Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers

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Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers Anna Godo a, Joan Blanco a, Francesca Vidal a, Mireia Sandalinas b, Elena Garcia-Guixé b, Ester Anton a,* a

Genetics of Male Fertility Group, Unitat de Biologia Cel·lular (Facultat de Biociències), Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain; b Reprogenetics Spain, Carrer Tuset, 23, 08006 Barcelona, Spain

* Corresponding author.

E-mail address: [email protected] (E Anton). Anna Godo obtained her degree in Biology in 2009 at the Universitat Pompeu Fabra, and her MSc in Cell Biology in 2010 at the Universitat Autònoma de Barcelona. Since then, she has been a PhD student at the Genetics of Male Fertility group of the Department of Cell Biology, Physiology and Immunology at the Universitat Autònoma de Barcelona. Her research has been mainly focused on male infertility cytogenetics in carriers of chromosomal rearrangements.

Abstract The aim of this study was to assess whether there is a relationship between numerical chromosome abnormalities and certain segregation modes in spermatozoa from Robertsonian translocation carriers. A sequential fluorescence in-situ hybridization protocol based on two successive hybridization rounds was performed on sperm samples from one t(13;22) and ten t(13;14) carriers. Patient inclusion criteria included the presence of a positive interchromosomal effect (ICE). In the first round, numerical abnormalities for chromosomes 15/22, 18, 21, X and Y were analysed. In the second round, the segregation outcome of the rearranged chromosomes was evaluated in the numerically abnormal spermatozoa detected in the first round, as well as in randomly assessed spermatozoa. Aneuploid spermatozoa showed statistical differences in all segregation modes when compared with randomly assessed spermatozoa: alternate (50.7% versus 84.3%), adjacent (36.6% versus 14.6%) and 3:0 (10.2% versus 1%). Diploid/multiple disomic spermatozoa showed differences in alternate (3.7% versus 84.3%) and 3:0 (67.6% versus 1%). We concluded that in Robertsonian translocation carriers that exhibit ICE, numerically abnormal spermatozoa preferentially contain unbalanced segregation products. This might be explained by heterosynapsis acting as a rescue mechanism that would lead to aberrant recombination, which is a predisposing factor for non-disjunction events. © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: ICE, Robertsonian translocation, segregation pattern, sequential FISH, spermatozoa

http://dx.doi.org/10.1016/j.rbmo.2015.04.003 1472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Anna Godo, et al., Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.04.003

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Introduction Heterozygous Robertsonian translocations are some of the most common structural reorganizations in humans and they are estimated to be present in about 1/1000 newborns (Gardner et al., 2011). Although carriers are usually phenotypically unaffected, many of them are detected when they ask for reproductive advice due to difficulties in achieving a pregnancy. Indeed, the incidence of Robertsonian translocations in infertile men is 0.9%, which is up to nine times higher than in the general population (Mau-Holzmann, 2005). It is well established that one of the main causes of the reduced fertility of heterozygote Robertsonian translocation carriers is the formation of chromosomally abnormal spermatozoa as a result of the malsegregation of the chromosomes involved in the reorganization. The derivative chromosome and the two normal homologues adopt a trivalent during prophase I to allow full pairing. The segregation of the chromosomes generates eight possible outcomes: one normal and one balanced product resulting from the alternate segregation, four unbalanced products with partial disomies and nullisomies resulting from adjacent segregations and two unbalanced products with partial disomies or nullisomies resulting from 3:0 segregation (Figure 1). To date, segregation

Figure 1 Prophase I trivalent configuration (1). Segregation modes at anaphase I: alternate segregation (2a), adjacent segregations (2b) and 3:0 segregation (2c). To simplify the interpretation of the scheme only one chromatid per chromosome is represented.

data from over 112 carriers of Robertsonian translocations using sperm fluorescence in-situ hybridization (FISH) have been published. Among them, 66 cases correspond to a t(13;14) reorganization (Anton et al., 2004; Brugnon et al., 2010; Chen et al., 2007; Escudero et al., 2000; Ferfouri et al., 2011; Frydman et al., 2001; Mahjoub et al., 2011; Morel et al., 2001; Nishikawa et al., 2007; Ogawa et al., 2000; Ogur et al., 2006; Pylyp et al., 2013; Roux et al., 2005) and 22 cases to a t(14;21) reorganization (Anton et al., 2010; Brugnon et al., 2010; Ferfouri et al., 2011; Honda et al., 2000; Nishikawa et al., 2007; Pylyp et al., 2013; Rousseaux et al., 1995). The remaining studies involve other rare combinations of acrocentric chromosomes (Acar et al., 2002; Anahory et al., 2005; Anton et al., 2010; Bernicot et al., 2012; Brugnon et al., 2010; Chen et al., 2007; Cinar et al., 2011; Ferfouri et al., 2011; Moradkhani et al., 2006; Nishikawa et al., 2007; Pylyp et al., 2013; Rogenhofer et al., 2012). As reviewed by Anton et al. (2010), the compiled data indicate that Robertsonian translocation carriers display a similar distribution of segregation products, irrespective of the chromosomes involved, with the main segregation outcome being alternate (84.5% ± 6.3), followed by adjacent (14.6% ± 5.8) segregation, and the rare occurrence of 3:0 disjunction (0.6% ± 0.7). Another factor that could influence the fertility of Robertsonian translocation carriers is the occurrence of the phenomenon referred to as interchromosomal effect (ICE). This phenomenon has been linked to the formation of numerical anomalies for chromosomes other than those involved in the translocation. ICE has been reported to be due to meiotic interferences caused by the reorganized chromosomes on the pairing and segregation of other chromosomes (Burgoyne et al., 2009). It has been related to the presence of asynaptic regions during trivalent formation in prophase I, which trigger the activation of the pachytene checkpoint. Heterosynapsis appears as a rescue mechanism to avoid asynapsis and it has been seen to occur preferentially between the trivalent and the sex chromosomes as well as between the trivalent and other acrocentric chromosomes (Guichaoua et al., 1990; Johanisson et al., 1987; Luciani et al., 1984; Navarro et al., 1991; Sciurano et al., 2007, 2011). Nevertheless, the occurrence of heterosynapsis during prophase I might predispose chromosomes to non-disjunction at anaphase I (Kurahashi et al., 2012; Sciurano et al., 2011; Tepperberg et al., 1999), and thus produce cells with numerical anomalies. A recent review reported that more than half of Robertsonian translocation carriers produce significantly increased percentages of gametes with numerical abnormalities unrelated to the rearranged chromosomes (Anton et al., 2011), which is an even higher frequency than that observed in reciprocal translocations and inversion carriers. A third factor that might affect the final outcome of spermatogenesis in Robertsonian translocation carriers is related to the meiotic checkpoints that act in response to meiotic disturbances. Although it has been shown that traces of unresolved double-strand DNA breaks or synapsis defects can trigger the pachytene checkpoint and induce apoptosis (Burgoyne et al., 2009; Odorisio et al., 1998; Roeder and Bailis, 2000), a certain number of cells can escape this programmed elimination. In fact, it has been observed that pachytene and spindle assembly checkpoints in murine models carrying multiple Robertsonian translocations present a low stringency in response to pairing defects or misaligned chromosomes (Eaker

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Seminogram

Normozoospermia Oligozoospermia – Oligoasthenoteratozoospermia Oligoasthenoteratozoospermia Teratozoospermia Teratozoospermia Normozoospermia Oligoasthenozoospermia Oligoteratozoospermia Oligoasthenozoospermia 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;14)(q10;10) 45,XY,der(13;22)(q10;10) P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Samples were fixed with methanol:acetic acid (3:1), spread on slides and processed for FISH, as previously described by the group (Sarrate and Anton, 2009). Later on, each sample

Karyotype

Sequential FISH protocol

Carrier

This study was undertaken on semen samples obtained from 11 Robertsonian translocation carriers (P1–P11; Table 1). Ten samples came from patients with a 45,XY,der(13;14)(q10;q10) karyotype and one sample was obtained from a 45,XY,der(13;22)(q10;q10) male. Besides the status of being Robertsonian translocation carriers, the inclusion criterion was to show positive ICE (a production of high rates of aneuploid/ diploid spermatozoa when compared with internal control data; Tables 2 and 3) ascertained in previous sperm FISH analyses. This allowed maximization of the number of spermatozoa with chromosomal abnormalities to be potentially analysable in each individual. This study was approved by our Institutional Ethics Committee Board on 19th September 2014 (reference: CEEAH 1883) and all donors signed the corresponding informed consent form.

Table 1

Biological samples

Karyotype, seminogram and probes used in FISH analyses.

Materials and methods

All probes are from Abbott Molecular, Inc., except Tel 15q from Kreatech Diagnostics (Amsterdam, The Netherlands). Seminograms were established according to World Health Organization (WHO) criteria (Cooper et al., 2010). SA = Spectrum Aqua; SG = Spectrum Green; SO = Spectrum Orange. FISH = fluorescence in situ hybridization; ICE = interchromosomal effect.

CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO) CEP 18 (SA)/CEP X (SG)/CEP Y (SO)

Probes used in the ICE studies

LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/LSI 22 BCR (SG) LSI 21 (SO)/Tel 15q (SA)

Probes used in the segregation studies

et al., 2001; Manterola et al., 2009). Also, the presence of apoptotic markers and high levels of DNA fragmentation have been observed in ejaculated spermatozoa from Robertsonian translocation carriers (Brugnon et al., 2006, 2010; Perrin et al., 2009). Altogether, this situation suggests that in some cases, checkpoints can detect meiotic abnormalities, but the elimination rate of these abnormal cells is not completely efficient. Previous studies by the present authors’ group have reported a relationship between the occurrence of ICE and unbalanced segregation modes in carriers of reciprocal translocations (Godo et al., 2013a, 2013b). The group concluded that heterosynapsis, driven by the incomplete pairing within the quadrivalent, influences the disjunction of the rearranged chromosomes and other bivalents, resulting in the concurrence of non-disjunction events in the same cell. Focusing on Robertsonian translocation carriers, our hypothesis is that, similarly to what has been observed in reciprocal translocation carriers, synaptic disturbances within the trivalent would affect later disjunction of chromosomes not involved in the rearrangement. Accordingly, the aim of the present study is to assess whether a relationship exists between numerical chromosome abnormalities derived from ICE and certain segregation modes of the rearranged chromosomes in Robertsonian translocation carriers. With this purpose in mind, a sequential FISH protocol has been performed that allows both the evaluation of numerical abnormalities and segregation analysis in the same sperm nuclei. The ultimate goal is to deepen the investigation of the cytogenetic characteristics of the gametes produced by translocation carriers and to increase the knowledge about the role of heterosynapsis in the meiotic behaviour of translocations.

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LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) LSI 13 (SG)/Tel 14q (SO) Tel 13q (SO)/LSI 22 BCR (SG)

Segregation and ICE interrelate in sperm from Roberstonian translocation carriers

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was subjected to a sequential FISH protocol as described in Godo et al. (2013a). Detailed information about the probes used is given in Table 1. In the first FISH round, two parallel FISH procedures were performed. Briefly, one of the trials was addressed to the evaluation of chromosomes 18, X and Y by using a panel containing these probes (AneuVysion DNA Multicolor Probe Kit, Abbott Molecular Inc., Des Plaines, IL, USA). The other trial was addressed to the evaluation of chromosomes 21 and 22 (LSI 21, Spectrum Orange; LSI 22 BCR, Spectrum Green, Abbott Molecular Inc.). Exceptionally in the individual P11, due to the implication of chromosome 22 in the translocation, the second panel was substituted by a combination of probes addressed to evaluate chromosome 15 (Tel 15q, Spectrum Aqua,. Kreatech Diagnostics) and chromosome 21 (LSI 21, Spectrum Orange, Abbott Molecular Inc.) (Table 1). Sperm nuclei were classified as disomic when two signals for a given chromosome and a single signal for the other chromosomes evaluated were observed. Nullisomic spermatozoa showed no signal for a given chromosome and a single signal for the other chromosomes evaluated. Nuclei with two signals for each chromosome were recorded as diploid/multiple disomic. The second FISH round performed on the same slides was addressed to evaluate the segregation products of the rearranged chromosomes and was accomplished as described in Godo et al. (2013a). The combination of probes used was in accordance with the characteristics of the reorganization (Table 1). This analysis was performed in the population of chromosomally abnormal sperm nuclei ascertained in the first round. The segregation outcome was also evaluated for every single carrier, in randomly assessed spermatozoa, in order to assess the general segregation behaviour of each reorganization. For each individual, 1000 sperm nuclei were analysed. Sperm nuclei were classified into each segregation mode according to the particular combination of signals.

Table 2 Carrier

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Controlsc

FISH analysis FISH evaluation was carried out using an Olympus BX-60 microscope (Olympus, Spain) equipped with a motorized stage, connected to the automatic Spot-Counting scan system (Spot AX software, Applied Imaging, Newcastle, U.K.), and with specific filters for 4,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC), Cy3 and Aqua. The workstation platform allowed all nuclei to be assessed and pictures to be taken, as well as enabling the relocalization of the nuclei of interest. Signal scoring was performed according to the strict analysis criteria described by the group referring to intensity, size and distribution of the signals (Blanco et al., 1996).

Statistical analysis Statistical analyses were carried out using GraphPad Prism v5.0 (GraphPad Software, Inc., La Jolla, USA). Statistical decisions were made at a 0.05 significance level. The chi-squared test was used for intra-individual comparison of diploid/multiple disomic frequencies obtained from the two parallel experiments undertaken in the first round of FISH. Regarding the segregation study, frequencies of each segregation mode reported in the randomly assessed spermatozoa were compared with frequencies found in spermatozoa carrying numerical abnormalities. This comparison was done at the population level using the Wilcoxon test and at an individual level using the chi-squared test.

Results Interchromosomal effect analysis The ICE analysis was performed on a total of 105,151 nuclei labelled with probes for chromosomes 18, X and Y (Table 2),

Frequencies of numerical chromosome abnormalities from the ICE study for chromosomes 18, X and Y. Chromosome 18

XY Chromosomes

Dis %

Null %

Dis %

Null %

0.15a 0.05 0.22a 0.11a 0.17a 0.09a 0.15a 0.15a 0.11a 0.10a 0.10a 0.03

0.04 0.04 0.11 0.05 0.45a 0.18a 0.14a 0.05 0.20a 0.03 0.02 0.07

0.22 0.40a 0.47a 0.18 1.21a 0.40a 0.52a 0.49a 0.75a 0.46a 0.41a 0.19

0.40 0.67 0.74 0.58 1.31a 0.45 0.26 0.28 0.91a 0.62 0.38 0.54

Dipl/md %

Total spermatozoa analysed

Total abnormal spermatozoa

0.69a,b 0.16 0.20 1.68a,b 0.47a,b 0.17 0.43a,b 0.52a,b 0.65a,b 0.28 0.53a 0.19

10,313 7910 5571 10,260 10,576 10,719 10,621 10,489 8043 10,355 10,294

154 105 96 266 383 138 160 156 210 154 148

Dipl/md = Diploidy/multiple disomies; Dis = Disomy; ICE = interchromosomal effect; Null = Nullisomy. a Statistically significant increase (P < 0.05) compared with control data. b Statistically significant differences (P < 0.05) compared with diploid frequency observed in ICE study for chromosomes 15, 21 and 22. c Data published in Sarrate et al. (2010).

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ARTICLE IN PRESS Segregation and ICE interrelate in sperm from Roberstonian translocation carriers Table 3 Carrier

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Controls

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Frequencies of numerical chromosome abnormalities from the ICE study for chromosomes 15, 21 and 22. Chromosome 15

Chromosome 21

Chromosome 22

Dis %

Null %

Dis %

Null %

Dis %

Null %

– – – – – – – – – – 0.14 0.09b

– – – – – – – – – – 0.06 0.06b

0.01 0.08 0.04 0.14a 0.09 0.05 0.77a 0.08 0.08 0.08 0.07 0.07c

0.16 0.08 0.24 0.36a 0.16 0.04 0.08 0.11 0.23 0.13 0.11 0.25c

0.05 0.03 0.01 0.10a 0.02 0.07 0.33a 0.24a 0.08 0.03 – 0.04b

0.04 0.02 0.16a 0.14a 0.36a 0.02 0.02 0.01 0.09 0.22a – 0.06b

Dipl/md %

Total sperm analysed

Total abnormal spermatozoa

0.10 0.08 0.25 1.24a 0.28 0.12 0.98a 0.30 0.30 0.34a 0.38a 0.23d

10,044 8644 10,159 10,163 10,248 10,610 10,416 11,183 10,084 10,155 10,001

36 26 70 201 93 31 226 82 78 81 76

Dipl/md = Diploidy/multiple disomies; Dis = Disomy; ICE = interchromosomal effect; Null = Nullisomy. a Statistically significant increase (P < 0.05) compared with control data. b Unpublished data based on the same population assessed in Sarrate et al. (2010). c Data published in Sarrate et al. (2010). d Diploidy/multiple disomy rates in control individuals have been calculated as the average of two FISH studies performed in each one of them.

and 111,707 nuclei labelled with probes for chromosomes 21 and 22 (samples P1–P10) or for chromosomes 15 and 21 (sample P11) (Table 3). The automatized system allowed the identification of 1950 aneuploid spermatozoa (disomic or nullisomic) and 1020 diploid/multiple disomic spermatozoa. Some individuals showed significantly different frequencies of diploid/multiple disomic spermatozoa in the two ICE studies (all P < 0.05). Specifically, P1, P4, P5, P8 and P9 presented higher diploid/ multiple disomic sperm rates in the 18-X-Y ICE study when compared with the diploid/multiple disomic sperm rates found in the 21–22 study. Conversely, P7 showed a higher incidence of diploid/multiple disomic rate in the 21–22 ICE study compared with the 18-X-Y study (Table 2).

Segregation of translocated chromosomes in randomly assessed spermatozoa Results from segregation analysis in randomly assessed spermatozoa in each carrier are detailed in Table 4. Alternate segregation had a mean frequency ± SD of 84.3% ± 3.7, while adjacent segregation was 14.6% ± 3.6 and 3:0 segregation was 1.0% ± 0.6. Signal combinations that could not be attributed to any of these segregations (categorized as ‘other’) were present in small percentages in all individuals (average of 0.1% ± 0.2). The majority of these ‘other’ combinations can be ascribed to non-disjunction events at meiosis II (Figure 2 and Table 4).

Segregation of translocated chromosomes in spermatozoa with numerical abnormalities Results from segregation analysis in aneuploid and diploid/ multiple disomic spermatozoa are detailed in Table 4. A total of 1691 aneuploid gametes were successfully relocalized and

reanalysed for the segregation content. The mean frequencies ± SD for the alternate, adjacent, 3:0 and ‘other’ segregation modes were 50.7% ± 11.7, 36.6% ± 7.7, 10.2% ± 6.5 and 2.5% ± 1.1, respectively (Figure 2 and Table 4). By comparing the distribution of the segregation modes obtained in aneuploid spermatozoa versus randomly assessed spermatozoa, significant differences in all segregation modes (all P < 0.05) were identified. At the individual level, balanced segregation was decreased in all carriers in favour of the unbalanced modes, which were significantly increased when compared with the randomly assessed sperm data (all P < 0.05; Figure 2 and Table 4). Regarding sperm nuclei categorized as diploid/multiple disomic, from the initial population of 1020 nuclei evaluated, a total of 876 were analysed in the second FISH round. They displayed a mean frequency ± SD for the alternate, adjacent, 3:0 and ‘other’ segregation modes of 3.7% ± 2.8, 19.2% ± 6.4, 67.6% ± 8.8 and 9.6% ± 5.7, respectively (Figure 2 and Table 4). At the individual level, alternate segregation was drastically reduced in all cases while 3:0 segregation was clearly increased. Adjacent segregation was significantly increased in carriers P1, P7 and P10 (P < 0.05; Table 4). At the population level, comparison of the segregation modes observed in diploid/multiple disomic spermatozoa versus randomly assessed spermatozoa showed similar results to those observed in aneuploid gametes (with the exception of the adjacent segregation): alternate segregation was significantly decreased, whereas 3:0 and ‘other’ segregation modes were significantly increased (all P < 0.05; Figure 2 and Table 4).

Discussion The aim of this study was to find out whether there is a relationship between the presence of numerical chromosome anomalies and unbalanced content of the rearranged chromosomes in spermatozoa from Robertsonian translocation

Please cite this article in press as: Anna Godo, et al., Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.04.003

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Aneuploid spermatozoa

Dipl/md spermatozoa

Randomly assessed spermatozoa

Aneuploid spermatozoa

Dipl/md spermatozoa

Randomly assessed spermatozoa

Aneuploid spermatozoa

Dipl/md spermatozoa

Randomly assessed spermatozoa

Aneuploid spermatozoa

Dipl/md spermatozoa

Total

Randomly assessed spermatozoa

Other %

Dipl/md spermatozoa

3:0 %

Aneuploid spermatozoa

Adjacent %

87.5 87.7 86.1 79.1 82.4 87.5 89.3 84.5 77.5 86.3 81.7 84.3 3.7

53.1a 59.8a 68.9a 42.3a 53.3a 50.0a 26.4a 40.2a 48.4a 60.5a 50.0a 50.7b 11.7

3.8a 0.0a 2.9a 3.2a 2.9a 2.2a 4.9a 0.0a 10.4a 3.3a 2.2a 3.7b 2.8

10.0 11.6 12.8 19.1 17.0 10.0 9.7 15.0 20.7 13.0 17.3 14.6 3.6

25.5a 32.2a 20.5a 41.5a 39.5a 33.1a 46.4a 43.6a 38.5a 32.7a 33.1a 36.6b 7.7

21.8a 16.7 23.5 18.1 18.6 14.6 32.0a 14.5 9.1a 25.0a 14.6 19.2 6.4

2.3 0.7 1.1 1.6 0.5 2.3 0.9 0.5 1.3 0.7 0.9 1.0 0.6

17.3a 5.7a 8.2a 14.6a 5.1a 13.7a 25.2a 13.7a 8.2a 5.6a 13.7a 10.2b 6.5

61.5a 83.3a 61.8a 69.4a 67.1a 78.7a 59.2a 78.3a 58.4a 61.7a 78.7a 67.6b 8.8

0.2 0.0 0.0 0.2 0.1 0.2 0.1 0.0 0.5 0.0 0.1 0.1 0.2

4.1a 2.3a 2.5a 1.5a 2.1a 3.2a 1.8a 2.6a 4.9a 1.2a 3.2a 2.5b 1.1

12.8a 0.0 11.8a 9.3a 11.4a 4.5a 3.9a 7.2a 22.1a 10.0a 4.5a 9.6b 5.7

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 11,000

98 87 122 130 375 131 163 117 182 162 124 1691

78 18 34 248 70 30 103 69 77 60 89 876

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P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Average SD

Alternate % Randomly assessed spermatozoa

Carrier

Frequencies of segregation modes in randomly assessed sperm, aneuploid and diploid/multiple disomic sperm populations.

Dipl/md = Diploid/multiple disomic. a Statistically significant difference (P < 0.05) compared with data from a randomly assessed sperm population of the same segregation mode and individual. b Statistically significant difference (P < 0.05) compared with the average data from randomly assessed sperm population of the same segregation mode.

A Godo et al.

Please cite this article in press as: Anna Godo, et al., Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.04.003

Table 4

ARTICLE IN PRESS Segregation and ICE interrelate in sperm from Roberstonian translocation carriers

Figure 2 Distribution of the mean frequencies of the segregation modes in the different sperm populations.

carriers. This was done by comparatively assessing the segregation modes achieved by the translocated chromosomes and the presence of numerical anomalies in the same sperm nuclei. The study was based on the assumption that if nothing differentially affects the progression through meiosis of numerically abnormal gametes, no differences would be expected in the segregation pattern of randomly assessed spermatozoa and aneuploid spermatozoa. A clear result inferred from the segregation analysis performed in randomly assessed spermatozoa is the homogeneity of segregation patterns observed in all individuals and the high number of normal/balanced gametes produced (84.3%). These results are in good agreement with previously published studies in Robertsonian translocation carriers and support the existence of a common segregation behaviour in this type of reorganization (Anton et al., 2010). On the other hand, the segregation patterns observed in numerically abnormal spermatozoa revealed significant variations when compared with the standard distribution, basically favouring the presence of all unbalanced products. This is particularly the case in aneuploid spermatozoa, in which the percentage of alternate segregation was only 50.7% whereas unbalanced segregation products reached 49.3% (adjacent segregation was the most common outcome among them, 36.6%). The main segregation mode observed in diploid/multiple disomic gametes was 3:0 (67.6%). This allowed the conclusion to be drawn that most spermatozoa classified as diploid/multiple disomic in the ICE analysis – in which only a few chromosomes were analysed – presented a true 2n chromosome content. Alternatively, the remaining gametes classified in this supposed diploid/multiple disomic population, but who presented segregation modes other than 3:0, are more likely to be carriers of multiple disomies (32.4%). There is evidence supporting this interpretation: the fact that diploid/ multiple disomic rates detected in the two studies of numerical abnormalities performed in each carrier were not equivalent in six cases (P1, P4, P5, P7, P8 and P9) reflects the presence of multiple disomies among the gametes initially classified as diploid/multiple disomic. It is important to mention that the percentage of alternate segregation among the spermatozoa that would contain

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multiple disomies (11.3%) also reveals a significant reduction (P = 0.001) compared with the values obtained in the randomly assessed spermatozoa. [Note: percentage estimated from Table 4 by considering that gametes showing a 3:0 segregation present a true diploid content (67.6% of 876 = 592 gametes). Accordingly, the rest of gametes in this category would carry multiple disomies (32.4% of 876 = 284 gametes). Then, by knowing that 3.7% of the diploid/md spermatozoa display an alternate content (32 gametes out of 876), we can estimate that 32 out of 284 (11.3%) of multiple disomic spermatozoa would carry an alternate segregation]. The phenomenon of the accumulation of numerical and structural abnormalities in the same nuclei may have different explanations. The starting point is the presence of asynaptic regions at prophase I, which is a common feature in Robertsonian translocation carriers (Sciurano et al., 2011). The presence of asynapsed regions within the reorganized chromosomes can be solved by heterologous pairing of the trivalent with the XY body (Luciani et al., 1984; Navarro et al., 1991); this is supported by the well-known homology of the heterochromatin blocks of the acrocentric chromosomes and the Y chromosome (Metzler-Guillemain et al., 1999). The heterologous pairing could also be a consequence of the segregation of active (synapsed) and silenced (unsynapsed) chromatin into separate nuclear domains (Sciurano et al., 2011). In any case, the presence of asynapsis and/or heterosynapsis may interfere with the correct establishment of crossovers. There are some studies of reciprocal translocation carriers that describe a significant reduction of the total number of chiasmata affecting both the reorganized and the non-reorganized chromosomes (Jiang et al., 2014; Leng et al., 2009; Pigozzi et al., 2005; Sun et al., 2005). As is already known, altered chiasmata distribution can lead to chromosome misalignments at the metaphase plate, causing disjunction errors at anaphase I (Eaker et al., 2001; Manterola et al., 2009). It would be very interesting to know whether the same phenomenon occurs in female meiosis. Unfortunately, there is only a single study that has assessed the segregation outcome and the presence of numerical abnormalities in the same human oocytes. In this study, Pujol et al. (2003) evaluated the content of eight different chromosomes in the first polar body from oocytes of two Robertsonian translocation carriers. The authors reported that 37% (7/19) of aneuploid oocytes showed a balanced content of the rearranged chromosome, while this rate was much higher in the population of euploid oocytes (75%, 3/4). Although this is a limited amount of data, both regarding the number of cells analysed and the number of individuals, the results are in accordance with the findings observed in the present study. Cytogenetic data from embryos obtained from male Robertsonian translocation carriers will also be of great interest. However, few preimplantation genetic diagnosis (PGD) studies report the results of the segregation outcome and the presence of numerical abnormalities in the same cells (Alfarawati et al., 2011; Colls et al., 2012; Fiorentino et al., 2011; Rius et al., 2011). These studies, performed either by comparative genomic hybridization (CGH) or microarrayCGH, report a similar range of alternate segregation in the euploid and aneuploid embryos from male Robertsonian translocation carriers. Although these results are not in the same line of the current study’s findings, a possible explanation for

Please cite this article in press as: Anna Godo, et al., Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.04.003

ARTICLE IN PRESS 8

A Godo et al.

the discrepancies could be based on the high percentage of aneuploid embryos observed in these studies (more than 50%). These figures could mask any evidence of a possible relationship between the presence of numerical abnormalities and a given segregation mode. In any case, the results obtained from the present study reinforce the need for more data to establish links between the cytogenetic abnormalities observed in spermatozoa, the consequences for the embryos, the PGD analysis outcome, and hence the reproductive prognosis of affected couples.

Conclusions Overall, this study clearly reflects an altered segregation pattern in aneuploid gametes from male Robertsonian translocation carriers, with an accumulation of numerical chromosome anomalies and unbalanced segregation products in the same sperm nucleus. We would like to point out that although only Robertsonian translocation carriers with a positive ICE were included in this study (and thus the conclusions obtained should be limited to Robertsonian translocation carriers that display this phenomenon), we believe that a similar condition would occur in the production of numerically abnormal spermatozoa in the remaining Robertsonian translocation carriers. This association relies on the fact that the features associated with the occurrence of numerical abnormalities are equivalent in all individuals and the differences between Robertsonian translocation carriers displaying positive ICE or negative ICE would only reflect the frequency with which the pairing anomalies appear. These results agree with previously published data from reciprocal translocation carriers and reinforce the hypothesis that the establishment of heterosynapsis at the pachytene stage might entail subsequent non-disjunction events, affecting the segregation of both the rearranged chromosomes and other bivalents. Further studies at the molecular level of meiocytes from reorganization carriers would be of great interest in confirming the involvement of heterosynapsis as a ‘cell rescue mechanism’ in the occurrence of nondisjunction events. Indeed, other consequences of heterosynapsis, such as a possible interference of the chromosome territories in the final organization of the sperm nucleus, should be taken into consideration. In this regard, studies of higher-order structures in sperm nuclei, as well as in different spermatogenic cells, would also help to shed more light on the effects of asynapsis/heterosynapsis through meiosis. Finally, a better understanding of the mechanisms and implications involved in non-disjunction events will contribute towards a better assessment of the genetic reproductive risk of Robertsonian translocation carriers.

Acknowledgements This work was supported by funding for the projects SAF201022241 (Ministerio de Ciencia e Innovación, España), UAB CF180034 (Universitat Autònoma de Barcelona) and SGR2014524 (Generalitat de Catalunya). AG is a recipient of an FIDGR grant (Generalitat de Catalunya). The funding sources had no involvement in the preparation of the manuscript. This manuscript has been proofread by Proof-Reading-Service.org.

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