Rare Robertsonian translocations and meiotic behaviour : sperm FISH analysis of t ( 13 ; 15 ) and t ( 14 ; 15 ) translocations : A Case Report

Institute of Human Genetics, CNRS, Department of Medical Genetics, CHU Montpellier, France, Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran, Laboratory of Cytogenetics, CHU Chambery, Laboratory of Cytogenetics, CHU Clermont-Ferrand and Laboratory of Biology of the Reproduction, CHU Montpellier, France To whom correspondence should be addressed at: CNRS UPR 1142, Institute of Human Genetics, 141 Rue de la Cardonille, F-34396 Montpellier Cedex 5, France. E-mail: franck.pellestor@igh.cnrs.fr


Introduction
Both Robertsonian translocations t(13;15) and t(14;15) are rare structural rearrangements whose meiotic pattern was seldom, if ever, analysed.Thanks to the introduction of in vitro humanhamster fertilization system (Humster test), and more recently to molecular cytogenetic techniques, several sperm studies have been performed in the human carriers of Robertsonian translocations.Most of them have focused on the two frequent Robertsonian translocations, i.e. the t(13;14) and the t(14;21).Only three cases of rare Robertsonian translocation were investigated using the Humster test, and fluorescence in-situ hybridization (FISH) analyses were performed in sperm from only three homologous t(21;21) Robertsonian translocations and one t(21;22) and from five men heterozygous for a t(13;22) and a t(13;15), respectively (Acar et al., 2002;Anahory et al., 2005;Rives et al., 2005;Brugnon et al., 2006;Ogur et al., 2006).All these studies have brought forth essential information concerning the chromosomal segregation of Robertsonian translocations in male carriers.In particular, they evidenced the similar meiotic behaviour of all Robertsonian translocations, with a high predominance of alternate segregation leading to normal or balanced complements.However, significant variations in the rates of imbalances were observed (from 10 to 25%), suggesting that the production of unbalanced gametes in infrequent Robertsonian translocations could be affected by the chromosomes involved and the locations of breakpoints.Consequently, there is a real need for data about the meiotic segregation of rare Robertsonian translocations and their risks of imbalance, especially because Robertsonian translocations are becoming one of the first indications for preimplantation genetic diagnosis (PGD).
In this study, we report the meiotic segregation pattern in sperm from two men heterozygous for a t(13;15) and two men carrying a t(14;15) Robertsonian translocation.

Patients
Four unrelated men, all carriers of Robertsonian translocation, were included in this study.The four subjects gave their informed consent before participation in the study, which was approved by the Ethical Board of the Montpellier University Hospital.
Patient A, a 35-year-old man, was referred for chromosomal investigation because of primary infertility.Severe oligoasthenozoospermia (sperm count 0.5 × 10 6 /ml, 32% normal morphology and 4% progressive motility) was found in two successive examinations.The cytogenetic analysis of cultured lymphoblasts revealed the existence of a t(13;15) Robertsonian translocation.
Patient B, a 35-year-old man, carrier of a t(13;15) Robertsonian translocation, was ascertained after his wife had a therapeutic firsttrimester termination because of fetus carrying an unbalanced form of the translocation.His spermiogram was normal with a sperm count of 70 × 10 6 /ml, 64% normal morphology and 55% progressive motility.
The sperm from three fertile men aged 26-33 years with a normal karyotype and normal sperm parameters were used as controls.

Sperm preparation
Each sperm sample was washed three times in 1 × phosphate-buffered saline (PBS) by centrifugation (300 g, 5 min) and fixed for 1 h in fresh fixative (3:1 methanol : glacial acetic acid) at -20°C.The sperm suspension was then dropped onto clean microscope slides and air-dried.Slides were aged 2 days at room temperature before use for in situ chromosomal labelling.
Before FISH procedure, the slides were immersed for 10 min in a pepsin solution (50 ng/ml in 0.01 M HCl) pre-warmed at 37°C, washed for 2 min in 1 × PBS and then dehydrated through an ethanol series (70, 90 and 100%) and air-dried.The sperm nucleus decondensation and DNA denaturation were performed by slide incubation in 0.5 M NaOH solution at room temperature for 8 min, followed by a wash in 2 × SSC, dehydration through an ethanol series and immersion in 70% formamide/2 × SSC solution 3 min at 73°C.Finally, the slides were washed in 2 × SSC, dehydrated through an ethanol series and air-dried.

FISH procedure
Two types of probes were used in this study.The first probe mixture was composed of commercial probes from Vysis, i.e. the chromosome 13 and 14 Vysis locus-specific probes (LSPs) labelled with Spectrum Orange and the chromosome 15 Vysis LSP labelled with Spectrum Green.The second probe mixture consisted of commercial whole chromosome painting (WCP) probes from Vysis, including chromosome 13 and 14 WCP probes (WCP 13 and WCP 14) labelled with Spectrum Orange and a chromosome 15 WCP probe (WCP 15) labelled with Spectrum Green.The probes were prepared according to the manufacturer's instructions.
Both WCP probes and LSPs were denatured separately for 7 min at 75°C in a water bath.Each probe mixture was applied to the denatured slides, covered with coverslips, sealed with rubber cement and then hybridized overnight in a dark, moist chamber at 37°C.After hybridization, the coverslips were gently removed and the slides washed in 0.4 × SSC/0.3%NP40, pH 7 for 2 min at 72°C, in 2 × SSC/0.1% NP40, pH 7 for 1 min at room temperature and finally mounted with 4′,6-diamidino-2-phenylindole (DAPI) (100 ng/ml) in antifade solution.The slides were analysed by two independent observers using a Leitz fluorescence microscope DMRA2 equipped with a filter set for fluorescein isothiocyanate (FITC), Texas Red, Aqua and DAPI/Texas Red/FITC.
Only individual and well-delineated sperm nuclei were scored.The scoring criteria were similar for WCP probes and LSPs.Briefly, overlapping sperm nuclei, disrupted nuclei or large nuclei with diffuse signals were not considered.Sperm nuclei were scored as having two identical signals when the two spots were of equal size and intensity and separated by at least the diameter of one hybridization domain.

Data analysis
The chi-square test was used to statistically analyse the segregation patterns observed in patients, compare the fluorescent phenotypes between the translocation carriers and the control subject and compare results between the two types of labelling procedures.Student's t-test was used to verify the homogeneity of segregation data between WCP and LSP assays.Differences were considered to be significant when P < 0.05.

Results
The probe specificity and efficiency were first determined in lymphocyte preparations from normal subjects.The hybridization efficiency values ranged from 99.5 to 99.9% according to the probes.No significant difference (P > 0.05) in the hybridization efficiency was noted between WCP probes and LSPs.When the hybridization efficiency of the probes was tested on sperm samples from the normal subjects, the hybridization efficiencies of the probe sets ranged from 99.2 to 99.8%.
A total of 26 277 sperm nuclei from the translocation carriers (from 891 to 5000) was analysed.In Patient A, only the WCP assay was performed because of the poor quality of the sperm sample obtained and the impossibility to get another sample from this patient.The results of the segregation analysis are summarized in Table I.
Both WCP probes and LSPs gave similar results (P > 0.05) in the four patients and the control (Figure 1A-F).The rate of normal and balanced spermatozoa resulting from the alternate mode of segregation ranged from 77.55 to 92.84%.The lowest frequency of alternate segregation was observed in the patients displaying the worst spermiogram (Patient A with oligoasthenozoospermia).
The proportion of unbalanced spermatozoa resulting from adjacent modes of segregation accounted for 6.75-20.43% of the scored cells.In both WCP and LSP assays, the imbalance patterns were similar, with an excess of disomy rates over the complementary frequencies of nullisomy (P < 0.05).The rate of spermatozoa displaying a fluorescent pattern corresponding to either 3:0 segregation or diploidy ranged from 0.29 to 1.01% (Figure 1D).
In the sperm samples from the control subjects, the mean frequencies of disomy and nullisomy for chromosomes 13, 14 and 15 ranged from 0.07 to 0.20% (Table II).These values were significantly lower (P < 0.05) than the rates of imbalances found in the four translocation carriers.The mean frequency of diploidy in these subjects was 0.06%.

Discussion
Although both t(13;15) and t(14;15) Robertsonian translocations are among the less frequent translocations found in humans, the segregation analysis of these two rearrangements With a frequency of 1/12 000, trisomy 13 (Patau's syndrome) is a severe disorder, for which ∼20% of cases result from a translocation.Retinoblastoma is associated with partial monosomy and trisomy 13, of which 20% are due to Robertsonian translocation.Both chromosomes 14 and 15 have imprinted regions, and consequently, uniparental disomy (UPD) for these chromosomes is a possible outcome, resulting in various syndromes.Maternal UPD for chromosome 15 led to Prader-Willi syndrome, whereas paternal UPD for chromosome 15 led to Angelman's syndrome.Also, both maternal and paternal UPDs for chromosome 14 result in distinct abnormal phenotypes.Most cases of UPD 14 are associated with a Robertsonian translocation (Kotzot and Utermann, 2005).The risk of UPD in the case of common t(13;14) translocation has been estimated to be ∼0.6%.The risk of UPD 15 linked to nonhomologous Robertsonian translocation could be at least as small.However, in the context of a rare Robertsonian translocation involving chromosomes 14 and 15, one can make no firm statement because of the paucity of available data.Consequently, a different propensity for UPD, perhaps higher, cannot be excluded.
Two different, but complementary FISH procedures have been used in this study to assess meiotic segregation patterns in sperm, i.e.WCP probes and LSPs.LSPs have been used in most of the previous sperm studies performed in normal donors and carriers of Robertsonian translocations.The use of LSP on human sperm can be limited by the DNA compaction and the lack of sperm nucleus decondensation, or by the small size of fluorescent signals in situ.On the contrary, signals generated by WCP probe are easier to detect in sperm nuclei.Rives et al. (1998Rives et al. ( , 1999)), who introduced this technique for sperm study, demonstrated that WCP labelling required moderate sperm decondensation to provide an efficient signal and that there was no significant variability in the rates of sperm disomy estimated by WCP probe in comparison with the frequent heterogeneity of disomy rates reported with centromeric probes or LSP.However, WCP signals can also easily be overlapped, which compels us to standardize fully the sperm decondensation protocol and the screening criteria.Thus, unlike Morel et al. (2001) who claimed that the WCP technique allows the in situ distinction of both normal and balanced spermatozoa, we have considered that WCP probe was not efficient enough to precisely estimate the proportion of normal and balanced nuclei, because a significant number of nuclei displayed partially overlapped WCP signals.However, the parallel use of these two types of probes allowed us to compare the efficiency of the two procedures and provided an internal control for an accurate analysis of meiotic segregation in human sperm.Both approaches gave similar results for adjacent and alternate patterns, which confirm the previous data on the competence of WCP on human sperm (Rives et al., 1998(Rives et al., , 2005)).
In the four carriers, the rate of sperm nuclei with normal and balanced patterns resulting from an alternate segregation is significantly higher than the rates of adjacent segregation leading to unbalanced gametes (Table I).This finding is in good agreement with results from previous sperm studies of both common and uncommon Robertsonian translocations (Rives et al., 2005;Roux et al., 2005;Ogur et al., 2006).Similar distribution of alternate and adjacent patterns in sperm of male carriers of Robertsonian translocations was observed in several mammalian species (Chayko and Martin-DeLeon, 1992;Tateno et al., 1994).The prevalence of alternate segregation does not appear to be influenced by the nature of acrocentric chromosomes involved in the rearrangement.Meiotic analyses of trivalent synaptonemal complexes performed in several male carriers of Robertsonian translocations have evidenced the preferential formation of cis-configurations, promoting the alternate segregation of translocated chromosomes (Luciani et al., 1984;  et al., 1991).These data support the hypothesis of a similar behaviour of all acrocentric chromosomes through male meiosis when involved in Robertsonian translocation, with a high prevalence of alternate segregation.This phenomenon could be less marked in female meiosis.Indeed, the few segregation studies performed in women carrying Robertsonian translocations (Munne et al., 2000;Durban et al., 2001) reported significantly higher rates of imbalance than those in men (mean values: 34 versus 13%).A similar higher production of alternates in gametes of female translocation carriers than in male carriers was also observed in the mouse and pig (Tease and Fisher, 1998;Pinton et al., 2005).This observation should be related to the fact that there are also more aneuploid gametes produced in female meiosis than in male meiosis in normal subjects (on average 22% in women versus 10% in men).These data suggest differences in the production and the outcome of imbalances according to the parental origin of the translocations.The weakness of the female meiosis checkpoint mechanisms has been evoked to explain this difference between male and female meioses (LeMaire-Adkins et al., 1997;Odorisio et al., 1998).Both impaired synapsis of homologous segments between the normal and the rearranged acrocentrics and association with the sex chromatin (Barr body and XY body) are fundamental elements in the disruption of gametogenesis.Spermatogenesis could be more sensitive than ovogenesis to these obstacles (Solari, 1999;Eaker et al., 2001).An epidemiological finding supporting this hypothesis is the different reproductive risk for male and female carriers of Robertsonian translocation.For instance, in female carriers of t(14;21), imbalances are detected in 15% of first-trimester pregnancies, whereas the same risk appears to be < 2% for male carriers (Boue and Gallano, 1984;Stene and Stengel-Rutkowski, 1988).Data obtained from PGD performed in Robertsonian translocation carriers have indicated high rates of unbalanced embryos (Conn et al., 1998;Alves et al., 2002).In this context, it would be interesting to know whether the translocations of maternal origin produce more unbalanced embryos than those of paternal origin.In addition, a distinction could be made between rare and frequent Robertsonian translocations, because an increased production of imbalances in cases of rare Robertsonian translocations cannot be excluded.Despite the similarity between frequent and infrequent Robertsonian translocations concerning the overall distribution of alternate and adjacent segregations, the risk of an excess of imbalances in rare rearrangements might be kept in mind and fully justifies the application of PGD in all couples with a rare Robertsonian translocation.Significant variations exist in the incidence and the distribution of imbalances produced in the four analysed translocations (Table I).A similar variability can be noted in the results of previous sperm studies of frequent and infrequent Robertsonian translocations, even in patients carrying identical rearrangements.Thus, in the 20 cases of t(13;14) reported, the frequencies of unbalanced spermatozoa ranged from 7.7 to 23.3%.In some studies, an excess of nullisomy for either one or both chromosomes involved in the translocations was also observed (Honda et al., 2000;Anton et al., 2004), whereas higher rate of disomic sperm was noted in this study and other reports (Frydman et al., 2001;Ogur et al., 2006).These differences in results might depend on technical aspects of sperm FISH studies such as types of probes, their efficiency on human sperm or the scoring criteria.However, such a variability emphasizes the possibility of variations in the formation and the meiotic behaviour of Robertsonian translocations, leading to discernible intra-or inter-individual fluctuations in the segregation pattern.Several authors have evoked the possibility of cell maturation arrest during the meiotic process to explain differences in production of disomic and nullisomic spermatozoa (Honda et al., 2000), but this finding might also be partially attributable to unequal hybridization efficiency (Rousseaux et al., 1995).

Navarro
A noticeable point is the variable alterations of spermatogenesis observed in Robertsonian translocation carriers, ranging from normal to azoospermia.Recently, an original approach for studying the correlation between the production of imbalances and infertility was developed by Brugnon et al. (2006) based on the parallel analysis of apoptosis markers and meiotic segregation in sperm samples from translocation carriers.The authors reported significant increases of DNA fragmentation and phosphatidylserine externalization in spermatozoa of Robertsonian and reciprocal translocation carriers.It was postulated that the association between the trivalent and XY body may lead to different levels of meiotic disturbance, and subsequently to germ cell deterioration or variable production of unbalanced gametes (Gabriel-Robez and Rumpler, 1996;Roux et al., 2005).In our study, the most elevated rate of imbalance (21.44%) was found in the patient presenting the worst sperm quality, i.e. the carrier of the t(13;15) with a severe oligoasthenozoospermia.The three other subjects displayed normal or subnormal spermiograms and low rates of imbalances.Similar observation can be made when comparing the t(13;15) carrier studied by Rives et al. (2005) with 18% of unbalanced spermatozoa and an oligoasthenozoospermia, and the t(13;15) carrier investigated by Pellestor (1990), presenting a normal spermiogram and only 10.4% of imbalances in sperm.These discrepancies justify the sperm analysis of each Robertsonian translocation carrier before assisted reproduction technique (ART) procedure.The accurate identification of an effective correlation between the production of unbalanced gametes and altered semen parameters is an important question, which requires the analysis of more rearrangements.An important factor to consider in this investigation might be the location of breakpoints in Robertsonian translocations.The identification and the comparison of breakpoints in various Robertsonian translocations have revealed the highly variable location of breakpoints in rare Robertsonian translocations, whereas the common t(13;14) and t(14;21) showed consistent breakpoint locations (Page et al., 1996).This finding has led to the hypothesis that different mechanisms of formation might exist for common and rare Robertsonian translocations, and such variations in breakpoints might result in the variable alterations of spermiogram observed in Robertsonian translocation carriers.