Methods. Patient. Sperm-FISH analysis. 110 Ferfouri et al.

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1 Molecular Human Reproduction, Vol.19, No.2 pp , 2013 Advanced Access publication on October 25, 2012 doi: /molehr/gas048 ORIGINAL RESEARCH Can one translocation impact the meiotic segregation of another translocation? A sperm-fish analysis of a 46,XY,t(1;16)(q21;p11.2),t(8;9) (q24.3;p24) patient and his 46,XY, t(8;9)(q24.3;p24) brother and cousin Fatma Ferfouri 1,2, Florence Boitrelle 1,2, Patrice Clément 3, Denise Molina Gomes 1,2, Jacqueline Selva 1,2, and François Vialard 1,2, * 1 Department of Reproductive Biology, Cytogenetics, Gynecology and Obstetrics, Poissy Medical Center, 10 rue du Champ-Gaillard, Poissy F-78303, France 2 EA 2493, University Versailles St-Quentin, Versailles F-78000, France 3 Laboratoire Clément, 8 Avenue Henri Barbusse, Le Blanc Mesnil F-93150, France *Correspondence address. Tel: ; Fax: ; fvialard@chi-poissy-st-germain.fr Submitted on July 20, 2012; resubmitted on October 16, 2012; accepted on October 16, 2012 abstract: Individuals with two independent chromosome rearrangements are rare and meiotic segregation studies are few. Two brothers (P1 and P2) and a cousin (P3) were karyotyped and found to have the same familial reciprocal translocation between the long arm of chromosome 8 and the short arm of chromosome 9: 46,XY,t(8;9)(q24.3;p24). In addition, one brother also had a different de novo reciprocal translocation between the long arm of chromosome 1 and the short arm of chromosome 16: 46,XY,t(1;16) (q21;p11.2)dn,t(8;9)(q24.3;p24)mat. Using locus-specific probes for segments involved in the translocations and for other chromosomes, sperm-fish analysis was used to investigate the products of meiotic segregation of the translocations and the possibility of an interchromosomal effect (ICE). Sperm nucleus fragmentation was also evaluated. For the t(8;9) translocation, the proportion of unbalanced products was higher for P1 (66.3%, P, ) than P2 (51.9%) and P3 (50.4%), and the proportion consistent with each meiosis I segregation mode was also different for P1. In addition, for P1, 61.6% of the products of the t(1;16) were unbalanced, and 85.6% of spermatozoa overall included both translocations. No evidence of an ICE was found and sperm nucleus fragmentation rates were similar. Our study suggests that co-segregation of the t(8;9) and the t(1;16) resulted in modifying the proportions of t(8;9) meiotic segregation products found in spermatozoa. This could be due to selection associated with meiotic checkpoints and germ cell death. Key words: double two-way complex chromosomal rearrangement / sperm FISH / sperm DNA fragmentation / interchromosomal effect Introduction Double, two-way complex chromosome rearrangements (CCRs) are rare events defined by at least two independent structural rearrangements, such as reciprocal or Robertsonian translocations (Bijlsma et al., 1978; Hansen et al., 1983; Watt and Couzin, 1983; Bell and Warburton, 1977) or inversions. Double, two-way CCRs are considered to be the simplest CCRs (Bass et al., 1985; Batanian and Eswara, 1998), when compared with three-way exchange CCRs (which involve three chromosomes and are generally hereditary; Meer et al., 1981; Farrell et al., 1994; Zahed et al., 1998) and exceptional CCRs (which are mainly de novo and characterized by one chromosome with at least two breakpoints). In general, CCR involves at least three breakpoints on two or more chromosomes, with an exchange of genetic material (Pai et al., 1980). In the literature, 255 CCR carriers have been identified (Zhang et al., 2009). About 70 75% of these CCRs are de novo (Pellestor et al., 2011a) and are found both in phenotypically normal and phenotypically abnormal patients (Madan et al., 1997; Patsalis, 2007). It is generally considered that the greater the number of breakpoints, the higher the risk of an abnormal phenotype (Pai et al., 1980). Most de novo CCRs are of paternal origin (Batista et al., 1994), whereas familial CCRs are primarily of maternal origin (Pellestor et al., 2011a). & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 110 Ferfouri et al. In males, CCRs can lead to infertility through the failure of spermatogenesis (Joseph and Thomas, 1982; Rodriguez et al., 1985; Siffroi et al., 1997; Coco et al., 2004; Sills et al., 2005; Bartels et al., 2007). Only 12 of the 130 reported male patients with CCRs were fertile (Grasshoff et al., 2003; Goumy et al., 2006). The meiotic segregation of CCRs frequently results in unbalanced gametes. In the event of fertilization, partial duplication/deletion causes recurrent spontaneous miscarriage or, in surviving infants, mental retardation and/or congenital abnormalities. To the best of our knowledge, only five studies of the meiotic segregation of CCRs have been reported. All were associated with a high unbalanced rates (ranging from 61.8 to 86.5%) (Pellestor et al., 2011a). Two other studies reported on chromosome segregation but not the overall CCR aneuploidy rates (Lu et al., 1994; Kirkpatrick and Ma, 2012). It was initially reported that the parents of children with Down s syndrome had a greater incidence of translocation (Lindenbaum et al., 1985). Hence, it was postulated that the rearrangement had an interchromosomal effect (ICE) on the segregation of chromosomes not involved in the abnormality. Even though some sperm-fish studies of single chromosome rearrangements have confirmed the existence on an ICE (Blanco et al., 2000; Kirkpatrick et al., 2008; Anton et al., 2010; Ferfouri et al., 2011), many others have not (Warburton, 1985; Martin et al., 1990; Schinzel et al., 1992; Douet-Guilbert et al., 2005). Hence, whether an ICE can occur in CCRs is still a matter of debate (Pellestor et al., 2011b; Kirkpatrick and Ma, 2012). Here, we studied meiotic segregation for a 46,XY,t(1;16) (q21;p11.2)dn,t(8;9)(q24.3;p24)mat double two-way CCR carrier, his brother and cousin. The two latter patients were heterozygous for the 46,XY,t(8;9)(q24.3;p24)mat. The present study sought to evaluate (i) the double two-way unbalanced rates, (ii) the potential impact of one translocation on the other and (iii) the presence or absence of an ICE on the chromosomes not involved in the chromosomal abnormalities (i.e. 7, 11, 12, 13, 15, 17, 18, 20, 21, X and Y). We also studied sperm nuclear DNA fragmentation, which is known to be high for single chromosome rearrangements (Brugnon et al., 2006, 2010; Perrin et al., 2009). Given the existence of an extensive family pedigree, we also assessed the potential correlation between the sperm aneuploidy rate and the observed, unbalanced inheritance of the t(8;9)(q24.3;p24). Patient A 34-year-old man (P1, propositus III-17) was referred to our lab as part of a family screening program for t(8;9)(q24.3;p24) (Fig. 1a). His mother was heterozygous for this abnormality. A 46,XY,t(1;16)(q21;p11.2),t(8;9)(q24.3;p24)mat CCR was identified. Given that the father s karyotype was normal, the second translocation was necessarily de novo (Fig. 1b). The same analysis had already been performed on P1 s 32-year-old brother (P2, III-18) and had detected a 46,XY,t(8;9)(q24.3;p24)mat familial translocation. During the P2 s wife first pregnancy, a prenatal diagnosis was performed. Balanced translocation was observed and a healthy daughter (IV-7) was delivered. However, the following pregnancy (IV-8) resulted in premature, spontaneous miscarriage at four weeks of gestation. The same familial translocation had been also Figure 1 The partial G-banded karyotype of a patient with a double, two-way CCR. (a) Familial t(8;9)(q24.3;p24)mat and (b) de novo t(1;16)(q21;p11.2)dn. detected in P1 and P2 s cousin (P3, III-12); the prenatal diagnosis performed for his partner s first pregnancy was negative for the familial translocation (IV-6). Both brothers and their cousin received genetic counseling and the family pedigree was established (Fig. 2). The inheritance of the t(8;9)(q24.3;p24) translocation was unbalanced in 5 of the 21 cases (23.8%), if one considers that the III-1 and IV-8 miscarriages were due to inheritance of an unbalanced translocation. After further genetic counseling, the patients provided their informed consent for an evaluation of sperm chromosome fragmentation [sperm-fish and a terminal deoxynucleotidyl transferasemediated dutp nick-end labeling (TUNEL) assay]. These cases were part of a broader study of genetic aspects of infertility that had been approved by the local investigational review board. Methods For each patient, a sperm analysis was performed according to the World Health Organization s criteria (WHO, 2010). After removal of seminal liquid, the spermatozoa were washed twice with sterile water (300 g for 10 min), fixed with Carnoy s solution and then spread on a slide for sperm-fish and DNA fragmentation assays. Sperm-FISH analysis Slides were prepared as previously described (Vialard et al., 2008). For hybridization efficiency, sperm FISH was used for chromosome-specific identification. Given that chromosome 1, 8, 9 and 16 specific probes did not appear to cross-hybridize with other chromosomes in situ, an accurate chromosome segregation analysis could be performed. Two different probe mixtures were used to assess: (i) the reciprocal translocation t(1;16)(q21;p11.2) segregation pattern, using a chromosome 1q12-specific probe [Kreatech SE 1 (1qh, Blue) Kreatech Diagnostics, Amsterdam, The Netherlands], a chromosome 16-specific probe [Vysis CEP16 (16q11.2, D16Z3, SpectrumGreen), Abbott Laboratories, Chicago, IL, USA] and the chromosome 1q long-arm subtelomeric probe [Vysis TelVysion 1q (1q44, D1S3738, SpectrumOrange), Abbott Laboratories]. (ii) the reciprocal translocation t(8;9)(q24.3;p24) segregation pattern, using a chromosome 8 centromeric probe [Vysis CEP8 (8p11. 1q11.1, D8Z2, SpectrumAqua), Abbott Laboratories], a chromosome 9 centromeric probe [Vysis CEP9 (9p11.1q11.1, SpectrumGreen),

3 Potential impact of one translocation on the other 111 Figure 2 The family pedigree. Patients with severe mental retardation are indicated by a top black square. Subjects who were heterozygous for t(8;9)(q24.3;p24) are indicated by a filled top right-hand quarter. The propositus (P1-III:17) carried a 46,XY,t(1;16)(q21;p11.2)dn,t(8;9)(q24.3;p24)- mat CCR and is indicated by the square with two filled quarters. His brother (P2-III:18) and cousin (P3-III:12) were heterozygous for the 46,XY,t(8;9)(q24.3;p24)mat translocation only. P1, P2 and P3 are indicated by arrows. Question marks correspond to subjects of interest who have not been karyotyped. Abbott Laboratories] and a chromosome 9p short-arm subtelomeric probe [Vysis TelVysion 9p (9p24.3, 305J7-T7, SpectrumOrange]. Copy number detection using locus-specific FISH probes cannot determine in all cases the actual mode of meiotic segregation which has produced every chromosome complement. We assumed that normal copy number was consistent with 2:2 alternate segregation following zero or an even number of crossovers in the interstitial segments and not 2:2 adjacent-1 segregation following an odd number, and that results indicating the deletion of one translocated segment and duplication of the other were consistent with 2:2 adjacent-1 segregation and not 2:2 alternate segregation following and an odd number of crossovers. The likelihood of obtaining a viable pregnancy was calculated with HC Forum software (Technidata France, Montbonnot, France). We also sought to identify an ICE with five mixtures containing specific probes for chromosomes not involved in the CCR (Abbott Laboratories). Probes specific for chromosomes 7 [Vysis CEP7 (7p11.1q11.1, D7Z1, SpectrumOrange)] and 15 [Vysis CEP15 (15p11.2, D15Z1, SpectrumGreen)]. Probes specific for chromosomes 11 [Vysis CEP11 (11p11.1q11.1, D11Z1, SpectrumOrange)] and 12 [Vysis CEP12 (12p11.1q11.1, D12Z3, SpectrumGreen)]. Probes specific for chromosomes 13 [Vysis LSI13 (13q14, RB1, SpectrumGreen)] and 21 [Vysis LSI21 (21q22.13q22.2, D21S342, D21S341 and D21S529, SpectrumOrange)]. Probes specific for chromosomes 17 [Vysis CEP17 (17p11.1q11.1, D17Z1, SpectrumAqua)] and 20 [Vysis CEP20 (20p11.1q11.1, D20Z1, SpectrumOrange)]. Probes specific for chromosomes X [Vysis CEPX (Xp11.1q11.1, DXZ1, SpectrumGreen)], Y [Vysis CEPY (Yp11.1q11.1, DYZ3, SpectrumOrange)] and 18 [Vysis CEP18 (18p11.1q11.1, D18Z1, SpectrumAqua)]. After codenaturation at 738C for 4 min, hybridization was carried out overnight at 378C. Slides were washed, counterstained with 4,6 -diamidino-2-phenylindole (DAPI) and then as previously reported (Vialard et al., 2008) using a Pathvysion Software Smart Capture FISH system, version 1.4 (Digital Scientific, Cambridge, UK). In line with previous reports (for review, Benet et al., 2005), we at least 1000 spermatozoa per slide. For ICE evaluation, aneuploidy rates were compared with three controls with the following mean (+ standard deviation) sperm parameters: sperm count per ejaculate: ; progressive motility: %; percentage with a typical morphology: %; sperm vitality: %. FISH interpretation criteria We considered that one spot corresponded to one chromosome. Two neighboring spots were considered to be distinct if the distance between them was greater than the spot diameter. In the study of reciprocal translocation segregation, normal, balanced spermatozoa (resulting from a 2:2 alternate segregation) were visualized by the presence of one spot per chromosome. Unbalanced spermatozoa (resulting from 2:2 adjacent, 3:1, 4:0 and other segregations) corresponded to all other combinations. ICE study In euploid spermatozoa, we considered that one spot corresponded to one chromosome. All other configurations were considered to be aneuploid cells. To avoid misinterpretation, one slide was used per probe mixture and rehybridization was not performed. P1 s overall balanced rate was calculated by multiplying the balanced proportion for each translocation. For ICE analysis, total aneuploidy rate has been defined as the sum of the results observed for each chromosome. TUNEL assay Slides were permeabilized with 0.1% SDS sodium citrate for 15 min. After two washes in phosphate-buffered saline (PBS), slides were incubated with the labeling solution (the fluorescein In Situ Cell Death Detection Kit from Roche Molecular Biochemicals, Rotkreuz, Switzerland) for 2 h at 378C. Next, slides were washed three times in PBS and after counterstaining with DAPI. Spermatozoa with DNA fragmentation fluoresced blue and green, as previously described (Frainais et al., 2010). One thousand spermatozoa were counted for each patient. Statistical analysis Using Statview software (SAS Institute, Cary, NC, USA), a x 2 test was used to compare patient and control aneuploidy rates and a Wilcoxon test was used to compare sperm DNA fragmentation rates. The threshold for statistical significance was set to P, 0.05.

4 112 Ferfouri et al. Results Sperm analysis P1: Normal sperm parameters were observed (sperm count: /ml; progressive motility: 35%; vitality: 83%; normal morphology: 19%). P2: Normal sperm parameters were observed (sperm count: /ml; progressive motility: 55%; vitality: 81%; normal morphology: 16%). P3: Normal sperm parameters were observed (sperm count: /ml; progressive motility: 50%; vitality: 73%; normal morphology: 20%). Segregation of the reciprocal translocation t(8;9)(q24.3;p24)mat P1: The principal segregation mode was 2:2 alternate; 36.7% of the spermatozoa (376 of 1024) had normal or balanced chromosomes. All other spermatozoa (63.3%) were unbalanced, with 2:2 adjacent-1 (22.6%), 2:2 adjacent-2 (20.2%) or 3:1 segregations (19.0%) (Table I). P2: The principal segregation mode was again 2:2 alternate; 48.1% of the spermatozoa had normal or balanced chromosomes. The rates were 27.4% for 2:2 adjacent-1, 15.3% for 2:2 adjacent-2 and 8.7% for 3:1 segregations. P3: The principal segregation mode was again 2:2 alternate; 49.6% of the spermatozoa had normal or balanced chromosomes. The rates were 29.6% for 2:2 adjacent-1, 16.2% for 2:2 adjacent-2 and 4.3% for 3:1 segregations. The proportion of the different products for the t(8;9) (q24.3;p24)mat differed significantly (Pearson s x 2,70 and P, ) when comparing P1 with P2 and P3, with a higher proportion of unbalanced spermatozoa (63.3 versus 51.9 and 50.4%, respectively; P, ), less 2:2 adjacent-1 segregation (22.6 versus 27.4 and 29.6%, respectively; P, ) and more 3:1 segregation (19.0 versus 8.7 and 4.3%; P, , respectively). The difference for the 2:2 adjacent-2 mode was not statistically significant for any of the inter-patient comparisons. When comparing P2 and P3, the proportion of the different products for the t(8;9)(q24.3;p24)mat differed significantly (Pearson s x 2 ¼ 18.6 and P ¼ ); this was mainly due to the difference in the unbalanced rate for the 2:2 adjacent-1 (P ¼ ) segregation mode. According to the HC Forum software and the sperm-fish results, the likelihood of obtaining a balanced viable pregnancy with this translocation was significantly lower for P1 than for P2 or P3, with rates of 53.9, 60.6 and 61.0%, respectively (P, 0.005). All these rates were significantly lower (P, 0.05) than the value estimated for the family pedigree (76.2%). Segregation of the reciprocal translocation t(1;16)(q21;p11.2)dn The preferred segregation mode was again 2:2 alternate (38.4%) (Table II). The rates for the other unbalanced segregations were 20.8% for 2:2 adjacent-1, 19.9% for 2:2 adjacent-2 and 20.0% for 3:1 segregations. Table I Spermatozoa meiotic segregation of the t(8;9)(q24.3;p24)mat. Segregation mode Genotype Spot color HC forum viability P1 P2 P3 P-value Number Rate Number of Rate Number of Rate of sperm (%) sperm (%) sperm (%)... Alternate 8, 9 or der8, AGR Yes a a a a:, der9 Adjacent I 8, der9 AR Yes b:, , der8 AGGR Yes c: ¼ Subtotal b b,c b,c Adjacent II 8, der8 AAG No d: NS 9, der9 GRR No Subtotal d d d 3:1 8 A No e,f:, , der8, der9 AGGRR Yes GR No , der8, der9 AAGR Yes der8 AG No , 9, der9 AGRR Yes der9 R No , 9, der8 AAGGR Yes Subtotal e e,f e,f Others No Total A, aqua; G, green; R, red.

5 Potential impact of one translocation on the other 113 For P1, the proportions of the different products consistent with the different modes were remarkably similar for the two translocations (Pearson s x 2 ¼ 2.338, 4 d.f., P ¼ ), despite the fact that the translocations are obviously very different; the t(1;16) was essentially whole-arm with breakpoints close to the centromeres, whereas the t(8;9) has very terminal breakpoints. The likelihood of obtaining a balanced viable fetus with this translocation (according to the HC Forum software) was 94.0%. Table II Spermatozoa meiotic segregation of the t(1;16)(q21;p11.2)dn. Segregation Genotype Spot Number Rate mode color of sperm (%)... 2:2 Alternate 1,16 or der1, AGR der16 2:2 Adjacent I 1, der16 AGRR , der1 AG Subtotal :2 Adjacent II 1, der1 AAR , der16 GGR Subtotal :1 1 AR , der1, AGGR der16 16 G , der1, AAGRR der16 der1 A , 16, der16 AGGRR der16 GR , 16, der1 AAGR Subtotal :0 and others Total 1027 A, aqua; G, green; R, red. P1 s overall euploidy rate The total sperm balanced rate for the CCR (i.e. the product of the balanced frequencies of each reciprocal translocation) was 14.1%. This was associated with a 50.7% likelihood of obtaining a balanced, viable fetus for P1. The interchromosomal effect For the ICE analysis (for chromosomes 7, 11, 12, 13, 15, 17, 18, 20, 21, X and Y), , , and spermatozoa were counted for P1, P2, P3 and three controls, respectively (Table III). The spermatozoa chromosome aneuploidy rates varied between 0.1 and 0.5% for P1, 0.1 and 0.3% for P2, 0.1 and 0.3% for P3 and 0.1 and 0.5% for the controls. None of the inter-subject differences were statistically significant. Likewise, P1, P2, P3 and the controls did not differ significantly in terms of the total aneuploidy rate (2.6, 2.1, 1.9 and 2.2%, respectively). Sperm DNA fragmentation The sperm DNA fragmentation rate was 3.0% for P1 (1015 spermatozoa ), 1.0% for P2 (1006 spermatozoa ) and 4.0% for P3 (1012 spermatozoa ). These values were significantly below our laboratory s normal cut-off value of 13%. Table III ICE analysis in spermatozoa; meiotic segregation of chromosomes not involved in either of the two translocations. Analyzed chromosome P1 P2 P3 Control (n 5 3) P-value Number of Rate Number of Rate Number of Rate Number of Rate sperm (%) sperm (%) sperm (%) sperm (%)... Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosome NS Chromosomes XY NS Global ICE NS aneuploidy rate

6 114 Ferfouri et al. Discussion In the present report, we first described meiotic segregation in (i) a patient with a double two-way CCR 46,XY,t(1;16)(q21;p11.2)dn, t(8;9)(q24.3;p24)mat and (ii) his brother and cousin, who were heterozygous for one of the reciprocal translocations [46,XY,t(8;9) (q24.3;p24)mat]. We then estimated the impact of t(1;16) (q21;p11.2) on t(8;9)(q24.3;p24) segregation by considering the complements in mature sperm and the likely mode of segregation. For P2 and P3, the chromosome unbalanced rate of 51.9 and 50.4% is in agreement with previously reported mean values of around 50% (Benet et al., 2005). Even though the proportions of the different translocation segregation products differed, this was mainly due to variations in the 2/2 adjacent-1 segregation; the rates for the other segregations were all similar. These results were consistent with previous data observed for a translocation involving submetacentric chromosomes with terminal or subterminal breakpoints, low 3:1 segregation and a majority of 2:2 adjacent-1 mode (considering only unbalanced modes) (Benet et al., 2005). Surprisingly, the proportions of the different products in P1 were remarkably similar when comparing his two very different translocations (Pearson s x 2 ¼ 2.338, P ¼ ). In light of previous reports and the results from P2 and P3, P1 s t(8;9) segregation mode was very different, with a higher proportion of 3:1 segregation modes and a lower proportion of 2:2 adjacent-1 segregation modes even though the latter difference might be due to a statistical fluke. Lastly, a higher proportion of meiotic abnormal segregation patterns (i.e. 2:2 adjacent-2 and 3:1) and a lower proportion of normal segregation patterns (i.e. 2:2 alternate and 2:2 adjacent-1) were observed for t(8;9). The five CCRs reported in the literature (Burns et al., 1986; Cifuentes et al., 1998; Loup et al., 2010; Ferfouri et al., 2012; Pellestor et al., 2011b) had unbalanced rates of between 61.8 and 86.5%. When considering double two-way translocations only, only one previous case has been reported (Burns et al., 1986); the unbalanced rate (86.3%) was similar to that observed for P1 (85.9%). Our new case confirms the high malsegregation rates observed for patients with CCRs especially for patients with a double two-way CCR with reciprocal translocations (with unbalanced rates of up to 80%). This value of 85.9% might be a slight overestimate. The total percentage of balanced gametes is probably higher than the sum of balanced spermatozoa detected for each translocation. To confirm these results, two subsequent rounds of FISH should be performed on the same slide (in order to analyze the same spermatozoa for each of the two translocations). Even though the translocation segregation pattern might vary from one patient to another with the same translocation (such as those heterozygous for a Robertsonian translocation; Roux et al., 2005), we hypothesize that the presence of both t(1;16)(q21;p11.2) and t(8;9)(q24.3;p24) may modify the proportion of the various products in the mature spermatozoa. This phenomenon might be linked to spatial crowding of the two quadrivalents and/or differences in germ cell apoptosis. Disrupted spermatogenesis in men who are heterozygous for a Robertsonian or reciprocal translocation has been postulated for many years and has been linked to an increase in the incidence of chromosome rearrangements in infertile populations (Bourrouillou et al., 1987; Van Assche et al., 1996; Martin, 2008). First meiotic analysis clearly revealed transient or permanent asynaptic segments in the spermatocytes of these individuals (Templado et al., 1984; Gabriel- Robez et al., 1986; Solari, 1999; Martin, 2008) and a XY body association of the rearranged chromosome during the pachytene stage. However, the basic molecular mechanism leading to spermatogenic failure has only recently been described. Studies in the pig and mouse (with optimal access to testicular material) have enabled these recent advances in our understanding of the functions and locations of specific meiotic proteins. First, asynaptic regions are associated with the presence of the DNA-damage response protein BRCA1, the kinase ATR and the variant histone c-h2ax in mouse pachytene spermatocytes (Turner et al., 2005) and lead to transcriptional silencing (Homolka et al., 2007). This correlates with failure to identify candidate genes for explaining spermatogenetic failure at the chromosome breakpoint (Bache et al., 2004). Secondly, heterosynapsis may be a way of escaping to apoptosis, as observed in the pig (Pinton et al., 2009). Furthermore, it has been postulated that the persistence of unrepaired double-strand breaks in the DNA of asynaptic segments leads to apoptosis (Burgoyne et al., 2009). Recently, a study of five cases (Sciurano et al., 2011) revealed the abnormal localization of the meiotic proteins in the asynaptic segments during the pachytene stage and the presence of the putative transcriptionally silenced chromatin domains in these same segments. The variable incidence of spermatocyte apoptosis in these patients is mainly due to the ability of heterosynapsis to successfully achieve the meiotic prophase and avoid the activation of a meiotic checkpoint for meiotic apoptosis (Odorisio et al., 1998) or damaging transcriptional inactivation (Martin, 2008). XY body association is mainly due to segregation of active and silenced chromatin into separate subnuclear compartments. Considering the sperm count differences between controls ( /ml), the single translocation heterozygotes ( /ml) and the double translocation heterozygote ( /ml), we hypothesized that both translocations lead to apoptosis but that the great majority of spermatocytes could achieve meiosis. However, in contrast to the situation seen in oligospermic patients who are heterozygous for a translocation (Brugnon et al., 2010; Perrin et al., 2011), no increase in the fragmentation rate was observed. Nevertheless, we could not rule out germ cell death, which could give rise to variations in the proportion of the different translocation products. A particular chromosome segregation may be associated with preferential apoptosis. The presence of two independent translocations may accentuate this variation and explain the observed difference between P1 and P2 or P3. In fact, it is still not known how translocation segregation is regulated. Animal models do not appear to be suitable for predicting translocation segregation. Differences are sometime very large, as in Robertsonian translocation (Pinton et al., 2009) or pericentric inversion (Massip et al., 2009) in the pig. When analyzing the extensive family pedigree with HC Forum software, the likelihood of obtaining an unbalanced fetus was calculated to be 23.8% (5 out of 21). This is a lower value than that obtained in P2 or P3 with respective rates at 39.4 and 39.0%, respectively. This difference may be due to (i) failure to the report miscarriages in this family, (ii) over-estimation of viability by considering only the spermatozoa unbalanced rate and the HC Forum database and/or (iii) apoptosis of unbalanced spermatozoa.

7 Potential impact of one translocation on the other 115 Despite the possible impact of the t(1;16)(q21;p11.2)dn on the meiotic segregation of t(8;9)(q24.3;p24)mat, the two reciprocal translocations in the CCR patient did not appear to affect the meiotic segregation of chromosomes not involved in the translocations. Aneuploidy rates per chromosome were similar in P1, P2, P3 and control patients. Similarly, there were no significant differences in the overall aneuploidy rate of chromosomes 7, 11, 12, 13, 15, 17, 18, 20, 21, X and Y when comparing P1 (2.6%), P2 (2.1%), P3 (1.9%) and the controls (2.2%). These values are similar to previously reported rates for control patients (Shi and Martin, 2001). Hence, there were no ICEs in these two patients with normal sperm parameters (P1 as a carrier of a double, two-way CCR and P2 and P3 as a carrier for only one reciprocal translocation). This situation differs slightly from that seen in patients who are normospermic and heterozygous for a Robertsonian translocation, where there appears to be an ICE on acrocentric chromosomes (Ferfouri et al., 2011). In contrast to Robertsonian translocations (which involve chromosomes known to be associated with the nucleolus and that may potentially interact with acrocentric chromosomes), none of the chromosomes involved here were acrocentric. This observation might account for the differing ICE results reported in the literature. Lastly, given that the same translocation was present in the three patients, we were able to assess the impact of a de novo translocation on the meiotic segregation of an inherited translocation. Our study suggests that co-segregation of the t(8;9) and the t(1;16) resulted in modifying the proportions of t(8;9) meiotic segregation products found in spermatozoa. Further studies are required to evaluate the reciprocal impact of double rearrangements. Acknowledgements We thanks the association AMMR-PSG for its financial help. Authors roles F.F.: performed sperm-fish analysis and wrote the paper. P.C.: performed karyotype analysis and helped to write the paper. F.B.: performed sperm analysis and wrote the paper. D.M.: performed genetic counseling and helped to write the paper. J.S.: laboratory director. F.V.: performed karyotype analysis, designed the study and wrote the paper. Funding No external funding was either sought or obtained for this study. Conflict of interest None declared. References Anton E, Blanco J, Vidal F. Meiotic behavior of three D;G Robertsonian translocations: segregation and interchromosomal effect. J Hum Genet 2010;55: Bache I, Assche EV, Cingoz S, Bugge M, Tumer Z, Hjorth M, Lundsteen C, Lespinasse J, Winther K, Niebuhr A et al. An excess of chromosome 1 breakpoints in male infertility. Eur J Hum Genet 2004;12: Bartels I, Starke H, Argyriou L, Sauter SM, Zoll B, Liehr T. 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