The rate of the 3:1 segregation was another astonishing point. Sperm bearing one chromosome were much more frequent than sperm bearing three chromosomes: 20% bore der(17) only versus 0.1% bore 17, der(22), 22 for example. A hybridization failure could explain these results. However only 3.3% of the control sperm showed abnormal results, which represent any abnormality, including hybridization failure. Nearly 45% of the patient s sperm showed only one hybridization symbol; thus this high percentage is not only related to hybridization failure. In addition, this particular point seemed in opposition with what is known about the conceptus products: autosomal monosomia, even partial, are very lethal. However, this fact could correspond to a recruitment bias: these kind of monosomia might be so lethal that they stopped their development very early and resulted in very early abortions which were not noticed. According to Durak et al. (Durak et al., 1999), who studied Table I. Different kinds of segregation from the t(17; 22) (q11; q22) carrier Segregation Sperm (n 700) First meiosis division Alternate 19% der(17), der(22) 17, 22 Adjacent % 17, der(22) der(17), 22 (3.2%) (9.7%) Adjacent 2 5.8% 17, der(17) 22, der(22) (4.3%) (1.5%) der(17) 17, der(22), 22 (20%) (0.1%) 17 der(17), der(22),22 3:1 46.8% (10.9%) (0.9%) 22 17, der(17), der(22) (9.6%) (0.1%) der(22) 17, der(17), 22 (4.4%) (0.8%) Second meiotic division Adjacent 1 followed by 2.1% der(17), 22, 22 der(17), der(17), 22 2nd meiosis abnormalities (1.3%) (0.8%) Adjacent 1 followed by two 0.1% der(17), der(17), 22, 22 2nd meiosis abnormalities (0.1%) 17, der(17), der(17), 22 (0.1%) 3:1 followed by 2nd 11.5% 22, 22 der(22), der(22) meiosis abnormalities (5.5%) (1.8%) 17, 17 der(17), der(17) (0.1%) (4%) Unexplained 1.8% 17, red spot der(22), red spot (1.5%) (0.3%) Figure 3. FISH on a metaphase spreading from peripheral blood sample of the patient. The normal chromosome 17 shows two spots: a green on the centromere and two red on the long arm (on the homolog loci on each chromatid). The der(17) shows only the green spot on the centromere. The red spots are translocated on der(22). Chromosome 22 shows two green spots located on the long arm of chromosome 22 (one on each chromatid locus). The der(22) shows four spots, a pair of green spots located as on a normal chromosome 22 and a pair of red spots on chromosome 17. Two normal chromosomes 9 show long arm terminal red spots. These probes belong to a kit used for Bcr [der(22) in our patient s translocation] and Abl genes location (see text). The Abl locus is painted in red, located on the terminal part of chromosome 9. Figure 4. The analysis of the colour and number of FISH spots on sperm nuclei shows: (1) a normal or balanced karyotype; (2) a nullisomy 22. The two spots are from the chromosome 17; (3) a nullisomy 17. One spot is from chromosome 22; (4) der(22) alone; (5) der(17) and chromosome 17; (6) two chromosomes 22; (7) der(22) and chromosome 22; (8) part of chromosome 17 alone, probable artefact. 329

6 A.Geneix et al. sperm from two translocation carriers, t(4; 8) (p15; p12) and t(15; 22) (q23; q13.2), the 3:1 segregation was rare and they only considered 2:2 segregation in their study. Therefore what they suggested cannot be compared with our results. A study of a balanced translocation t(2;3) (p24;q26) (Martin, 1994) where, in these sperm, the segregation 3:1 was only present at the rate of 1.2 versus 50% in ours and even 58% if we also consider the 3:1 segregation followed by a second meiosis abnormality. The alternate segregation, adjacent 1 segregation and adjacent 2 segregation were at the rate of 55.4, 3.6 and 7.2% versus 20, 13 and 6% in ours respectively. Other results (Estop et al., 1997; Hummelen et al., 1997; Martini et al., 1998; Mercier et al., 1998; Honda et al., 1999) were in agreement with those of Martin (Martin, 1994). In contrast, in a case of another reciprocal translocation t(11; 22) described by Estop et al. (Estop et al., 1999), the 3:1 segregation was found at a higher rate (40.1%) than the adjacent 1 (17.6%) and adjacent 2 (12.5%) segregation. Furthermore, in a case of (5; 7) translocation, the rate of 3:1 segregation was very close to the adjacent 2 segregation rate: 17 versus 16.6% (Estop et al., 1995). These authors considered these results as unusual. Finally, according to Honda et al. (Honda et al., 2000), meiotic segregation analysis of a Robertsonian translocation carrier revealed a rate of unbalanced sperm of 88.4%, significantly different from the theoretical frequency of 33.3%. Hence, we believe that in these cases of balanced translocation, no general features can be drawn. Each one must be considered as a particular case. The chromosomes and the genes implied that their breakpoints are likely to be one of the important points, although it is currently not possible to elucidate this fully. Thus, genetic counselling remains an important step before assisted reproductive technologies, if needed, are performed for these couples. In addition, our results show that genetic counselling must be done with care, in view of the different conclusions that have been reached by various investigators. To date, the history of recurrent miscarriages with a clear cytogenetic origin is relevant for pre-implantation genetic diagnosis. This kind of assisted reproductive technology could help the couple to have the desired pregnancy with fewer risks than for a naturally conceived pregnancy. FISH techniques can successfully be used, giving an individual result on very few embryonic cells and allowing the medical team to select an embryo or embryos devoid of the investigated chromosomal abnormalities. In conclusion, our study shows that a higher incidence of the nullisomia compared with the disomia is found in the sperm of a t(17; 22) (q11; q12) balanced translocation carrier. This is not in agreement with the well-known higher incidence of trisomia than of monosomia (except for the gonosomes) in ongoingpregnancies. Hence, as no general features can be drawn for the distribution of the sperm karyotypes of balanced translocation bearing men, the FISH technique is an important way to estimate their distribution and to help the medical team to adapt genetic counselling. 330 Acknowledgements We thank Mr Moyse, supervisor in the cytogenetics laboratory, for his helpful assistance. References Cifuentes, P., Navarro, J., Blanco, J. et al. (1999) Cytogenetic analysis of sperm chromosomes and sperm nuclei in a male heterozygous for a reciprocal translocation t(5;7) (q21;q32) by in situ hybridization. Eur. J. Hum. Genet., 7, Colls, P., Martinez-Pasarell, O., Pérez, M.M. et al. (1998) Cytogenetic analysis of spermatoza in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23). Mol. Hum. Reprod., 4, David, G., Bisson, J.P., Czyglick, F. et al. (1975) Anomalies morphologiques du spermatozoïde humain. 1. Proposition pour un système de classification. J. Gynecol. Obstet. Biol. Reprod., 4, (Suppl.), Downie, S.E., Flaherty, S.P. and Matthews, C.D. (1997) Detection of chromosomes and estimation of aneuploidy in human spermatoza using fluorescence in-situ hybridization. Mol. Hum. Reprod., 3, Durak, B., Ozön, Y.H., Ozdemir, M. et al. (1999) FISH analysis with locusspecific probes in sperm from two translocation carrier men. Clin. Genet., 56, Egozcue, S., Blanco, J., Vendrell, J.M. et al. (2000) Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatoza and recurrent abortion. Hum. Reprod. Update, 6, Estop, A.M., Van Kirk, V. and Cieply, K. (1995) Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7) and t(10;12), by spermatoza chromosome studies and a review of the literature. Cytogenet. Cell. Genet., 70, Estop. A.M., Cieply, K.M. and Aston, C.E. (1997) The meiotic segregation pattern of a reciprocal translocation t(10;12) (q26.1;p13.3) by fluorescence in situ hybridization spermatoza analysis. Eur. J. Hum. Genet., 5, Estop, A.M., Cieply, K.M., Munne, S. and Feingold, E. (1999) Multicolor fluorescence in situ hybridization analysis of the spermatoza of a male heterozygous for a reciprocal translocation t(11;22) (q23;q11). Hum. Genet., 104, Giltay, J.C., Kastrop, P.M.M., Tiemessen, C.H.J. et al. (1999) Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH. Cytogenet. Cell. Genet., 84, Guttenbach, M., 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, Honda, H., Miharu, N., Ohashi, Y. et al. (1999) Analysis of segregation and aneuploidy in two reciprocal translocation carriers, t(3;9) (q26.2;q32) and t(3;9) (p25;q32), by triple-color fluorescence in situ hybridization. Hum. Genet., 105, Honda, H., Miharu, N., Samura, O. et al. (2000) Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization. Hum. Genet., 106, Hummelen, P.V., Manchester, D., Lowe, X. and Wyrobek, A.J. (1997) Meiotic segregation, recombination and gamete aneuploidy assessed in a t(1;10) (p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm. Am. J. Hum. Genet., 61, Lim, A., Fong, Y. and Yu, S. (1999) Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46, XY mosaic Klinefelter patient. Hum. Genet., 104, Martin, R.H. (1994) Sperm chromosome complements in a man heterozygous for a reciprocal translocation t(2;3) (q24;p26). Hum. Reprod., 9, Martini E., Speel, E.J.M., Geraedts, J.P.M. et al. 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7 Sperm analysis by FISH Robbins, W.A., Segraves, R., Pinkel, D. and Wyrobek, A.J. (1993) Detection of aneuploid human sperm by fluorescence in situ hybridization: evidence for a donor difference in frequency of sperm disomic for chromosomes I and Y. Am. J. Hum. Genet., 52, Sloter, E.D., Lowe, X., Moore, D.H. et al. (2000) Multicolor FISH analysis of chromosomal breaks, duplications, deletions and numerical abnormalities in the sperm of healthy men. Am. J. Hum. Genet., 67, Spriggs, E.L., Martin, R.H. and Hulten, M. (1992) Sperm chromosome complements from two human reciprocal translocation heterozygotes. Hum Genet., 88, Ushijima, C., Kumasako, Y., Kihaile, P.E. et al. (2000) Analysis of chromosomal abnormalities in human spermatoza using multi-colour fluorescence in-situ hybridization. Hum. Reprod., 15, World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction, 3 rd edn Springer Verlag, Berlin. Submitted on February 22, 2001; resubmitted on July 2, 2001; accepted on October 1,